HDX and In Silico Docking Reveal that Excipients Stabilize G-CSF via a Combination of Preferential Exclusion and Specific Hotspot Interactions

Assuring the stability of therapeutic proteins is a major challenge in the biopharmaceutical industry, and a better molecular understanding of the mechanisms through which formulations influence their stability is an ongoing priority. While the preferential exclusion effects of excipients are well known, the additional presence and impact of specific protein–excipient interactions have proven to be more elusive to identify and characterize. We have taken a combined approach of in silico molecular docking and hydrogen deuterium exchange-mass spectrometry (HDX-MS) to characterize the interactions between granulocyte colony-stimulating factor (G-CSF), and some common excipients. These interactions were related to their influence on the thermal-melting temperatures (Tm) for the nonreversible unfolding of G-CSF in liquid formulations. The residue-level interaction sites predicted in silico correlated well with those identified experimentally and highlighted the potential impact of specific excipient interactions on the Tm of G-CSF

Label-free segmentation of co-cultured cells on a nanotopographical gradient

The function and fate of cells is influenced by many different factors, one of which is surface topography of the support culture substrate. Systematic studies of nanotopography and cell response have typically been limited to single cell types and a small set of topographical variations. Here, we show a radical expansion of experimental throughput using automated detection, measurement, and classification of co-cultured cells on a nanopillar array where feature height changes continuously from planar to 250 nm over 9 mm. Individual cells are identified and characterized by more than 200 descriptors, which are used to construct a set of rules for label-free segmentation into individual cell types. Using this approach we can achieve label-free segmentation with 84% confidence across large image data sets and suggest optimized surface parameters for nanostructuring of implant devices such as vascular stents

Identification of Schistosoma mansoni microRNAs

Background: MicroRNAs (miRNAs) constitute a class of single-stranded RNAs which play a crucial role in regulating development and controlling gene expression by targeting mRNAs and triggering either translation repression or messenger RNA (mRNA) degradation. miRNAs are widespread in eukaryotes and to date over 14,000 miRNAs have been identified by computational and experimental approaches. Several miRNAs are highly conserved across species. In Schistosoma, the full set of miRNAs and their expression patterns during development remain poorly understood. Here we report on the development and implementation of a homology-based detection strategy to search for miRNA genes in Schistosoma mansoni. In addition, we report results on the experimental detection of miRNAs by means of cDNA cloning and sequencing of size-fractionated RNA samples. Results: Homology search using the high-throughput pipeline was performed with all known miRNAs in miRBase. A total of 6,211 mature miRNAs were used as reference sequences and 110 unique S. mansoni sequences were returned by BLASTn analysis. The existing mature miRNAs that produced these hits are reported, as well as the locations of the homologous sequences in the S. mansoni genome. All BLAST hits aligned with at least 95% of the miRNA sequence, resulting in alignment lengths of 19-24 nt. Following several filtering steps, 15 potential miRNA candidates were identified using this approach. By sequencing small RNA cDNA libraries from adult worm pairs, we identified 211 novel miRNA candidates in the S. mansoni genome. Northern blot analysis was used to detect the expression of the 30 most frequent sequenced miRNAs and to compare the expression level of these miRNAs between the lung stage schistosomula and adult worm stages. Expression of 11 novel miRNAs was confirmed by northern blot analysis and some presented a stage-regulated expression pattern. Three miRNAs previously identified from S. japonicum were also present in S. mansoni. Conclusion: Evidence for the presence of miRNAs in S. mansoni is presented. The number of miRNAs detected by homology-based computational methods in S. mansoni is limited due to the lack of close relatives in the miRNA repository. In spite of this, the computational approach described here can likely be applied to the identification of pre-miRNA hairpins in other organisms. Construction and analysis of a small RNA library led to the experimental identification of 14 novel miRNAs from S. mansoni through a combination of molecular cloning, DNA sequencing and expression studies. Our results significantly expand the set of known miRNAs in multicellular parasites and provide a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites

The importance and clinical relevance of surfaces in tissue culture

Cell and tissue culture has evolved from the use of simple glassware for the propagation of cells and tissues into a comprehensive platform for interrogating complex biological systems, directing cell fate and deriving products with clinical and therapeutic value. However, despite significant advances, current in vitro culture approaches remain limited in their capacity to model the clinical/biological complexities of disease, in part at least due to the deficiencies of existing culture materials. The challenge is therefore to identify innovative materials-based solutions that have greater control over cells in vitro, while better representing biological systems in vivo. Such platforms would be suitable for biomarker discovery and tissue engineering applications. This review examines the development of tissue culture materials, advances in our understanding of cell-surface interactions and the application of this knowledge towards the development of new approaches for better examining biological events

Electron beam computed tomography (EBCT) funding protocol

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Genetic testing of children for adult-onset conditions: opinions of the British adult population and implications for clinical practice

This study set out to explore the attitudes of a representative sample of the British public towards genetic testing in children to predict disease in the future. We sought opinions about genetic testing for adult-onset conditions for which no prevention/treatment is available during childhood, and about genetic 'carrier' status to assess future reproductive risks. The study also examined participants' level of agreement with the reasons professional organisations give in favour of deferring such testing. Participants (n=2998) completed a specially designed questionnaire, distributed by email. Nearly half of the sample (47%) agreed that parents should be able to test their child for adult-onset conditions, even if there is no treatment or prevention at time of testing. This runs contrary to professional guidance about genetic testing in children. Testing for carrier status was supported by a larger proportion (60%). A child's future ability to decide for her/himself if and when to be tested was the least supported argument in favour of deferring testing.European Journal of Human Genetics advance online publication, 5 November 2014; doi:10.1038/ejhg.2014.221

Thermoresponsive polymer micropatterns fabricated by dip-pen nanolithography for a highly controllable substrate with potential cellular applications

We report a novel approach for patterning thermoresponsive hydrogels based on N,N-diethylacrylamide (DEAAm) and bifunctional Jeffamine ED-600 by dip-pen nanolithography (DPN). The direct writing of micron-sized thermoresponsive polymer spots was achieved with efficient control over feature size. A Jeffamine-based ink prepared through the combination of organic polymers, such as DEAAm, in an inorganic silica network was used to print thermosensitive arrays on a thiol-silanised silicon oxide substrate. The use of a Jeffamine hydrogel, acting as a carrier matrix, allowed a reduction in the evaporation of ink molecules with high volatility, such as DEAAm, and facilitated the transfer of ink from tip to substrate. The thermoresponsive behaviour of polymer arrays which swell/de-swell in aqueous solution in response to a change in temperature was successfully characterised by atomic force microscopy (AFM) and Raman spectroscopy: a thermally-induced change in height and hydration state was observed, respectively. Finally, we demonstrate that cells can adhere to and interact with these dynamic features and exhibit a change in behaviour when cultured on the substrates above and below the transition temperature of the Jeffamine/DEAAm thermoresponsive hydrogels. This demonstrates the potential of these micropatterned hydrogels to act as a controllable surface for cell growth

Rational substrate and enzyme engineering of transketolase for aromatics

The uses of 3-formylbenzoic acid and 4-formylbenzoic acid as molecular probes along with previous and new transketolase mutants revealed the factors governing the rate of reaction between transketolase and aromatic aldehydes. The novel α,α-dihydroxyketones were produced at 15 to 30-fold higher yields and up to 250-fold higher specific activities with D469T TK when compared to those obtained for benzaldehyde

Effects of hydroxyapatite and PDGF concentrations on osteoblast growth in a nanohydroxyapatite-polylactic acid composite for guided tissue regeneration

The technique of guided tissue regeneration (GTR) has evolved over recent years in an attempt to achieve periodontal tissue regeneration by the use of a barrier membrane. However, there are significant limitations in the currently available membranes and overall outcomes may be limited. A degradable composite material was investigated as a potential GTR membrane material. Polylactic acid (PLA) and nanohydroxyapatite (nHA) composite was analysed, its bioactive potential and suitability as a carrier system for growth factors were assessed. The effect of nHA concentrations and the addition of platelet derived growth factor (PDGF) on osteoblast proliferation and differentiation was investigated. The bioactivity was dependent on the nHA concentration in the films, with more apatite deposited on films containing higher nHA content. Osteoblasts proliferated well on samples containing low nHA content and differentiated on films with higher nHA content. The composite films were able to deliver PDGF and cell proliferation increased on samples that were pre absorbed with the growth factor. nHA–PLA composite films are able to deliver active PDGF. In addition the bioactivity and cell differentiation was higher on films containing more nHA. The use of a nHA–PLA composite material containing a high concentration of nHA may be a useful material for GTR membrane as it will not only act as a barrier, but may also be able to enhance bone regeneration by delivery of biologically active molecules