A study of a proline specific seprase activity from mammalian serum

Seprase or Fibroblast activation protein (FAP) is an integral membrane serine peptidase, which has been shown to have gelatinase activity. It appears to act as a proteolytically active 170-kDa dimer, consisting of two 97-kDa subunits. It is a member of the group type II integral serine proteases, which include dipeptidyl peptidase IV (DPPIV/CD26) and related type II trans-membrane prolyl serine peptidases, which exert their mechanisms of action on the cell surface. DPPIV and Seprase exhibit multiple functions due to their abilities to form complexes with each other and to interact with other membrane-associated molecules. Localization of these protease complexes at cell surface protrusions, called invadopodia, may have a prominent role in processing soluble factors and in the degradation of extracellular matrix components that are essential to the cellular migration and matrix invasion that occur during tumour invasion, metastasis and angiogenesis. Seprase was isolated and purified from bovine serum, yielding a specific activity of 166.41 units/mg. The purified soluble Seprase was shown to have both exopeptidase and endopeptidase activity. The soluble form of the glycoprotein was shown to bind to the Wheat Germ Agglutinin (WGA)-Lectin Affinity Chromatography. Biochemical studies show that Seprase has a pH optimum of 8.0 and it is thermostable (up to 40 °C). The second order rate constant, k2 for DFP inhibition of Seprase was determined to be 3.31 x 103 Af V 7. Positional scanning of the Pi dipeptide library determined that Seprase has a marked preference for proline in the Si subsite. Data obtained during screening o f the P2 sub-library revealed a much broader specificity in the S2 binding pocket of bovine Seprase. In the S2 subsites, this study shows that Seprase has a preference for Nor leucine, Alanine, Leucine, Glycine, Arginine, Methionine and does not tolerate aromatic, strongly basic or acidic residues. The kinetic constant Km determined for purified Seprase was 82.10¡jM. Seprase was found to have an average kCat / Km ratio of 1.17 x 105 M 1 s'1 for cleavage o f the fluorimetric substrate Z-Gly- Pro-AMC. Tissue localisation studies identified Seprase activity in bovine large intestine, serum, kidney, liver and spleen and also in the breast cancer cell line Hs578T. Seprase was successfully cloned into prokaryotic and eukaryotic expression systems. Clinical studies on serum samples from breast cancer patients indicated that Seprase levels are elevated in patients with invasive ductal carcinoma

The livelihood impacts of cash transfers in sub-Saharan Africa: beneficiary perspectives from six countries

Cash transfers (CTs) are a social protection mechanism to reduce the poorest households’ vulnerability to shocks and build human capital by smoothing consumption and sustaining expenditure on education and social welfare. Our study examines whether and how CTs go beyond welfare objectives to promote livelihoods. Presenting a cross-case analysis using original qualitative data on beneficiary perspectives from six African countries - Kenya, Ethiopia, Malawi, Lesotho, Zimbabwe and Ghana – we explore CT livelihood impacts within household economies and social networks, paying attention to gender issues. We find that a small but predictable flow of cash improves strategic livelihood choices and stimulates productive investments, including through positive effects on beneficiary entry into risk-sharing arrangements and networks for economic collaboration. Levels of household vulnerability and labour constraint nevertheless significantly mediate the ability of CTs to consolidate present livelihood outcomes. The varying availability of economic opportunities and effective programme implementation also shape livelihood impact. Incorporating beneficiary perspectives brings to the fore the multi-dimensionality of CT effects on experiences of poverty and deprivation, including gender dynamics and intangibles such as dignity and respect; they add powerful realism to the influence of the CT on both immediate survival and livelihood choices. Beyond this, they confirm wider knowledge on productive impact and bring nuance to the conditions under which, and mechanisms by which beneficiaries’ use CTs to build productive capability and assets and to make strategic livelihood choices

Relationship of Metabolic Syndrome With Incident Aortic Valve Calcium and Aortic Valve Calcium Progression: The Multi-Ethnic Study of Atherosclerosis (MESA)*

ObjectiveMetabolic syndrome (MetS) has been associated with increased prevalence of aortic valve calcium (AVC) and with increased progression of aortic stenosis. The purpose of this study was to determine whether MetS is associated with increased risks for the development of new ("incident") AVC or for progression of established AVC as assessed by CT.Research design and methodsThe relationships of MetS or its components as well as of diabetes to risks for incident AVC or AVC progression were studied among participants with CT scans performed at baseline and at either year 2 or year 3 examinations in the Multi-Ethnic Study of Atherosclerosis (MESA).ResultsOf 5,723 MESA participants meeting criteria for inclusion, 1,674 had MetS by Adult Treatment Panel III criteria, whereas 761 had diabetes. Among the 5,123 participants without baseline AVC, risks for incident AVC, adjusted for time between scans, age, sex, race/ethnicity, LDL cholesterol, lipid-lowering medications, and smoking, were increased significantly for MetS (odds ratio [OR] 1.67 [95% CI 1.21-2.31]) or diabetes (2.06 [1.39-3.06]). In addition, there was an increase in incident AVC risk with increasing number of MetS components. Similar results were found using the International Diabetes Federation MetS criteria. Among the 600 participants (10.5%) with baseline AVC, neither MetS nor diabetes was associated with AVC progression.ConclusionsIn the MESA cohort, MetS was associated with a significant increase in incident ("new") AVC, raising the possibility that MetS may be a potential therapeutic target to prevent AVC development

Beef production from feedstuffs conserved using new technologies to reduce negative environmental impacts

End of project reportMost (ca. 86%) Irish farms make some silage. Besides directly providing feed for livestock, the provision of grass silage within integrated grassland systems makes an important positive contribution to effective grazing management and improved forage utilisation by grazing animals, and to effective feed budgeting by farmers. It can also contribute to maintaining the content of desirable species in pastures, and to livestock not succumbing to parasites at sensitive times of the year. Furthermore, the optimal recycling of nutrients collected from housed livestock can often be best achieved by spreading the manures on the land used for producing the conserved feed. On most Irish farms, grass silage will remain the main conserved forage for feeding to livestock during winter for the foreseeable future. However, on some farms high yields of whole-crop (i.e. grain + straw) cereals such as wheat, barley and triticale, and of forage maize, will be an alternative option provided that losses during harvesting, storage and feedout are minimised and that input costs are restrained. These alternative forages have the potential to reliably support high levels of animal performance while avoiding the production of effluent. Their production and use however will need to advantageously integrate into ruminant production systems. A range of technologies can be employed for crop production and conservation, and for beef production, and the optimal options need to be identified. Beef cattle being finished indoors are offered concentrate feedstuffs at rates that range from modest inputs through to ad libitum access. Such concentrates frequently contain high levels of cereals such as barley or wheat. These cereals are generally between 14% to 18% moisture content and tend to be rolled shortly before being included in coarse rations or are more finely processed prior to pelleting. Farmers thinking of using ‘high-moisture grain’ techniques for preserving and processing cereal grains destined for feeding to beef cattle need to know how the yield, conservation efficiency and feeding value of such grains compares with grains conserved using more conventional techniques. European Union policy strongly encourages a sustainable and multifunctional agriculture. Therefore, in addition to providing European consumers with quality food produced within approved systems, agriculture must also contribute positively to the conservation of natural resources and the upkeep of the rural landscape. Plastics are widely used in agriculture and their post-use fate on farms must not harm the environment - they must be managed to support the enduring sustainability of farming systems. There is an absence of information on the efficacy of some new options for covering and sealing silage with plastic sheeting and tyres, and an absence of an inventory of the use, re-use and post-use fate of plastic film on farms. Irish cattle farmers operate a large number of beef production systems, half of which use dairy bred calves. In the current, continuously changing production and market conditions, new beef systems must be considered. A computer package is required that will allow the rapid, repeatable simulation and assessment of alternate beef production systems using appropriate, standardised procedures. There is thus a need to construct, evaluate and utilise computer models of components of beef production systems and to develop mathematical relationships to link system components into a network that would support their integration into an optimal system model. This will provide a framework to integrate physical and financial on-farm conditions with models for estimating feed supply and animal growth patterns. Cash flow and profit/loss results will be developed. This will help identify optimal systems, indicate the cause of failure of imperfect systems and identify areas where applied research data are currently lacking, or more basic research is required

Examining the impact of a universal social and emotional learning intervention (Passport) on internalising symptoms and other outcomes among children, compared to the usual school curriculum: study protocol for a school-based cluster randomised trial.

Background: School-based universal social and emotional learning (SEL) interventions implemented during the transition to adolescence may be efficacious in preventing the development of mental health difficulties. This protocol describes a two-arm parallel cluster randomised controlled trial to investigate the impact of a universal SEL intervention (Passport, compared to usual provision) on internalising symptoms (primary outcome), emotion regulation, well-being, loneliness, social support, bullying, academic attainment, and health-related quality of life in English primary school pupils aged 9-11 years. A developer-led trial demonstrated the feasibility, acceptability, and utility of Passport; this will be the first independent trial.Methods: Sixty primary schools will be recruited across the Greater Manchester city region and surrounding areas, involving 2400 pupils aged 8-9 at baseline. Schools will be allocated to the intervention arm to implement Passport over 18 weekly sessions or to the control arm to implement the usual school curriculum. Random allocation will be at school level following completion of baseline measures, with minimisation to ensure balance across trial arms in school size and free school meal eligibility. Measures will be collected at baseline, post-intervention (12 months post-baseline), and at 12 months follow-up (24 months post-baseline). The primary outcome analysis (intervention effects on internalising symptoms at post-intervention) will comprise a two-level (school, child) hierarchical linear model, following the intention-to-treat principle. Additional analyses will be undertaken to assess intervention effects on secondary outcomes, maintenance effects for all outcomes, intervention compliance moderator effects, subgroup moderator effects, and mechanisms underpinning intervention effects on the primary outcome. A mixed-methods implementation and process evaluation will examine factors that influence implementation, and a health economic evaluation will assess the cost-effectiveness of the intervention.Discussion: Findings will provide educators with crucial knowledge of whether and how increasing emotion regulation through a universal intervention impacts internalising symptoms and a range of related outcomes. Findings will also inform policy related to the promotion of mental health among children and young people. If the intervention is found to be efficacious in reducing internalising symptoms and is also cost-effective, it may offer high potential as a preventative intervention for widespread implementation.Trial Registration: ISRCTN12875599; registered on 24 November 2022.</p

Examining the impact of a universal social and emotional learning intervention (Passport) on internalising symptoms and other outcomes among children, compared to the usual school curriculum:study protocol for a school-based cluster randomised trial

BACKGROUND: School-based universal social and emotional learning (SEL) interventions implemented during the transition to adolescence may be efficacious in preventing the development of mental health difficulties. This protocol describes a two-arm parallel cluster randomised controlled trial to investigate the impact of a universal SEL intervention (Passport, compared to usual provision) on internalising symptoms (primary outcome), emotion regulation, well-being, loneliness, social support, bullying, academic attainment, and health-related quality of life in English primary school pupils aged 9-11 years. A developer-led trial demonstrated the feasibility, acceptability, and utility of Passport; this will be the first independent trial.METHODS: Sixty primary schools will be recruited across the Greater Manchester city region and surrounding areas, involving 2400 pupils aged 8-9 at baseline. Schools will be allocated to the intervention arm to implement Passport over 18 weekly sessions or to the control arm to implement the usual school curriculum. Random allocation will be at school level following completion of baseline measures, with minimisation to ensure balance across trial arms in school size and free school meal eligibility. Measures will be collected at baseline, post-intervention (12 months post-baseline), and at 12 months follow-up (24 months post-baseline). The primary outcome analysis (intervention effects on internalising symptoms at post-intervention) will comprise a two-level (school, child) hierarchical linear model, following the intention-to-treat principle. Additional analyses will be undertaken to assess intervention effects on secondary outcomes, maintenance effects for all outcomes, intervention compliance moderator effects, subgroup moderator effects, and mechanisms underpinning intervention effects on the primary outcome. A mixed-methods implementation and process evaluation will examine factors that influence implementation, and a health economic evaluation will assess the cost-effectiveness of the intervention.DISCUSSION: Findings will provide educators with crucial knowledge of whether and how increasing emotion regulation through a universal intervention impacts internalising symptoms and a range of related outcomes. Findings will also inform policy related to the promotion of mental health among children and young people. If the intervention is found to be efficacious in reducing internalising symptoms and is also cost-effective, it may offer high potential as a preventative intervention for widespread implementation.TRIAL REGISTRATION: ISRCTN12875599; registered on 24 November 2022.</p

The role of molecular testing and enzyme analysis in the management of hypomorphic citrullinemia

No Abstract.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69185/1/33256_ftp.pd

Beef production from feedstuffs conserved using new technologies to reduce negative environmental impacts

End of Project ReportThe three separate components with parallel objectives to this programme were to: 1. Develop technologies for conserving and optimally feeding alternative/complimentary feedstuffs to grass silage. 2. Quantify the use and re-use of plastic sheeting or film used to seal ensiled feedstuffs or mulch maize, and evaluate some new options. 3. Develop computer programs that will facilitate investigating prototype models of forage-based beef production systems

Tissue sampling methods and standards for vertebrate genomics

The recent rise in speed and efficiency of new sequencing technologies have facilitated high-throughput sequencing, assembly and analyses of genomes, advancing ongoing efforts to analyze genetic sequences across major vertebrate groups. Standardized procedures in acquiring high quality DNA and RNA and establishing cell lines from target species will facilitate these initiatives. We provide a legal and methodological guide according to four standards of acquiring and storing tissue for the Genome 10K Project and similar initiatives as follows: four-star (banked tissue/cell cultures, RNA from multiple types of tissue for transcriptomes, and sufficient flash-frozen tissue for 1 mg of DNA, all from a single individual); three-star (RNA as above and frozen tissue for 1 mg of DNA); two-star (frozen tissue for at least 700 μg of DNA); and one-star (ethanol-preserved tissue for 700 μg of DNA or less of mixed quality). At a minimum, all tissues collected for the Genome 10K and other genomic projects should consider each species’ natural history and follow institutional and legal requirements. Associated documentation should detail as much information as possible about provenance to ensure representative sampling and subsequent sequencing. Hopefully, the procedures outlined here will not only encourage success in the Genome 10K Project but also inspire the adaptation of standards by other genomic projects, including those involving other biota

Anxiety and depression following critical illness: A comparison of the recovery trajectories of patients and caregivers

Background: Following critical illness, family members are often required to adopt caregiving responsibilities. Anxiety and depression are common long term problems for both patients and caregivers. However, at present, it is not known how the trajectories of these symptoms compare between patients and caregivers. Objectives: The aim of this study was to investigate and compare the trajectories of anxiety and depression in patients and caregivers in the first year following critical illness. Methods: This study analyses data from a prospective multicentre cohort study of patients and caregivers who underwent a complex recovery intervention following critical illness. Paired patients and caregivers were recruited. The Hospital Anxiety and Depression Scale was used to evaluate symptoms of anxiety and depression at three timepoints: baseline; 3 months; and 12 months in both patient and caregivers. A linear mixed-effects regression model was used to evaluate the trajectories of these symptoms over the first year following critical illness. Results: 115 paired patients and caregivers, who received the complex recovery intervention, were recruited. There was no significant difference in the relative trajectory of depressive symptoms between patients and caregivers in the first 12 months following critical illness (p = 0.08). There was, however, a significant difference in the trajectory of anxiety symptoms between patients and caregivers during this time period (p = 0.04), with caregivers seeing reduced resolution of symptoms in comparison to patients. Conclusions: Following critical illness, symptoms of anxiety and depression are common in both patients and caregivers. The trajectory of symptoms of depression was similar between caregivers and patients; however, there was a significantly different recovery trajectory in symptoms of anxiety. Further research is required to understand the recovery pathway of caregivers in order to design effective interventions