Showing papers by "Douglas B. Kell published in 2019"

Bacterial Dysbiosis and Translocation in Psoriasis Vulgaris

Abstract: Psoriasis vulgaris is a chronic inflammatory skin condition, associated with both a physical and a psychological burden. Our understanding of the etiology of this disease remains incomplete. Conventionally, psoriasis has been viewed as a condition that manifests solely in the skin. However, the systemic inflammatory nature of this disease has been confirmed by the presence of a wide array of dysregulated cytokines and inflammatory markers in the serum of these patients. Both dysregulated gut and skin microbiomes have been found in association with psoriasis. An evident association also exists between inflammatory bowel disease and this condition. Regarding the skin microbiome, changes have been observed in the relative abundance of Firmicutes, Actinobacteria, and Proteobacteria. Additionally, Staphylococcus and Streptococcus spp. were detected more frequently in lesional skin. Alterations in the gut microbiome have been characterized by a decrease in the Bacteroidetes phylum and an increase in the Faecalibacterium genus. We suggest that dysbiosis of the skin and gut microbiota may contribute to psoriasis, by promoting the translocation of microbes from these sites into the bloodstream. Consistent with the Iron Dysregulation and Dormant Microbes hypothesis, these microorganisms are in a physiologically dormant state, but may be awakened periodically and shed their cell wall components, such as lipopolysaccharide and lipoteichoic acid. Both of these inflammagens may contribute significantly to maintaining a chronic inflammatory state in the host, such as is seen in individuals diagnosed with psoriasis.

Serum amyloid A binds to fibrin(ogen), promoting fibrin amyloid formation

Abstract: Complex associations exist between inflammation and thrombosis, with the inflammatory state tending to promote coagulation. Fibrinogen, an acute phase protein, has been shown to interact with the amyloidogenic s-amyloid protein of Alzheimer’s disease. However, little is known about the association between fibrinogen and serum amyloid A (SAA), a highly fibrillogenic protein that is one of the most dramatically changing acute phase reactants in the circulation. To study the role of SAA in coagulation and thrombosis, in vitro experiments were performed where purified human SAA, in concentrations resembling a modest acute phase response, was added to platelet-poor plasma (PPP) and whole blood (WB), as well as purified and fluorescently labelled fibrinogen. Results from thromboelastography (TEG) suggest that SAA causes atypical coagulation with a fibrin(ogen)-mediated increase in coagulation, but a decreased platelet/fibrin(ogen) interaction. In WB scanning electron microscopy analysis, SAA mediated red blood cell (RBC) agglutination, platelet activation and clumping, but not platelet spreading. Following clot formation in PPP, the presence of SAA increased amyloid formation of fibrin(ogen) as determined both with auto-fluorescence and with fluorogenic amyloid markers, under confocal microcopy. SAA also binds to fibrinogen, as determined with a fluorescent-labelled SAA antibody and correlative light electron microscopy (CLEM). The data presented here indicate that SAA can affect coagulation by inducing amyloid formation in fibrin(ogen), as well as by propelling platelets to a more prothrombotic state. The discovery of these multiple and complex effects of SAA on coagulation invite further mechanistic analyses.

Parkinson’s disease : a systemic inflammatory disease accompanied by bacterial inflammagens

Abstract: Parkinson's disease (PD) is a well-known neurodegenerative disease with a strong association established with systemic inflammation. Recently, the role of the gingipain protease group from Porphyromonas gingivalis was implicated in Alzheimer's disease and here we present evidence, using a fluorescent antibody to detect gingipain R1 (RgpA), of its presence in a PD population. To further elucidate the action of this gingipain, as well as the action of the lipopolysaccharide (LPS) from P. gingivalis, low concentrations of recombinant RgpA and LPS were added to purified fluorescent fibrinogen. We also substantiate previous findings regarding PD by emphasizing the presence of systemic inflammation via multiplex cytokine analysis, and demonstrate hypercoagulation using thromboelastography (TEG), confocal and electron microscopy. Biomarker analysis confirmed significantly increased levels of circulating proinflammatory cytokines. In our PD and control blood analysis, our results show increased hypercoagulation, the presence of amyloid formation in plasma, and profound ultrastructural changes to platelets. Our laboratory analysis of purified fibrinogen with added RgpA, and/or LPS, showed preliminary data with regards to the actions of the protease and the bacterial membrane inflammagen on plasma proteins, to better understand the nature of established PD.

A brain-permeable inhibitor of the neurodegenerative disease target kynurenine 3-monooxygenase prevents accumulation of neurotoxic metabolites

Abstract: Dysregulation of the kynurenine pathway (KP) leads to imbalances in neuroactive metabolites associated with the pathogenesis of several neurodegenerative disorders, including Huntington's disease (HD). Inhibition of the enzyme kynurenine 3-monooxygenase (KMO) in the KP normalises these metabolic imbalances and ameliorates neurodegeneration and related phenotypes in several neurodegenerative disease models. KMO is thus a promising candidate drug target for these disorders, but known inhibitors are not brain permeable. Here, 19 new KMO inhibitors have been identified. One of these (1) is neuroprotective in a Drosophila HD model but is minimally brain penetrant in mice. The prodrug variant (1b) crosses the blood-brain barrier, releases 1 in the brain, thereby lowering levels of 3-hydroxykynurenine, a toxic KP metabolite linked to neurodegeneration. Prodrug 1b will advance development of targeted therapies against multiple neurodegenerative and neuroinflammatory diseases in which KP likely plays a role, including HD, Alzheimer's disease, and Parkinson's disease.

Engineering the Yeast Saccharomyces cerevisiae for the Production of L-(+)-Ergothioneine.

Abstract: L-(+)-Ergothioneine (ERG) is an unusual, naturally occurring antioxidant nutraceutical that has been shown to help reduce cellular oxidative damage. Humans do not biosynthesise ERG, but acquire it from their diet; it exploits a specific transporter (SLC22A4) for its uptake. ERG is considered to be a nutraceutical and possible vitamin that is involved in the maintenance of health, and seems to be at too low a concentration in several diseases in vivo. Ergothioneine is thus a potentially useful dietary supplement. Present methods of commercial production rely on extraction from natural sources or on chemical synthesis. Here we describe the engineering of the baker's yeast Saccharomyces cerevisiae to produce ergothioneine by fermentation in defined media. After integrating combinations of ERG biosynthetic pathways from different organisms, we screened yeast strains for their production of ERG. The highest-producing strain was also engineered with known ergothioneine transporters. The effect of amino acid supplementation of the medium was investigated and the nitrogen metabolism of S. cerevisiae was altered by knock-out of TOR1 or YIH1. We also optimized the media composition using fractional factorial methods. Our optimal strategy led to a titer of 598 ± 18 mg/L ergothioneine in fed-batch culture in 1 L bioreactors. Because S. cerevisiae is a GRAS ("generally recognized as safe") organism that is widely used for nutraceutical production, this work provides a promising process for the biosynthetic production of ERG.

Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli

Abstract: It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient high-throughput assay for xenobiotic uptake in individual cells. We used high-throughput flow cytometry to assess the ability of individual gene knockout strains of E coli to take up two membrane-permeable, cationic fluorescent dyes, namely the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in either direction (increased or decreased); knockouts of known influx and efflux transporters behaved as expected, giving credence to the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar results were obtained with SYBR Green (the range being approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains (although the membrane potential is presumably the same in each case). Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of putatively broad and presently unknown specificity, and that the very large range between the ‘lowest’ and the ‘highest’ levels of uptake, even in knockouts of just single genes, implies strongly that phospholipid bilayer transport is indeed negligible. This work also casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

The role and robustness of the Gini coefficient as an unbiased tool for the selection of Gini genes for normalising expression profiling data

Abstract: We recently introduced the Gini coefficient (GC) for assessing the expression variation of a particular gene in a dataset, as a means of selecting improved reference genes over the cohort (‘housekeeping genes’) typically used for normalisation in expression profiling studies. Those genes (transcripts) that we determined to be useable as reference genes differed greatly from previous suggestions based on hypothesis-driven approaches. A limitation of this initial study is that a single (albeit large) dataset was employed for both tissues and cell lines. We here extend this analysis to encompass seven other large datasets. Although their absolute values differ a little, the Gini values and median expression levels of the various genes are well correlated with each other between the various cell line datasets, implying that our original choice of the more ubiquitously expressed low-Gini-coefficient genes was indeed sound. In tissues, the Gini values and median expression levels of genes showed a greater variation, with the GC of genes changing with the number and types of tissues in the data sets. In all data sets, regardless of whether this was derived from tissues or cell lines, we also show that the GC is a robust measure of gene expression stability. Using the GC as a measure of expression stability we illustrate its utility to find tissue- and cell line-optimised housekeeping genes without any prior bias, that again include only a small number of previously reported housekeeping genes. We also independently confirmed this experimentally using RT-qPCR with 40 candidate GC genes in a panel of 10 cell lines. These were termed the Gini Genes. In many cases, the variation in the expression levels of classical reference genes is really quite huge (e.g. 44 fold for GAPDH in one data set), suggesting that the cure (of using them as normalising genes) may in some cases be worse than the disease (of not doing so). We recommend the present data-driven approach for the selection of reference genes by using the easy-to-calculate and robust GC.

Parkinson’s disease: a systemic inflammatory disease accompanied by bacterial inflammagens

Abstract: Parkinson’s disease (PD) is a well-known neurodegenerative disease. Recently, the role of gingipains from Porphyromonas gingivalis was implicated in Alzheimer’s disease. Here we present evidence of systemic inflammation, accompanied by hypercoagulation; we also show that ginipains from P. gingivalis and its LPS may foster abnormal clotting, and that ginipains are present in PD blood, and thus that ginipain’s action on blood may be relevant to PD pathology. Bloods from both PD and healthy blood samples were analysed using thromboelastography (TEG), confocal and electron microscopies, and for cytokine and other circulating biomarkers. We also probed PD and healthy plasma clots with a polyclonal antibody for the bacterial protease, gingipain R1, from P. gingivalis. Low concentrations of recombinant gingipain R1 were also added to purified fluorescent fibrinogen. TEG, fibrin(ogen) amyloid formation and platelet ultrastructure analysis confirmed profound hypercoagulation, while the biomarker analysis confirmed significantly increased levels of circulating proinflammatory cytokines. We provide evidence for the presence of the protease, gingipain R1 in PD blood, implicating inflammatory microbial cell wall products in PD.

Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth.

Abstract: Rapid changes in the number and flow cytometric behaviour of cells of E. coli taken from a stationary phase and inoculated into rich medium.Cells of E. coli were grown in LB medium, taken from a stationary phase of 2-4 h, and re-inoculated into fresh media at a concentration (105 ml-1 or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energization (using a carbocyanine dye) and DNA distribution. It transpired that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4 h, the true lag phase could be less than 15-20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of re-inoculation, providing the possibility of a very rapid antibiotic susceptibility test on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential urinary tract infection (UTI) samples taken from a doctor's surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.

Engineering the yeast Saccharomyces cerevisiae for the production of L-(+)-ergothioneine

Abstract: L-(+)-Ergothioneine is an unusual, naturally occurring antioxidant nutraceutical that has been shown to help reduce cellular oxidative damage. Humans do not biosynthesise it, so can acquire it only from their diet; it exploits a specific transporter (SLC22A4) for its uptake. ERG is considered to be a nutraceutical and possible vitamin that is involved in the maintenance of health, and seems to be at too low a concentration in several diseases in vivo. Ergothioneine is thus a potentially useful dietary supplement. Present methods of commercial production rely on extraction from natural sources or on chemical synthesis. Here we describe the engineering of the baker’s yeast Saccharomyces cerevisiae to produce ergothioneine by fermentation in defined media. After integrating combinations of ERG biosynthetic pathways from different organisms, we screened yeast strains for their production of ERG. The highest-producing strain was also engineered with known ergothioneine transporters. The effect of amino acid supplementation of the medium was investigated and the nitrogen metabolism of S. cerevisiae was altered by knock-out of TOR1 or YIH1. We also optimized the media composition using fractional factorial methods. Our optimal strategy led to a titer of 598 ± 18 mg/L ergothioneine in fed-batch culture in bioreactors. Because S. cerevisiae is a GRAS (‘generally recognised as safe’) organism that is widely used for nutraceutical production, this work provides a promising process for the biosynthetic production of ERG.

GeneORator: An Effective Strategy for Navigating Protein Sequence Space More Efficiently through Boolean OR-Type DNA Libraries

Abstract: Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional site-directed methods implicitly utilize the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of genetic variants as the number of mutations increases. We introduce GeneORator, a novel strategy for creating DNA libraries based on the Boolean logical OR operator. Here, a single library is divided into many subsets, each containing different combinations of the desired mutations. Consequently, the effect of adding more mutations on the number of genetic combinations is additive (Boolean OR logic) and not exponential (AND logic). We demonstrate this strategy with large-scale mutagenesis studies, using monoamine oxidase-N ( Aspergillus niger) as the exemplar target. First, we mutated every residue in the secondary structure-containing regions (276 out of a total 495 amino acids) to screen for improvements in kcat. Second, combinatorial OR-type libraries permitted screening of diverse mutation combinations in the enzyme active site to detect activity toward novel substrates. In both examples, OR-type libraries effectively reduced the number of variants searched up to 1010-fold, dramatically reducing the screening effort required to discover variants with improved and/or novel activity. Importantly, this approach enables the screening of a greater diversity of mutation combinations, accessing a larger area of a protein's sequence space. OR-type libraries can be applied to any biological engineering objective requiring DNA mutagenesis, and the approach has wide ranging applications in, for example, enzyme engineering, antibody engineering, and synthetic biology.

A Possible Role of Amyloidogenic Blood Clotting in the Evolving Haemodynamics of Female Migraine-With-Aura: Results From a Pilot Study

Abstract: Introduction: Migraine is a debilitating primary headache disorder with a poorly understood aetiology. An extensive body of literature supports the theory of migraine as a systemic vascular inflammatory disorder characterised by endothelial dysfunction. It is also well-known that chronic inflammation results in an excessive burden of oxidative stress and therefore cellular dysfunction. In this study the effects of excessive oxidative stress through the phases of female migraine-with-aura (FMA) were evaluated by examining the health of the systems of haemostasis. Methods: Blood was obtained from 11 FMA patients at baseline and during the headache phase of migraine, as well as from 8 healthy age-matched female controls. Samples were analysed using thromboelastography (TEG) to evaluate viscoelastic profiles, light microscopy for erythrocyte morphology, Scanning Electron Microscopy (SEM) for erythrocyte and fibrin clot structure, confocal microscopy for β-amyloid detection in fibrin clots. Results: Viscoelastic profiles from platelet poor plasma showed decreased clot reaction times in FMA at baseline (95% CI [5.56, 8.41]) vs. control (95% CI [7.22, 11.68]); as well as decreased time to maximum thrombus generation for the same comparison (95% CI [6.78, 10.20] vs. [8.90, 12.96]). Morphological analysis of erythrocytes indicated widespread macrocytosis, poikilocytosis and eryptosis in the migraineurs. Analysis of fibrin networks indicated that this hypercoagulability may be a result of aberrant fibrin polymerisation kinetics caused by the adoption of a β-amyloid conformation of fibrin(ogen). Conclusion: The results reaffirm the hypercoagulable state in migraine, and would suggest that this state is most likely a result of a systemic inflammatory state which induces oxidative damage to both erythrocytes and fibrin(ogen) in female episodic migraine-with-aura. Furthermore, if the amylodogenic changes to fibrin(ogen) were observed in a larger cohort, this would support theories of micro-embolisation in migraine-with-aura.

Generation of a Small Library of Natural Products Designed to Cover Chemical Space Inexpensively

Abstract: Natural products space includes at least 200,000 compounds and the structures of most of these compounds are available in digital format. Previous analyses showed (i) that although they were capable of taking up synthetic pharmaceutical drugs, such exogenous molecules were likely the chief ‘natural’ substrates in the evolution of the transporters used to gain cellular entry by pharmaceutical drugs, and (ii) that a relatively simple but rapid clustering algorithm could produce clusters from which individual elements might serve to form a representative library covering natural products space. This exploited the fact that the larger clusters were likely to be formed early in evolution (and hence to have been accompanied by suitable transporters), so that very small clusters, including singletons, could be ignored. In the latter work, we assumed that the molecule chosen might be that in the middle of the cluster. However, this ignored two other criteria, namely the commercial availability and the financial cost of the individual elements of these clusters. We here develop a small representative library in which we to seek to satisfy the somewhat competing criteria of coverage (‘representativeness’), availability and cost. It is intended that the library chosen might serve as a testbed of molecules that may or may not be substrates for known or orphan drug transporters. A supplementary spreadsheet provides details, and their availability via a particular supplier.

Structural similarities between some common fluorophores used in biology and marketed drugs, endogenous metabolites, and natural products

Abstract: Background It is known that at least some fluorophores can act as ‘surrogate’ substrates for solute carriers (SLCs) involved in pharmaceutical drug uptake, and this promiscuity is taken to reflect at least a certain structural similarity. As part of a comprehensive study seeking the ‘natural’ substrates of ‘orphan’ transporters that also serve to take up pharmaceutical drugs into cells, we have noted that many drugs bear structural similarities to natural products. A cursory inspection of common fluorophores indicates that they too are surprisingly ‘drug-like’, and they also enter at least some cells. Some are also known to be substrates of efflux transporters. Consequently, we sought to assess the structural similarity of common fluorophores to marketed drugs, endogenous mammalian metabolites, and natural products. We used a set of some 150 fluorophores. Results The great majority of fluorophores tested exhibited significant similarity (Tanimoto similarity > 0.75) to at least one drug as judged via descriptor properties (especially their aromaticity, for identifiable reasons that we explain), by molecular fingerprints, by visual inspection, and via the “quantitative estimate of drug likeness” technique. It is concluded that this set of fluorophores does overlap a significant part of both drug space and natural products space. Consequently, fluorophores do indeed offer a much wider opportunity than had possibly been realised to be used as surrogate uptake molecules in the competitive or trans-stimulation assay of membrane transporter activities.

The role and robustness of the Gini coefficient as an unbiased tool for the selection of Gini genes for normalising expression profiling data

Abstract: We recently introduced the Gini coefficient (GC) for assessing the expression variation of a particular gene in a dataset, as a means of selecting improved reference genes over the cohort (‘housekeeping genes’) typically used for normalisation in expression profiling studies. Those genes (transcripts) that we determined to be useable as reference genes differed greatly from previous suggestions based on hypothesis-driven approaches. A limitation of this initial study is that a single (albeit large) dataset was employed for both tissues and cell lines. We here extend this analysis to encompass seven other large datasets. Although their absolute values differ a little, the Gini values and median expression levels of the various genes are well correlated with each other between the various cell line datasets, implying that our original choice of the more ubiquitously expressed low-Gini-coefficient genes was indeed sound. In tissues, the Gini values and median expression levels of genes showed a greater variation, with the GC of genes changing with the number and types of tissues in the data sets. In all data sets, regardless of whether this was derived from tissues or cell lines, we also show that the GC is a robust measure of gene expression stability. Using the GC as a measure of expression stability we illustrate its utility to find tissue- and cell line-optimised housekeeping genes without any prior bias, that again include only a small number of previously reported housekeeping genes. We also independently confirmed this experimentally using RT-qPCR with 40 candidate GC genes in a panel of 10 cell lines. These were termed the Gini Genes. In many cases, the variation in the expression levels of classical reference genes is really quite huge (e.g. 44 fold for GAPDH in one data set), suggesting that the cure (of using them as normalising genes) may in some cases be worse than the disease (of not doing so). We recommend the present data-driven approach for the selection of reference genes by using the easy-to-calculate and robust GC.

Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli

Abstract: We used high-throughput flow cytometry to assess the ability of individual gene knockout strains of E coli to take up two membrane-permeable, cationic fluorescent dyes, viz the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by say a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in both directions (increased or decreased); knockouts in known influx and efflux transporters behaved as expected, giving confidence in the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar findings were made with SYBR Green (the range being some 69-fold), though despite it too containing a benzimidazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains. Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of possibly broad and presently unknown specificity. This casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.