Showing papers by "Douglas B. Kell published in 2015"

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

Abstract: The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the ‘search space’ of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (Kd) and catalytic (kcat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving kcat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the ‘best’ amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole, simultaneously, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape navigation, informed by sequence-activity relationships and coupled to the emerging methods of synthetic biology, offers scope for the development of novel biocatalysts that are both highly active and robust.

The dormant blood microbiome in chronic, inflammatory diseases

Abstract: Blood in healthy organisms is seen as a ‘sterile’ environment: it lacks proliferating microbes. Dormant or not-immediately-culturable forms are not absent, however, as intracellular dormancy is well established. We highlight here that a great many pathogens can survive in blood and inside erythrocytes. ‘Non-culturability’, reflected by discrepancies between plate counts and total counts, is commonplace in environmental microbiology. It is overcome by improved culturing methods, and we asked how common this would be in blood. A number of recent, sequence-based and ultramicroscopic studies have uncovered an authentic blood microbiome in a number of non-communicable diseases. The chief origin of these microbes is the gut microbiome (especially when it shifts composition to a pathogenic state, known as ‘dysbiosis’). Another source is microbes translocated from the oral cavity. ‘Dysbiosis’ is also used to describe translocation of cells into blood or other tissues. To avoid ambiguity, we here use the term ‘atopobiosis’ for microbes that appear in places other than their normal location. Atopobiosis may contribute to the dynamics of a variety of inflammatory diseases. Overall, it seems that many more chronic, non-communicable, inflammatory diseases may have a microbial component than are presently considered, and may be treatable using bactericidal antibiotics or vaccines.

Molecular phenotyping of a UK population: defining the human serum metabolome.

Abstract: Phenotyping of 1,200 ‘healthy’ adults from the UK has been performed through the investigation of diverse classes of hydrophilic and lipophilic metabolites present in serum by applying a series of chromatography–mass spectrometry platforms. These data were made robust to instrumental drift by numerical correction; this was prerequisite to allow detection of subtle metabolic differences. The variation in observed metabolite relative concentrations between the 1,200 subjects ranged from less than 5 % to more than 200 %. Variations in metabolites could be related to differences in gender, age, BMI, blood pressure, and smoking. Investigations suggest that a sample size of 600 subjects is both necessary and sufficient for robust analysis of these data. Overall, this is a large scale and non-targeted chromatographic MS-based metabolomics study, using samples from over 1,000 individuals, to provide a comprehensive measurement of their serum metabolomes. This work provides an important baseline or reference dataset for understanding the ‘normal’ relative concentrations and variation in the human serum metabolome. These may be related to our increasing knowledge of the human metabolic network map. Information on the Husermet study is available at http://www.husermet.org/ . Importantly, all of the data are made freely available at MetaboLights ( http://www.ebi.ac.uk/metabolights/ ).

On the translocation of bacteria and their lipopolysaccharides between blood and peripheral locations in chronic, inflammatory diseases: the central roles of LPS and LPS-induced cell death

Abstract: We have recently highlighted (and added to) the considerable evidence that blood can contain dormant bacteria. By definition, such bacteria may be resuscitated (and thus proliferate). This may occur under conditions that lead to or exacerbate chronic, inflammatory diseases that are normally considered to lack a microbial component. Bacterial cell wall components, such as the endotoxin lipopolysaccharide (LPS) of Gram-negative strains, are well known as potent inflammatory agents, but should normally be cleared. Thus, their continuing production and replenishment from dormant bacterial reservoirs provides an easy explanation for the continuing, low-grade inflammation (and inflammatory cytokine production) that is characteristic of many such diseases. Although experimental conditions and determinants have varied considerably between investigators, we summarise the evidence that in a great many circumstances LPS can play a central role in all of these processes, including in particular cell death processes that permit translocation between the gut, blood and other tissues. Such localised cell death processes might also contribute strongly to the specific diseases of interest. The bacterial requirement for free iron explains the strong co-existence in these diseases of iron dysregulation, LPS production, and inflammation. Overall this analysis provides an integrative picture, with significant predictive power, that is able to link these processes via the centrality of a dormant blood microbiome that can resuscitate and shed cell wall components.

A 'rule of 0.5' for the metabolite-likeness of approved pharmaceutical drugs.

Abstract: We exploit the recent availability of a community reconstruction of the human metabolic network (‘Recon2’) to study how close in structural terms are marketed drugs to the nearest known metabolite(s) that Recon2 contains. While other encodings using different kinds of chemical fingerprints give greater differences, we find using the 166 Public MDL Molecular Access (MACCS) keys that 90 % of marketed drugs have a Tanimoto similarity of more than 0.5 to the (structurally) ‘nearest’ human metabolite. This suggests a ‘rule of 0.5’ mnemonic for assessing the metabolite-like properties that characterise successful, marketed drugs. Multiobjective clustering leads to a similar conclusion, while artificial (synthetic) structures are seen to be less human-metabolite-like. This ‘rule of 0.5’ may have considerable predictive value in chemical biology and drug discovery, and may represent a powerful filter for decision making processes.

Poorly controlled type 2 diabetes is accompanied by significant morphological and ultrastructural changes in both erythrocytes and in thrombin-generated fibrin: implications for diagnostics

Abstract: We have noted in previous work, in a variety of inflammatory diseases, where iron dysregulation occurs, a strong tendency for erythrocytes to lose their normal discoid shape and to adopt a skewed morphology (as judged by their axial ratios in the light microscope and by their ultrastructure in the SEM). Similarly, the polymerization of fibrinogen, as induced in vitro by added thrombin, leads not to the common ‘spaghetti-like’ structures but to dense matted deposits. Type 2 diabetes is a known inflammatory disease. In the present work, we found that the axial ratio of the erythrocytes of poorly controlled (as suggested by increased HbA1c levels) type 2 diabetics was significantly increased, and that their fibrin morphologies were again highly aberrant. As judged by scanning electron microscopy and in the atomic force microscope, these could be reversed, to some degree, by the addition of the iron chelators deferoxamine (DFO) or deferasirox (DFX). As well as their demonstrated diagnostic significance, these morphological indicators may have prognostic value.

Viscoelastic and ultrastructural characteristics of whole blood and plasma in Alzheimer-type dementia, and the possible role of bacterial lipopolysaccharides (LPS)

Abstract: // Janette Bester 1 , Prashilla Soma 1 , Douglas B. Kell 2 and Etheresia Pretorius 1 1 Department of Physiology, Faculty of Health Sciences, University of Pretoria, Arcadia, South Africa 2 School of Chemistry and The Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK Correspondence to: Etheresia Pretorius, email: // Douglas B. Kell, email: // Keywords : Alzheimer-type dementia, iron levels, lipopolysaccharides, scanning electron microscopy, thromboelastography®, Gerotarget Received : September 24, 2015 Accepted : September 28, 2015 Published : October 10, 2015 Abstract Alzheimer-type dementia (AD) is a neurodegenerative disorder and the most common form of dementia. Patients typically present with neuro- and systemic inflammation and iron dysregulation, associated with oxidative damage that reflects in hypercoagulability. Hypercoagulability is closely associated with increased fibrin(ogen) and in AD patients fibrin(ogen) has been implicated in the development of neuroinflammation and memory deficits. There is still no clear reason precisely why (a) this hypercoagulable state, (b) iron dysregulation and (c) increased fibrin(ogen) could together lead to the loss of neuronal structure and cognitive function. Here we suggest an alternative hypothesis based on previous ultrastructural evidence of the presence of a (dormant) blood microbiome in AD. Furthermore, we argue that bacterial cell wall components, such as the endotoxin lipopolysaccharide (LPS) of Gram-negative strains, might be the cause of the continuing and low-grade inflammation, characteristic of AD. Here, we follow an integrated approach, by studying the viscoelastic and ultrastructural properties of AD plasma and whole blood by using scanning electron microscopy, Thromboelastography (TEG ® ) and the Global Thrombosis Test (GTT ® ). Ultrastructural analysis confirmed the presence and close proximity of microbes to erythrocytes. TEG ® analysis showed a hypercoagulable state in AD. TEG ® results where LPS was added to naive blood showed the same trends as were found with the AD patients, while the GTT ® results (where only platelet activity is measured), were not affected by the added LPS, suggesting that LPS does not directly impact platelet function. Our findings reinforce the importance of further investigating the role of LPS in AD.

The simultaneous occurrence of both hypercoagulability and hypofibrinolysis in blood and serum during systemic inflammation, and the roles of iron and fibrin(ogen)

Abstract: Although the two phenomena are usually studied separately, we summarise a considerable body of literature to the effect that a great many diseases involve (or are accompanied by) both an increased tendency for blood to clot (hypercoagulability) and the resistance of the clots so formed (hypofibrinolysis) to the typical, ‘healthy’ or physiological lysis. We concentrate here on the terminal stages of fibrin formation from fibrinogen, as catalysed by thrombin. Hypercoagulability goes hand in hand with inflammation, and is strongly influenced by the fibrinogen concentration (and vice versa); this can be mediated via interleukin-6. Poorly liganded iron is a significant feature of inflammatory diseases, and hypofibrinolysis may change as a result of changes in the structure and morphology of the clot, which may be mimicked in vitro, and may be caused in vivo, by the presence of unliganded iron interacting with fibrin(ogen) during clot formation. Many of these phenomena are probably caused by electrostatic changes in the iron–fibrinogen system, though hydroxyl radical (OH˙) formation can also contribute under both acute and (more especially) chronic conditions. Many substances are known to affect the nature of fibrin polymerised from fibrinogen, such that this might be seen as a kind of bellwether for human or plasma health. Overall, our analysis demonstrates the commonalities underpinning a variety of pathologies as seen in both hypercoagulability and hypofibrinolysis, and offers opportunities for both diagnostics and therapies.

Individuality, phenotypic differentiation, dormancy and ‘persistence’ in culturable bacterial systems: commonalities shared by environmental, laboratory, and clinical microbiology

Abstract: For bacteria, replication mainly involves growth by binary fission. However, in a very great many natural environments there are examples of phenotypically dormant, non-growing cells that do not replicate immediately and that are phenotypically ‘nonculturable’ on media that normally admit their growth. They thereby evade detection by conventional culture-based methods. Such dormant cells may also be observed in laboratory cultures and in clinical microbiology. They are usually more tolerant to stresses such as antibiotics, and in clinical microbiology they are typically referred to as ‘persisters’. Bacterial cultures necessarily share a great deal of relatedness, and inclusive fitness theory implies that there are conceptual evolutionary advantages in trading a variation in growth rate against its mean, equivalent to hedging one’s bets. There is much evidence that bacteria exploit this strategy widely. We here bring together data that show the commonality of these phenomena across environmental, laboratory and clinical microbiology. Considerable evidence, using methods similar to those common in environmental microbiology, now suggests that many supposedly non-communicable, chronic and inflammatory diseases are exacerbated (if not indeed largely caused) by the presence of dormant or persistent bacteria (the ability of whose components to cause inflammation is well known). This dormancy (and resuscitation therefrom) often reflects the extent of the availability of free iron. Together, these phenomena can provide a ready explanation for the continuing inflammation common to such chronic diseases and its correlation with iron dysregulation. This implies that measures designed to assess and to inhibit or remove such organisms (or their access to iron) might be of much therapeutic benefit.

Event-based text mining for biology and functional genomics

Abstract: The assessment of genome function requires a mapping between genome-derived entities and biochemical reactions, and the biomedical literature represents a rich source of information about reactions between biological components. However, the increasingly rapid growth in the volume of literature provides both a challenge and an opportunity for researchers to isolate information about reactions of interest in a timely and efficient manner. In response, recent text mining research in the biology domain has been largely focused on the identification and extraction of ‘events’, i.e. categorised, structured representations of relationships between biochemical entities, from the literature. Functional genomics analyses necessarily encompass events as so defined. Automatic event extraction systems facilitate the development of sophisticated semantic search applications, allowing researchers to formulate structured queries over extracted events, so as to specify the exact types of reactions to be retrieved. This article provides an overview of recent research into event extraction. We cover annotated corpora on which systems are trained, systems that achieve state-of-the-art performance and details of the community shared tasks that have been instrumental in increasing the quality, coverage and scalability of recent systems. Finally, several concrete applications of event extraction are covered, together with emerging directions of research.

The apparent permeabilities of Caco-2 cells to marketed drugs: magnitude, and independence from both biophysical properties and endogenite similarities.

Abstract: We bring together fifteen, nonredundant, tabulated collections (amounting to 696 separate measurements) of the apparent permeability (P app) of Caco-2 cells to marketed drugs. While in some cases there are some significant interlaboratory disparities, most are quite minor. Most drugs are not especially permeable through Caco-2 cells, with the median P app value being some 16 ⋅ 10(-6) cm s(-1). This value is considerably lower than those (1,310 and 230 ⋅ 10(-6) cm s(-1)) recently used in some simulations that purported to show that P app values were too great to be transporter-mediated only. While these values are outliers, all values, and especially the comparatively low values normally observed, are entirely consistent with transporter-only mediated uptake, with no need to invoke phospholipid bilayer diffusion. The apparent permeability of Caco-2 cells to marketed drugs is poorly correlated with either simple biophysical properties, the extent of molecular similarity to endogenous metabolites (endogenites), or any specific substructural properties. In particular, the octanol:water partition coefficient, logP, shows negligible correlation with Caco-2 permeability. The data are best explained on the basis that most drugs enter (and exit) Caco-2 cells via a multiplicity of transporters of comparatively weak specificity.

Understanding the foundations of the structural similarities between marketed drugs and endogenous human metabolites

Abstract: Background. A recent comparison showed the extensive similarities between the structural properties of metabolites in the reconstructed human metabolic network (‘endogenites’) and those of successful, marketed drugs (‘drugs’). Results. Clustering indicated the related but differential population of chemical space by endogenites and drugs. Differences between the drug-endogenite similarities resulting from various encodings and judged by Tanimoto similarity could be related simply to the fraction of the bitstrings set to 1. By extracting drug/endogenite substructures, we develop a novel family of fingerprints, the Drug Endogenite Substructure (DES) encodings, based on the ranked frequency of the various substructures. These provide a natural assessment of drug-endogenite likeness, and may be used as descriptors with which to derive quantitative structure-activity relationships (QSARs). Conclusions. ‘Drug-endogenite likeness’ seems to have utility, and leads to a simple, novel and interpretable substructure-based molecular encoding for cheminformatics.

Software review: the KNIME workflow environment and its applications in genetic programming and machine learning

Abstract: Software comes in various forms, from the hair shirt style of the command line to fully blown, GUI-based commercial offerings. The former tends to give its users more control, but disenfranchises many other potential users who cannot themselves program yet who might otherwise benefit from it. A kind of halfway house is represented by software environments that provide both flexibility (power) and ease of use. A particular subset is represented by Workflow environments, in which loosely coupled, individual processing nodes can be ?bolted together? to permit complex computational operations. Taverna [7] (http://?www.?taverna.?org.?uk/?) is a very well known scientific workflow system, especially in bioinformatics. It is a fully open environment, freely available, and workflows can be shared via its sister site myExperiment http://?www.?myexperiment.?org/?. It has some extensions for cheminformatics [2]. A particular strength is the means by which it can use Web services to link federated Web-based resources, a particular feature of bioinformatics.

The virtue of innovation: innovation through the lenses of biological evolution

Abstract: We rehearse the processes of innovation and discovery in general terms, using as our main metaphor the biological concept of an evolutionary fitness landscape Incremental and disruptive innovations are seen, respectively, as successful searches carried out locally or more widely They may also be understood as reflecting evolution by mutation (incremental) versus recombination (disruptive) We also bring a platonic view, focusing on virtue and memory We use ‘virtue’ as a measure of efforts, including the knowledge required to come up with disruptive and incremental innovations, and ‘memory’ as a measure of their lifespan, ie how long they are remembered Fostering innovation, in the evolutionary metaphor, means providing the wherewithal to promote novelty, good objective functions that one is trying to optimize, and means to improve one's knowledge of, and ability to navigate, the landscape one is searching Recombination necessarily implies multi- or inter-disciplinarity These principles are generic to all kinds of creativity, novel ideas formation and the development of new products and technologies

The transporter-mediated cellular uptake of pharmaceutical drugs is based on their metabolite-likeness and not on their bulk biophysical properties: Towards a systems pharmacology ☆

Abstract: Several recent developments are brought together: (i) the new availability of a consensus, curated human metabolic network reconstruction (Recon2), approximately a third of whose steps are represented by transporters, (ii) the recognition that most successful (marketed) drugs, as well as natural products, bear significant similarities to the metabolites in Recon2, (iii) the recognition that to get into and out of cells such drugs hitchhike on the transporters that are part of normal intermediary metabolism, and the consequent recognition that for intact biomembrane Phospholipid Bilayer diffusion Is Negligible (PBIN), and (iv) the consequent recognition that we need to exploit this and to use more phenotypic assays to understand how drugs affect cells and organisms. I show in particular that lipophilicity is a very poor predictor of drug permeability, and that we need to (and can) bring together our knowledge of both pharmacology and systems biology modelling into a new systems pharmacology.

Individuality, phenotypic differentiation, dormancy and ‘persistence’ in culturable bacterial systems: commonalities shared by environmental, laboratory, and clinical microbiology [version 2; referees: 2 approved, 1 approved with reservations]

Abstract: For bacteria, replication mainly involves growth by binary fission. However, in a very great many natural environments there are examples of phenotypically dormant, non-growing cells that do not replicate immediately and that are phenotypically 'nonculturable' on media that normally admit their growth. They thereby evade detection by conventional culture-based methods. Such dormant cells may also be observed in laboratory cultures and in clinical microbiology. They are usually more tolerant to stresses such as antibiotics, and in clinical microbiology they are typically referred to as 'persisters'. Bacterial cultures necessarily share a great deal of relatedness, and inclusive fitness theory implies that there are conceptual evolutionary advantages in trading a variation in growth rate against its mean, equivalent to hedging one's bets. There is much evidence that bacteria exploit this strategy widely. We here bring together data that show the commonality of these phenomena across environmental, laboratory and clinical microbiology. Considerable evidence, using methods similar to those common in environmental microbiology, now suggests that many supposedly non-communicable, chronic and inflammatory diseases are exacerbated (if not indeed largely caused) by the presence of dormant or persistent bacteria (the ability of whose components to cause inflammation is well known). This dormancy (and resuscitation therefrom) often reflects the extent of the availability of free iron. Together, these phenomena can provide a ready explanation for the continuing inflammation common to such chronic diseases and its correlation with iron dysregulation. This implies that measures designed to assess and to inhibit or remove such organisms (or their access to iron) might be of much therapeutic benefit.

Alignment by numbers: sequence assembly using compressed numerical representations

Abstract: Motivation: DNA sequencing instruments are enabling genomic analyses of unprecedented scope and scale, widening the gap between our abilities to generate and interpret sequence data. Established methods for computational sequence analysis generally use nucleotide-level resolution of sequences, and while such approaches can be very accurate, increasingly ambitious and data-intensive analyses are rendering them impractical for applications such as genome and metagenome assembly. Comparable analytical challenges are encountered in other data-intensive fields involving sequential data, such as signal processing, in which dimensionality reduction methods are routinely used to reduce the computational burden of analyses. We therefore seek to address the question of whether it is possible to improve the efficiency of sequence alignment by applying dimensionality reduction methods to numerically represented nucleotide sequences. Results: To explore the applicability of signal transformation and dimensionality reduction methods to sequence assembly, we implemented a short read aligner and evaluated its performance against simulated high diversity viral sequences alongside four existing aligners. Using our sequence transformation and feature selection approach, alignment time was reduced by up to 14-fold compared to uncompressed sequences and without reducing alignment accuracy. Despite using highly compressed sequence transformations, our implementation yielded alignments of similar overall accuracy to existing aligners, outperforming all other tools tested at high levels of sequence variation. Our approach was also applied to the de novo assembly of a simulated diverse viral population. Our results demonstrate that full sequence resolution is not a prerequisite of accurate sequence alignment and that analytical performance can be retained and even enhanced through appropriate dimensionality reduction of sequences.

Why does serum contain ferritin

Abstract: We thank the Biotechnology and Biological Sciences Research Council (grant BB/L025752/1) as well as the National Research Foundation (NRF) of South Africa for supporting this collaboration.