236 research outputs found
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
Temporal and embryonic lineage-dependent regulation of human vascular SMC development by NOTCH3.
Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8-9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner.This project was supported by a grant from British Heart
Foundation (BHF) (NH/11/1/28922) and the National Institute
for Health Research Cambridge Biomedical Research
Centre. William G Bernard’s studentship is funded by the
BHF (FS/11/77/29327). Dr Sanjay Sinha is a BHF Clinical
Senior Research Fellow (FS/13/29/30024).This is the final version of the article. It was first published by Mary Ann Liebert Inc at http://online.liebertpub.com/doi/10.1089/scd.2014.052
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 2â„¢ (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
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