Respiratory heat and moisture loss in health, asthma and chronic obstructive pulmonary disease (COPD)

It was hypothesized that Respiratory heat and moisture loss (RHML) would be altered in patients with Asthma and Chronic obstructive pulmonary disease (COPD) due to the effects of airway inflammation and re-modeling. By creating a novel device incorporating humidity, temperature and flow sensors, RHML was measured in 25 normal controls, 33 asthmatics and 17 patients with COPD. In normal subjects RHML was found to be dependent on breathing pattern as defined by tidal volume and minute ventilation whereas no association was found between RHML and body surface area or forced expiratory volume in one second (FEV1). At matched breathing patterns asthmatics whether in exacerbation or stable showed a small but significant increase in RHML compared to controls (exacerbation asthmatics -93.2 ± 0.8 (SD), p=0.003, stable asthma - 89.3 ± 7.4, p=0.025 and controls 85 ± 4.3 Joules/L). No significant difference was found in RHML between the asthmatics with an exacerbation and those with stable disease. COPD patients showed no significant difference in RHML (stable group- 83 ± 4.8, p=0.23 and exacerbation group - 81 ± 5.8 Joules/L, p=0.06) compared to controls or between exacerbation and stable groups. Evaporative heat loss was the major heat transfer modality (up to 3-times the dry convective heat loss). It can be concluded that asthma is associated with a measurable increase in heat and moisture loss in breath and that this may reflect the inflammatory and vascular changes known to occur in the asthmatic airway. Further longitudinal studies are required to assess whether the technique developed in this study can provide a practical means to measure inflammation in asthma

Development of a monoclonal anti-ADAMTS-5 antibody that specifically blocks the interaction with LRP1

The potent aggrecanase ADAMTS-5 is constitutively secreted by chondrocytes, but it is rapidly endocytosed in normal cartilage via the cell surface endocytic receptor LRP1. Therefore it is difficult to detect the total ADAMTS-5 activity produced. In this study, we isolated a monoclonal anti-ADAMTS-5 antibody 1B7 that blocks LRP1-mediated internalization without affecting the aggrecanolytic activity. Addition of 1B7 to cultured human chondrocytes revealed the full aggrecanolytic activity of ADAMTS-5 generated by the cells. 1B7 is a useful tool to estimate the ADAMTS-5 activity and to identify its potential roles in the tissues

Urban grasslands support threatened water voles

Urbanisation is often linked with habitat loss and a reduction in species richness but some species may be able to adapt to urban environments. Water voles Arvicola amphibius, a rapidly declining species in the UK, have recently been recorded in isolated grassland habitats in Glasgow, Scotland’s largest city (human population 1.2 million). The aim of this study was to determine the distribution and habitat characteristics of water vole populations occupying these dry grasslands. Field work was undertaken from March to October 2014 in a 34 km2 study area located 3 km east of the city centre. Field sign transects recorded water vole presence in 21/65 (32%) and 19/62 (31%) surveyed sites in spring and autumn, respectively. Vole occupancy increased with distance from water and was greatest in parkland, followed by sites with rank vegetation and roadside habitats. Occupancy was lower where signs of predators were recorded but surprisingly occupancy was found to be greater in the most disturbed sites, perhaps linked to the fact that many of these sites were public parks containing suitable grassland. Sites occupied by water voles were classed as neutral grasslands with species composition dominated by two main species. The number of grassland sites occupied by water voles, especially within public areas suggests that careful management of these urban grassland habitats will benefit the conservation of this highly threatened species in the UK

Collaborative systems for enhancing the analysis of social surveys: the grid enabled specialist data environments

This paper describes a group of online services which are designed to support social survey research and the production of statistical results. The 'Grid Enabled Specialist Data Environment' (GESDE) services constitute three related systems which offer facilities to search for, extract and exploit supplementary data and metadata concerned with the measurement and operationalisation of survey variables. The services also offer users the opportunity to deposit and distribute their own supplementary data resources for the benefit of dissemination and replication of the details of their own analysis. The GESDE services focus upon three application areas: specialist data relating to the measurement of occupations; educational qualifications; and ethnicity (including nationality, language, religion, national identity). They identify information resources related to the operationalisation of variables which seek to measure each of these concepts - examples include coding frames, crosswalk and translation files, and standardisation and harmonisation recommendations. These resources constitute important supplementary data which can be usefully exploited in the analysis of survey data. The GESDE services work by collecting together as much of this supplementary data as possible, and making it searchable and retrievable to others. This paper discusses the current features of the GESDE services (which have been designed as part of a wider programme of ‘e-Science’ research in the UK), and considers ongoing challenges in providing effective support for variable-oriented statistical analysis in the social sciences

Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

BACKGROUND: In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. RESULTS: Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. CONCLUSIONS: By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli

UNLV College of Education Multicultural & Diversity Newsletter

Each morning I wound my way up the steep hill along the deeply rutted dirt path, exchanging daily maaa\u27s with five bleating sheep and shouting out, ¡Hola! in response to the children who gleefully identified me as ¡Gringa! Women and children, colorful bowls of cooked maize balanced atop their heads, sauntered to and from Maria Elena\u27s where their maize would be ground; at home the dough would be shaped and flattened into tortillas, the mainstay of every meal in the small Guatemalan village of San Juan

Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands

BACKGROUND: G-protein-coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However, purification of GPCRs for biochemical study is difficult and current methods of studying receptor-ligand interactions involve in vitro systems. Caenorhabditis elegans is a soil-dwelling, bacteria-feeding nematode that uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds, making this an ideal system for studying in vivo GPCR-ligand interactions. We sought to test this by functionally expressing two medically important mammalian GPCRs, somatostatin receptor 2 (Sstr2) and chemokine receptor 5 (CCR5) in the gustatory neurons of C. elegans. RESULTS: Expression of Sstr2 and CCR5 in gustatory neurons allow C. elegans to specifically detect and respond to somatostatin and MIP-1α respectively in a robust avoidance assay. We demonstrate that mammalian heterologous GPCRs can signal via different endogenous G(α )subunits in C. elegans, depending on which cells it is expressed in. Furthermore, pre-exposure of GPCR transgenic animals to its ligand leads to receptor desensitisation and behavioural adaptation to subsequent ligand exposure, providing further evidence of integration of the mammalian GPCRs into the C. elegans sensory signalling machinery. In structure-function studies using a panel of somatostatin-14 analogues, we identified key residues involved in the interaction of somatostatin-14 with Sstr2. CONCLUSION: Our results illustrate a remarkable evolutionary plasticity in interactions between mammalian GPCRs and C. elegans signalling machinery, spanning 800 million years of evolution. This in vivo system, which imparts novel avoidance behaviour on C. elegans, thus provides a simple means of studying and screening interaction of GPCRs with extracellular agonists, antagonists and intracellular binding partners

A simple vector system to improve performance and utilisation of recombinant antibodies

BACKGROUND: Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. RESULTS: We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs). Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3). The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3)) was investigated and found to be inferior to periplasmic expression in BL21 (DE3) cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner), bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule), or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and immunohistochemistry. CONCLUSION: Nine scFv expression vectors have been generated and tested. Three vectors showed utility for expression of functional scFv fragments. One vector, pSANG14-3F, produces scFv-alkaline phosphatase fusion molecules which offers a simple, convenient and sensitive way of determining the reactivity of recombinant antibody fragments in a variety of common assay systems