Interrogating fragments using a protein thermal shift assay

Protein thermal shift is a relatively rapid and inexpensive technique for the identification of low molecular weight compound interactions with protein targets. An increase in the melting temperature of the target protein in the presence of a test ligand is indicative of a promising ligand-protein interaction. Due to its simplicity, protein thermal shift is an attractive method for screening libraries and validating hits in drug discovery programs. The methodology has been used successfully in high throughput screens of small molecule libraries, and its application has been extended to report on protein-drug-like-fragment interactions. Here, we review how protein thermal shift has been employed recently in fragment-based drug discovery (FBDD) efforts, and highlight its application to protein-protein interaction targets. Multiple validation of fragment hits by independent means is paramount to ensure efficient and economical progress in a FBDD campaign. We discuss the applicability of thermal shift assays in this light, and discuss more generally what one does when orthogonal approaches disagree

Backbone and side chain H-1, N-15 and C-13 assignments for the oxidised and reduced forms of the oxidoreductase protein DsbA from Staphylococcus aureus

The function and dynamics of the thiol-disulfide oxidoreductase DsbA in the low-GC gram positive bacterium, Staphylococcus aureus, are yet to be elucidated. Here we report 13C, 15N and 1H assignments for the oxidised and reduced forms of SaDsbA as a prelude to further studies on the enzyme

Structural studies of MUC1 core related peptides

Polymorphic epithelial mucins are large complex glycoproteins which consist of a single polypeptide backbone, a large domain of which is usually made up of degenerate tandem repeats. One such molecule MUC1 is expressed at the surface of human mammary cells and is developmentally regulated and aberrantly expressed in tumours. These mucins have been identified as the target antigens for a number of murine monoclonal antibodies raised against a variety of immunogens including human milk products and breast tumour extracts. The anti-MUC1 antibodies have been shown to display tumour reactivity and have been used both as agents for imaging and in immunoassays to assess tumour burden and response to therapy. A number of these antibodies have been found to define epitopes within the tandem repeat of the protein core of the MUC1. Hydropathicity calculations and secondary structure predictions on the twenty amino acid tandem repeat of the MUC1 core protein have identified a hydrophilic domain Pro-Asp-Thr-Arg-Pro-Ala-Pro, which has a high probability of turn formation. It is within this domain that the epitopes of the anti-MUC1 antibodies are found. High field N.M.R. studies undertaken on an antigenic twenty amino acid peptide corresponding to the tandem repeat sequence of MUC1 in dimethyl sulphoxide have identified the presence of a type-I beta-turn in the region Pro-Asp-Thr-Arg. This turn overlaps the epitopes of all of the MUC1 antibodies characterised to date. In an attempt to identify more precisely the conformational requirements for binding to two anti-MUC1 antibodies, HMFG1 and HMFG2, the solution structures of several MUC1 core related peptides in dimethyl sulphoxide have been investigated. All of the peptides studied have been found to contain either beta-turns or modified turns which overlap their hydrophilic epitope domains. While these observations may provide some explanation for the observed reactivity of the different peptides, they give little insight into the precise structural requirements for antibody binding. Due to the flexibility of linear peptides in solution it is not possible to define the side chain conformations which are crucial to the processes of antibody recognition. In order to define the bound conformations of antigenic peptides using N.M.R. it is necessary to undertake experiments in the presence of antibody. Initial experiments have been performed in order to determine the conformation of the MUC 1 core related twenty amino acid peptide when bound to the anti-mucin antibody C595. In addition DNA coding for the variable domains of C595 has been cloned and sequenced in order to facilitate both expression of recombinant antibody binding fragments and the modelling of the binding site. These studies should provide a clearer understanding of the structural basis of antibody recognition of the peptides, and may give an insight into the specificity of anti-MUC1 antibodies for malignant cells

Structural studies of MUC1 core related peptides

Polymorphic epithelial mucins are large complex glycoproteins which consist of a single polypeptide backbone, a large domain of which is usually made up of degenerate tandem repeats. One such molecule MUC1 is expressed at the surface of human mammary cells and is developmentally regulated and aberrantly expressed in tumours. These mucins have been identified as the target antigens for a number of murine monoclonal antibodies raised against a variety of immunogens including human milk products and breast tumour extracts. The anti-MUC1 antibodies have been shown to display tumour reactivity and have been used both as agents for imaging and in immunoassays to assess tumour burden and response to therapy. A number of these antibodies have been found to define epitopes within the tandem repeat of the protein core of the MUC1. Hydropathicity calculations and secondary structure predictions on the twenty amino acid tandem repeat of the MUC1 core protein have identified a hydrophilic domain Pro-Asp-Thr-Arg-Pro-Ala-Pro, which has a high probability of turn formation. It is within this domain that the epitopes of the anti-MUC1 antibodies are found. High field N.M.R. studies undertaken on an antigenic twenty amino acid peptide corresponding to the tandem repeat sequence of MUC1 in dimethyl sulphoxide have identified the presence of a type-I beta-turn in the region Pro-Asp-Thr-Arg. This turn overlaps the epitopes of all of the MUC1 antibodies characterised to date. In an attempt to identify more precisely the conformational requirements for binding to two anti-MUC1 antibodies, HMFG1 and HMFG2, the solution structures of several MUC1 core related peptides in dimethyl sulphoxide have been investigated. All of the peptides studied have been found to contain either beta-turns or modified turns which overlap their hydrophilic epitope domains. While these observations may provide some explanation for the observed reactivity of the different peptides, they give little insight into the precise structural requirements for antibody binding. Due to the flexibility of linear peptides in solution it is not possible to define the side chain conformations which are crucial to the processes of antibody recognition. In order to define the bound conformations of antigenic peptides using N.M.R. it is necessary to undertake experiments in the presence of antibody. Initial experiments have been performed in order to determine the conformation of the MUC 1 core related twenty amino acid peptide when bound to the anti-mucin antibody C595. In addition DNA coding for the variable domains of C595 has been cloned and sequenced in order to facilitate both expression of recombinant antibody binding fragments and the modelling of the binding site. These studies should provide a clearer understanding of the structural basis of antibody recognition of the peptides, and may give an insight into the specificity of anti-MUC1 antibodies for malignant cells

Amelioration of root disease of subterranean clover (Trifolium subterraneum) by mineral nutrients

Subterranean clover (Trifolium subterraneum) is a key pasture legume across southern Australia and elsewhere. Decline in subterranean clover pastures was first recognised in Australia during the 1960s and manifests as an increase in weeds and a decrease in desirable legume species. While both root disease and poor nutrition contribute to subterranean clover pasture decline, the relationships between root disease and nutrition have not been determined. The objective of this study was to define these relationships. Field experiments were undertaken to determine the nutritional and pathogen status of soils and subterranean clover from three Western Australian field sites. Subsequently, controlled environment experiments were undertaken to determine the relative severities of tap and lateral root disease and growth of plants when soil cores taken from these three field sites were amended with a complete nutrient solution or a range of individual macro- or micronutrient treatments. Application of a ‘Hoaglands’ complete nutrient solution decreased the severity of tap root disease by an average of 45% and lateral root disease by 32%. Amendment with K alone reduced the severity of tap root disease an average of 32%; while the application of N alone reduced the severity of tap root disease by 33% and lateral root disease by 27%. Application of Hoaglands, K, N or Zn increased shoot and root dry weight, while Mo only increased shoot dry weight. This is the first report to show that mineral nutrients can substantially ameliorate root disease in subterranean clover. The results demonstrate that while root disease limits plant growth, improvement in the nutritional status of nutrient-impoverished soils can significantly reduce root disease. There is significant potential to incorporate nutrient amendments into an integrated and more sustainable approach to better manage root disease and to increase productivity of pasture legumes where soils are inherently nutrient deficient in one or more nutrients

Influence of positional correlations on the propagation of waves in a complex medium with polydisperse resonant scatterers

We present experimental results on a model system for studying wave propagation in a complex medium exhibiting low frequency resonances. These experiments enable us to investigate a fundamental question that is relevant for many materials, such as metamaterials, where low-frequency scattering resonances strongly influence the effective medium properties. This question concerns the effect of correlations in the positions of the scatterers on the coupling between their resonances, and hence on wave transport through the medium. To examine this question experimentally, we measure the effective medium wave number of acoustic waves in a sample made of bubbles embedded in an elastic matrix over a frequency range that includes the resonance frequency of the bubbles. The effective medium is highly dispersive, showing peaks in the attenuation and the phase velocity as functions of the frequency, which cannot be accurately described using the Independent Scattering Approximation (ISA). This discrepancy may be explained by the effects of the positional correlations of the scatterers, which we show to be dependent on the size of the scatterers. We propose a self-consistent approach for taking this "polydisperse correlation" into account and show that our model better describes the experimental results than the ISA

Effect of Polyethylene Glycol 3350 on the Handling Properties of Low Salt Wheat Dough Formulations

The effect of polyethylene glycol (PEG) 3350 addition (3%, flour wt. basis) on the properties of dough made from two Canadian Western Red Spring wheat cultivars (Triticum aestivum L. ‘Harvest’ and ‘Pembina’) differing in dough mixing requirements and dough-handling properties was investigated in a low salt dough formulation (1% NaCl, flour wt. basis). PEG was added for experimental purposes to alter water mobility to better understand underlining mechanisms, however would not be used in real bread formulations. For cultivar Harvest, but not Pembina, dough stickiness was reduced by the addition of PEG. Dough freezable water content decreased with the addition of PEG for both cultivars. Rheological measurements showed that PEG increased dough stiffness as measured by the complex modulus |G*|. Creep measurements indicated that the relative elastic component (Jel) increased whereas maximum deformation (Jmax) decreased with the addition of PEG for cultivar Harvest only. Dough made with a weaker cultivar (Harvest) with the addition of PEG performed similarly to dough made with a stronger cultivar (Pembina) without PEG. Results indicate that in a low sodium environment, availability of water is critically important for controlling a number of properties that relate closely to dough machinability, especially in a weaker wheat cultivar

Births and their outcomes by time, day and year: a retrospective birth cohort data linkage study

Background Studies of daily variations in the numbers of births in England and Wales since the 1970s have found a pronounced weekly cycle, with numbers of daily births being highest from Tuesdays to Fridays and lowest at weekends and on public holidays. Mortality appeared to be higher at weekends. As time of birth was not included in national data systems until 2005, there have been no previous analyses by time of day. Objectives To link data from birth registration and birth notification to data about care during birth and any subsequent hospital admissions and to quality assure the linkage. To use the linked data to analyse births and their outcomes by time of day, day of the week and year of birth. Design A retrospective birth cohort analysis of linked routine data. Setting England and Wales. Outcome measures Mortality of babies and mothers, and morbidity recorded at birth and any subsequent hospital admission. Population and data sources Birth registration and notification records of 7,013,804 births in 2005–14, already linked to subsequent death registration records for babies, children and women who died within 1 year of giving birth, were provided by the Office for National Statistics. Stillbirths and neonatal deaths data from confidential enquiries for 2005–9 were linked to the registration records. Data for England were linked to Hospital Episode Statistics (HES) and data for Wales were linked to the Patient Episode Database for Wales and the National Community Child Health Database. Results Cross-sectional analysis of all births in England and Wales showed a regular weekly cycle. Numbers of births each day increased from Mondays to Fridays. Numbers were lowest at weekends and on public holidays. Overall, numbers of births peaked between 09.00 and 12.00, followed by a much smaller peak in the early afternoon and a decrease after 17.00. Numbers then increased from 20.00, peaking at around 03.00–05.00, before falling again after 06.00. Singleton births after spontaneous onset and birth, including births in freestanding midwifery units and at home, were most likely to occur between midnight and 06.00, peaking at 04.00–06.00. Elective caesarean births were concentrated in weekday mornings. Births after induced labours were more likely to occur at hours around midnight on Tuesdays to Saturdays, irrespective of the mode of birth. Limitations The project was delayed by data access and information technology infrastructure problems. Data from confidential enquiries were available only for 2005–9 and some HES variables were incomplete. There was insufficient time to analyse the mortality and morbidity outcomes. Conclusions The timing of birth varies by place of birth, onset of labour and mode of birth. These patterns have implications for midwifery and medical staffing. Future work An application has now been submitted for funding to analyse the mortality outcomes and further funding will be sought to undertake the other outstanding analyses. Funding This project was funded by the National Institute for Health Research (NIHR) Health Services and Delivery Research programme and will be published in full in Health Services and Delivery Research; Vol. 7, No. 18. See the NIHR Journals Library website for further project information

Small heat-shock proteins interact with a flanking domain to suppress polyglutamine aggregation

Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal cell death in nine diseases, including Huntington disease and the spinocerebellar ataxias (SCAs). There is evidence that sHsps have a role in suppression of polyQ-induced neurodegeneration; for example, the sHsp alphaB-crystallin (αB-c) has been identified as a suppressor of SCA3 toxicity in a Drosophila model. However, the molecular mechanism for this suppression is unknown. In this study we tested the ability of αB-c to suppress the aggregation of a polyQ protein. We found that αB-c does not inhibit the formation of SDS-insoluble polyQ fibrils. We further tested the effect of αB-c on the aggregation of ataxin-3, a polyQ protein that aggregates via a two-stage aggregation mechanism. The first stage involves association of the N-terminal Josephin domain followed by polyQ-mediated interactions and the formation of SDS-resistant mature fibrils. Our data show that αB-c potently inhibits the first stage of ataxin-3 aggregation; however, the second polyQ-dependent stage can still proceed. By using NMR spectroscopy, we have determined that αB-c interacts with an extensive region on the surface of the Josephin domain. These data provide an example of a domain/region flanking an amyloidogenic sequence that has a critical role in modulating aggregation of a polypeptide and plays a role in the interaction with molecular chaperones to prevent this aggregation

TRH: Pathophysiologic and clinical implications

Thyrotropin releasing hormone is thought to be a tonic stimulator of the pituitary TSH secretion regulating the setpoint of the thyrotrophs to the suppressive effect of thyroid hormones. The peptide stimulates the release of normal and elevated prolactin. ACTH and GH may increase in response to exogenous TRH in pituitary ACTH and GH hypersecretion syndromes and in some extrapituitary diseases. The pathophysiological implications of extrahypothalamic TRH in humans are essentially unknown. The TSH response to TRH is nowadays widely used as a diganostic amplifier in thyroid diseases being suppressed in borderline and overt hyperthyroid states and increased in primary thyroid failure. In hypothyroid states of hypothalamic origin, TSH increases in response to exogenous TRH often with a delayed and/or exaggerated time course. But in patients with pituitary tumors and suprasellar extension TSH may also respond to TRH despite secondary hypothyroidism. This TSH increase may indicate a suprasellar cause for the secondary hypothyroidism, probably due to portal vessel occlusion. The TSH released in these cases is shown to be biologically inactive