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Showing papers by "Douglas B. Kell published in 2014"


Journal ArticleDOI
TL;DR: It is argued here that serum ferritin arises from damaged cells, and is thus a marker of cellular damage, and therefore why it correlates with the presence and/or severity of numerous diseases.
Abstract: 'Serum ferritin' presents a paradox, as the iron storage protein ferritin is not synthesised in serum yet is to be found there. Serum ferritin is also a well known inflammatory marker, but it is unclear whether serum ferritin reflects or causes inflammation, or whether it is involved in an inflammatory cycle. We argue here that serum ferritin arises from damaged cells, and is thus a marker of cellular damage. The protein in serum ferritin is considered benign, but it has lost (i.e. dumped) most of its normal complement of iron which when unliganded is highly toxic. The facts that serum ferritin levels can correlate with both disease and with body iron stores are thus expected on simple chemical kinetic grounds. Serum ferritin levels also correlate with other phenotypic readouts such as erythrocyte morphology. Overall, this systems approach serves to explain a number of apparent paradoxes of serum ferritin, including (i) why it correlates with biomarkers of cell damage, (ii) why it correlates with biomarkers of hydroxyl radical formation (and oxidative stress) and (iii) therefore why it correlates with the presence and/or severity of numerous diseases. This leads to suggestions for how one might exploit the corollaries of the recognition that serum ferritin levels mainly represent a consequence of cell stress and damage.

446 citations


Journal ArticleDOI
TL;DR: There are now metabolic network models; the metabolome is represented by their nodes, and the consensus network Recon2 represents the present state of the art, and has predictive power.

159 citations


Journal ArticleDOI
TL;DR: The view that Phospholipid Bilayer diffusion Is Negligible (PBIN) provides a starting hypothesis for assessing cellular drug uptake that is much better supported by the available evidence, and is both more productive and more predictive.
Abstract: One approach to experimental science involves creating hypotheses, then testing them by varying one or more independent variables and assessing the effects of this variation on the processes of interest. We use this strategy to compare the intellectual status and available evidence for two models or views of mechanisms of transmembrane drug transport into intact biological cells. One (BDII) asserts that lipoidal phospholipid Bilayer Diffusion Is Important, while a second (PBIN) proposes that in normal intact cells Phospholipid Bilayer diffusion Is Negligible (i.e. may be neglected quantitatively), because evolution selected against it, and with transmembrane drug transport being effected by genetically encoded proteinaceous carriers or pores, whose ‘natural’ biological roles and substrates are based in intermediary metabolism. Despite a recent review elsewhere, we can find no evidence able to support BDII as we can find no experiments in intact cells in which phospholipid bilayer diffusion was either varied independently or measured directly (although there are many papers where it was inferred by seeing a covariation of other dependent variables). By contrast, we find an abundance of evidence showing cases in which changes in the activities of named and genetically identified transporters led to measurable changes in the rate or extent of drug uptake. PBIN also has considerable predictive power, and accounts readily for the large differences in drug uptake between tissues, cells and species, in accounting for the metabolite-likeness of marketed drugs, in pharmacogenomics, and in providing a straightforward explanation for the late-stage appearance of toxicity and of lack of efficacy during drug discovery programmes despite macroscopically adequate pharmacokinetics. Consequently, the view that Phospholipid Bilayer diffusion Is Negligible (PBIN) provides a starting hypothesis for assessing cellular drug uptake that is much better supported by the available evidence,

142 citations


Journal ArticleDOI
TL;DR: Inflammatory biomarkers such as ferritin and fibrinogen are themselves inflammatory, creating a positive feedback that exacerbates disease progression, and may provide novel, inexpensive and useful biomarkers of the therapeutic benefits of successful treatments.
Abstract: Most non-communicable diseases involve inflammatory changes in one or more vascular systems, and there is considerable evidence that unliganded iron plays major roles in this Most studies concentrate on biochemical changes, but there are important biophysical correlates Here we summarize recent microscopy-based observations to the effect that iron can have major effects on erythrocyte morphology, on erythrocyte deformability and on both fibrinogen polymerization and the consequent structure of the fibrin clots formed, each of which contributes significantly and negatively to such diseases We highlight in particular type 2 diabetes mellitus, ischemic thrombotic stroke, systemic lupus erythematosus, hereditary hemochromatosis and Alzheimer's disease, while recognizing that many other diseases have co-morbidities (and similar causes) Inflammatory biomarkers such as ferritin and fibrinogen are themselves inflammatory, creating a positive feedback that exacerbates disease progression The biophysical correlates we describe may provide novel, inexpensive and useful biomarkers of the therapeutic benefits of successful treatments

126 citations


Journal ArticleDOI
30 Oct 2014
TL;DR: It is shown that the RBCs of PD patients are indeed rather dramatically deranged in their morphology, exhibiting eryptosis (a kind of programmed cell death), and this morphological indicator may have useful diagnostic and prognostic significance.
Abstract: A major trend in recent Parkinson's disease (PD) research is the investigation of biological markers that could help in identifying at-risk individuals or to track disease progression and response to therapies. Central to this is the knowledge that inflammation is a known hallmark of PD and of many other degenerative diseases. In the current work, we focus on inflammatory signalling in PD, using a systems approach that allows us to look at the disease in a more holistic way. We discuss cyclooxygenases, prostaglandins, thromboxanes and also iron in PD. These particular signalling molecules are involved in PD pathophysiology, but are also very important in an aberrant coagulation/hematology system. We present and discuss a hypothesis regarding the possible interaction of these aberrant signalling molecules implicated in PD, and suggest that these molecules may affect the erythrocytes of PD patients. This would be observable as changes in the morphology of the RBCs and of PD patients relative to healthy controls. We then show that the RBCs of PD patients are indeed rather dramatically deranged in their morphology, exhibiting eryptosis (a kind of programmed cell death). This morphological indicator may have useful diagnostic and prognostic significance.

81 citations


Journal ArticleDOI
09 Jan 2014-PLOS ONE
TL;DR: Findings are consistent with the view that the aberrant morphology of the HH and HF erythrocytes is caused, at least in part, by unliganded iron, whether derived directly via raised ferritin levels or otherwise, and that lowering it or affecting the consequences of its action may be of therapeutic benefit.
Abstract: It is well-known that individuals with increased iron levels are more prone to thrombotic diseases, mainly due to the presence of unliganded iron, and thereby the increased production of hydroxyl radicals. It is also known that erythrocytes (RBCs) may play an important role during thrombotic events. Therefore the purpose of the current study was to assess whether RBCs had an altered morphology in individuals with hereditary hemochromatosis (HH), as well as some who displayed hyperferritinemia (HF). Using scanning electron microscopy, we also assessed means by which the RBC and fibrin morphology might be normalized. An important objective was to test the hypothesis that the altered RBC morphology was due to the presence of excess unliganded iron by removing it through chelation. Very striking differences were observed, in that the erythrocytes from HH and HF individuals were distorted and had a much greater axial ratio compared to that accompanying the discoid appearance seen in the normal samples. The response to thrombin, and the appearance of a platelet-rich plasma smear, were also markedly different. These differences could largely be reversed by the iron chelator desferal and to some degree by the iron chelator clioquinol, or by the free radical trapping agents salicylate or selenite (that may themselves also be iron chelators). These findings are consistent with the view that the aberrant morphology of the HH and HF erythrocytes is caused, at least in part, by unliganded (‘free’) iron, whether derived directly via raised ferritin levels or otherwise, and that lowering it or affecting the consequences of its action may be of therapeutic benefit. The findings also bear on the question of the extent to which accepting blood donations from HH individuals may be desirable or otherwise.

66 citations


Journal ArticleDOI
TL;DR: SpeedyGenes, a PCR-based method for the synthesis of diverse protein libraries that includes an error-correction procedure, enables the efficient synthesis of large genes for use directly in functional screening and its first application for directed evolution is demonstrated.
Abstract: The de novo synthesis of genes is becoming increasingly common in synthetic biology studies. However, the inherent error rate (introduced by errors incurred during oligonucleotide synthesis) limits its use in synthesising protein libraries to only short genes. Here we introduce SpeedyGenes, a PCR-based method for the synthesis of diverse protein libraries that includes an error-correction procedure, enabling the efficient synthesis of large genes for use directly in functional screening. First, we demonstrate an accurate gene synthesis method by synthesising and directly screening (without pre-selection) a 747 bp gene for green fluorescent protein (yielding 85% fluorescent colonies) and a larger 1518 bp gene (a monoamine oxidase, producing 76% colonies with full catalytic activity, a 4-fold improvement over previous methods). Secondly, we show that SpeedyGenes can accommodate multiple and combinatorial variant sequences while maintaining efficient enzymatic error correction, which is particularly crucial for larger genes. In its first application for directed evolution, we demonstrate the use of SpeedyGenes in the synthesis and screening of large libraries of MAO-N variants. Using this method, libraries are synthesised, transformed and screened within 3 days. Importantly, as each mutation we introduce is controlled by the oligonucleotide sequence, SpeedyGenes enables the synthesis of large, diverse, yet controlled variant sequences for the purposes of directed evolution.

42 citations


Journal ArticleDOI
03 Sep 2014-PLOS ONE
TL;DR: Substantial differences in metabolite profiles were apparent between the 2 ‘at-risk’ groups and controls, particularly in concentrations of phospholipids, and impaired mitochondrial fatty acid oxidation may initiate the observed perturbations in lipid metabolism.
Abstract: Background: Blood-vessel dysfunction arises before overt hyperglycemia in type-2 diabetes (T2DM). We hypothesised that a metabolomic approach might identify metabolites/pathways perturbed in this pre-hyperglycemic phase. To test this hypothesis and for specific metabolite hypothesis generation, serum metabolic profiling was performed in young women at increased, intermediate and low risk of subsequent T2DM. Methods: Participants were stratified by glucose tolerance during a previous index pregnancy into three risk-groups: overt gestational diabetes (GDM; n=18); those with glucose values in the upper quartile but below GDM levels (UQ group; n=45); and controls (n=43, below the median glucose values). Follow-up serum samples were collected at a mean 22 months postnatally. Samples were analysed in a random order using Ultra Performance Liquid Chromatography coupled to an electrospray hybrid LTQ-Orbitrap mass spectrometer. Statistical analysis included principal component (PCA) and multivariate methods. Findings: Significant between-group differences were observed at follow-up in waist circumference (86, 95%CI (79–91) vs 80 (76–84) cm for GDM vs controls, p,0.05), adiponectin (about 33% lower in GDM group, p=0.004), fasting glucose, postprandial glucose and HbA1c, but the latter 3 all remained within the ‘normal’ range. Substantial differences in metabolite profiles were apparent between the 2 ‘at-risk’ groups and controls, particularly in concentrations of phospholipids (4 metabolites with p#0.01), acylcarnitines (3 with p#0.02), short- and long-chain fatty acids (3 with p,=0.03), and diglycerides (4 with p#0.05). Interpretation: Defects in adipocyte function from excess energy storage as relatively hypoxic visceral and hepatic fat, and impaired mitochondrial fatty acid oxidation may initiate the observed perturbations in lipid metabolism. Together with evidence from the failure of glucose-directed treatments to improve cardiovascular outcomes, these data and those of others indicate that a new, quite different definition of type-2 diabetes is required. This definition would incorporate disturbed lipid metabolism prior to hyperglycemia.

41 citations


Journal ArticleDOI
TL;DR: GeneGenie allows for the design of oligonucleotide cohorts encoding the gene sequence optimized for expression in any suitable host through an intuitive, easy-to-use web interface, and provides support for mutagenesis or directed evolution studies.
Abstract: GeneGenie, a new online tool available at http://www.gene-genie.org, is introduced to support the design and self-assembly of synthetic genes and constructs. GeneGenie allows for the design of oligonucleotide cohorts encoding the gene sequence optimized for expression in any suitable host through an intuitive, easy-to-use web interface. The tool ensures consistent oligomer overlapping melting temperatures, minimizes the likelihood of misannealing, optimizes codon usage for expression in a selected host, allows for specification of forward and reverse cloning sequences (for downstream ligation) and also provides support for mutagenesis or directed evolution studies. Directed evolution studies are enabled through the construction of variant libraries via the optional specification of ‘variant codons’, containing mixtures of bases, at any position. For example, specifying the variant codon TNT (where N is any nucleotide) will generate an equimolar mixture of the codons TAT, TCT, TGT and TTT at that position, encoding a mixture of the amino acids Tyr, Ser, Cys and Phe. This facility is demonstrated through the use of GeneGenie to develop and synthesize a library of enhanced green fluorescent protein variants.

36 citations


Journal ArticleDOI
TL;DR: The results show how cells rearrange their metabolism to cope with the problems that arise from the loss of these drug-resistance genes, which likely evolved to combat chemical attack from bacterial or fungal competitors.
Abstract: Multiple drug resistance (MDR) in yeast is effected by two major superfamilies of membrane transporters: the major facilitator superfamily (MFS) and the ATP-binding cassette (ABC) superfamily. In the present work, we investigated the cellular responses to disruptions in both MFS (by deleting the transporter gene, QDR3) and ABC (by deleting the gene for the Pdr3 transcription factor) transporter systems by growing diploid homozygous deletion yeast strains in glucose- or ammonium-limited continuous cultures. The transcriptome and the metabolome profiles of these strains, as well as the flux distributions in the optimal solution space, reveal novel insights into the underlying mechanisms of action of QDR3 and PDR3. Our results show how cells rearrange their metabolism to cope with the problems that arise from the loss of these drug-resistance genes, which likely evolved to combat chemical attack from bacterial or fungal competitors. This is achieved through the accumulation of intracellular glucose, glycerol, and inorganic phosphate, as well as by repurposing genes that are known to function in other parts of metabolism in order to minimise the effects of toxic compounds.

14 citations