Comparison of the pH- and thermally-induced fluctuations of a therapeutic antibody Fab fragment by molecular dynamics simulation

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions. A deeper understanding of the drivers of instability across different stress conditions, will enable the engineering of more robust protein scaffolds. We compared the impacts of low pH and high temperature stresses on the structure of a humanized antibody fragment (Fab) A33, using atomistic molecular dynamics simulations, using a recent 2.5 Å crystal structure. This revealed that low-pH induced the loss of native contacts in the domain CL. By contrast, thermal stress led to 5–7% loss of native contacts in all four domains, and simultaneous loss of >30% of native contacts in the VL-VH and CL-CH interfaces. This revealed divergent destabilising pathways under the two different stresses. The underlying cause of instability was probed using FoldX and Rosetta mutation analysis, and packing density calculations. These agreed that mutations in the CL domain, and CL-CH1 interface have the greatest potential for stabilisation of Fab A33. Several key salt bridge losses underpinned the conformational change in CL at low pH, whereas at high temperature, salt bridges became more dynamic, thus contributing to an overall destabilization. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR) to solvent, which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, and the VH and CL domains as interesting future targets for engineering stability to both pH- and thermal-stresses simultaneously

Complete analysis of the H5 hemagglutinin and N8 neuraminidase phylogenetic trees reveals that the H5N8 subtype has been produced by multiple reassortment events

The analysis of the complete H5 hemagglutinin and H8 neuraminidase phylogenetic trees presented in this paper shows that the H5N8 avian influenza has been generated by multiple reassortment events. The H5N8 strain does not have a single origin and is produced when the H5 hemagglutinin and N8 neuraminidase re-assort from other H5 and N8 containing strains. While it was known that there had been a re-assortment to incorporate the Guangdong H5 hemagglutinin at the start of the Korean outbreak, the results show that there have also been multiple reassortment events amongst the non-Korean sequences

Modelling and optimisation of the one-pot, multi-enzymatic synthesis of chiral amino-alcohols based on microscale kinetic parameter determination

Advances in synthetic biology are facilitating the de novo design of complex, multi-step enzymatic conversions for industrial organic synthesis. This work describes the integration of multi-step enzymatic pathway construction with enzyme kinetics and bioreactor modelling, in order to optimise the synthesis of chiral amino-alcohols using engineered Escherichia coli transketolases (TK) and the Chromobacterium violaceum transaminase (TAm). The specific target products were (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT). Kinetic models and parameters for each of the enzymatic steps were first obtained using automated microwell experiments. These identified the TK-catalysed conversions as being up to 25 times faster than the subsequent TAm conversions and inhibition of TAm by the amino-donor used, (S)-(−)-α-methylbenzylamine (MBA), as limiting the overall conversion yields. In order to better ‘match’ the relative rates of the two enzymes an E. coli expression system, based on two compatible plasmids, was constructed to produce both enzymes in a single host. By control of induction time and temperature it was possible to produce six times more recombinant TAm than TK to help balance the reaction rates. To overcome MBA inhibition and an unfavourable reaction equilibrium, fed-batch addition of the amino-donor was introduced as well as the use of isopropylamine as an alternate amino-donor. Adopting these strategies, and using the kinetic models to optimise feeding strategies, the one pot syntheses of APD and ABT were successfully scaled-up to preparative scales. Excellent agreement was found between the kinetic profiles and yields predicted and those achieved experimentally at the larger scale. In this case the integration of these multi-disciplinary approaches enabled us to achieve up to a 6 fold greater yield using concentrations an order of magnitude higher than in previous preparative scale batch bioconversions carried out sequentially

Novel statistical approaches for non-normal censored immunological data: analysis of cytokine and gene expression data

Background: For several immune-mediated diseases, immunological analysis will become more complex in the future with datasets in which cytokine and gene expression data play a major role. These data have certain characteristics that require sophisticated statistical analysis such as strategies for non-normal distribution and censoring. Additionally, complex and multiple immunological relationships need to be adjusted for potential confounding and interaction effects. Objective: We aimed to introduce and apply different methods for statistical analysis of non-normal censored cytokine and gene expression data. Furthermore, we assessed the performance and accuracy of a novel regression approach in order to allow adjusting for covariates and potential confounding. Methods: For non-normally distributed censored data traditional means such as the Kaplan-Meier method or the generalized Wilcoxon test are described. In order to adjust for covariates the novel approach named Tobit regression on ranks was introduced. Its performance and accuracy for analysis of non-normal censored cytokine/gene expression data was evaluated by a simulation study and a statistical experiment applying permutation and bootstrapping. Results: If adjustment for covariates is not necessary traditional statistical methods are adequate for non-normal censored data. Comparable with these and appropriate if additional adjustment is required, Tobit regression on ranks is a valid method. Its power, type-I error rate and accuracy were comparable to the classical Tobit regression. Conclusion: Non-normally distributed censored immunological data require appropriate statistical methods. Tobit regression on ranks meets these requirements and can be used for adjustment for covariates and potential confounding in large and complex immunological datasets

Engineered coatings for titanium implants to present ultralow doses of BMP-7

The ongoing research to improve the clinical outcome of titanium implants has resulted in the implemetation of multiple approches to deliver osteogenic growth factors accelerating and sustaining osseointegration. Here we show the presentation of human bone morphogenetic protein 7 (BMP-7) adsorbed to titanium discs coated with poly(ethyl acrylate) (PEA). We have previously shown that PEA promotes fibronectin organization into nanonetworks exposing integrin- and growth-factor-binding domains, allowing a synergistic interaction at the integrin/growth factor receptor level. Here, titanium discs were coated with PEA and fibronectin and then decorated with ng/mL doses of BMP-7. Human mesenchymal stem cells were used to investigate cellular responses on these functionalized microenvironments. Cell adhesion, proliferation, and mineralization, as well as osteogenic markers expression (osteopontin and osteocalcin) revealed the ability of the system to be more potent in osteodifferentiation of the mesenchymal cells than combinations of titanium and BMP-7 in absence of PEA coatings. This work represents a novel strategy to improve the biological activity of titanium implants with BMP-7

A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells

Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN) or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions

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

Nanotopographical induction of osteogenesis through adhesion, bone morphogenic protein cosignaling, and regulation of microRNAs

It is emerging that nanotopographical information can be used to induce osteogenesis from mesenchymal stromal cells from the bone marrow and it is hoped that this nanoscale bioactivity can be utilized to engineer next generation implants. However, the osteogenic mechanism of surfaces is currently poorly understood. In this report, we investigate mechanism and implicate bone morphogenic protein (BMP) in up-regulation of RUNX2 and show that RUNX2 and its regulatory miRNAs are BMP sensitive. Our data demonstrates that osteogenic nanotopography promotes co-localization of intergrins and BMP2 receptors in order to enhance osteogenic activity and that vitronectin is important in this interface. This provides insight that topographical regulation of adhesion can have effects on signaling cascades outside of cytoskeletal signaling and that adhesions can have roles in augmenting BMP signaling

Applications of Direct Injection Soft Chemical Ionisation-Mass Spectrometry for the Detection of Pre-blast Smokeless Powder Organic Additives

Analysis of smokeless powders is of interest from forensics and security perspectives. This article reports the detection of smokeless powder organic additives (in their pre-detonation condition), namely the stabiliser diphenylamine and its derivatives 2-nitrodiphenylamine and 4-nitrodiphenylamine, and the additives (used both as stabilisers and plasticisers) methyl centralite and ethyl centralite, by means of swab sampling followed by thermal desorption and direct injection soft chemical ionisation-mass spectrometry. Investigations on the product ions resulting from the reactions of the reagent ions H3O+ and O2+ with additives as a function of reduced electric field are reported. The method was comprehensively evaluated in terms of linearity, sensitivity and precision. For H3O+, the limits of detection (LoD) are in the range of 41-88 pg of additive, for which the accuracy varied between 1.5 and 3.2%, precision varied between 3.7 and 7.3% and linearity showed R20.9991. For O2+, LoD are in the range of 72 to 1.4 ng, with an accuracy of between 2.8 and 4.9% and a precision between 4.5 and 8.6% and R20.9914. The validated methodology was applied to the analysis of commercial pre-blast gun powders from different manufacturers.(VLID)4826148Accepted versio