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Neuronal lesions in Parkinson’s disease (PD) are commonly associated with α-synuclein (α-Syn)-induced cell damage that are present both in the central and peripheral nervous systems of patients, with the enteric nervous system also being especially vulnerable. Here, we bring together evidence that the development and presence of PD depends on specific sets of interlinking factors that include neuroinflammation, systemic inflammation, α-Syn-induced cell damage, vascular dysfunction, iron dysregulation, and gut and periodontal dysbiosis. We argue that there is significant evidence that bacterial inflammagens fuel this systemic inflammation, and might be central to the development of PD. We also discuss the processes whereby bacterial inflammagens may be involved in causing nucleation of proteins, including of α-Syn. Lastly, we review evidence that iron chelation, pre-and probiotics, as well as antibiotics and faecal transplant treatment might be valuable treatments in PD. A most important consideration, however, is that these therapeutic options need to be validated and tested in randomized controlled clinical trials. However, targeting underlying mechanisms of PD, including gut dysbiosis and iron toxicity, have potentially opened up possibilities of a wide variety of novel treatments, which may relieve the characteristic motor and nonmotor deficits of PD, and may even slow the progression and/or accompanying gut-related conditions of the disease.

Affordable and automated cloning platforms are essential to many synthetic biology studies. However, the traditional E. coli-based cloning is a major bottleneck as it requires heat shock or electroporation implemented in the robotic workflows. To overcome this problem, we explored bacterial natural transformation for automatic DNA cloning and engineering. Recombinant plasmids are efficiently generated from Gibson or overlap extension PCR (OE-PCR) products by simply adding the DNA into Acinetobacter baylyi ADP1 cultures. No DNA purification, competence induction, or special equipment is required. Up to 10,000 colonies were obtained per microgram of DNA, while the number of false positive colonies was low. Wecloned and engineered 21 biosynthetic gene clusters (BGCs) of various types, with length from 1.5 to 19 kb and GC content from 35% to 72%. One of them, a nucleoside BGC, showed antibacterial activity. Furthermore, the method was easily transferred to a low-cost benchtop robot with consistent cloning efficiency. Thus, this automatic natural transformation (ANT) cloning provides an easy, robust, and affordable platform for high throughput DNA engineering.

Neuronal lesions in Parkinson’s disease (PD) are commonly associated with α-synuclein (α-Syn)-induced cell damage that are present both in the central and peripheral nervous systems of patients, with the enteric nervous system also being especially vulnerable. Here we bring together evidence that the development and presence of PD depends on specific sets of interlinking factors that include neuro-inflammation, systemic inflammation, α-Syn-induced cell damage, vascular dysfunction, iron dysregulation, gut and periodontal dysbiosis. We argue that there is significant evidence that bacterial inflammagens fuel this systemic inflammation, and might be central to the development of PD. We also discuss the processes whereby lipopolysaccharides may be involved in causing nucleation of proteins, including of α-Syn. Lastly, we review evidence that pre-and probiotics, as well as antibiotics and faecal transplant treatment might be valuable treatments in PD. A most important consideration, however, is that these therapeutic options need to be validated and tested in randomized controlled clinical trials. However, targeting underlying mechanisms of PD, including gut dysbiosis and iron toxicity, have potentially opened up possibilities of a wide variety of novel treatments which may relieve the characteristic non-motor deficits of PD, and may even slow the progression and/or accompanying gut-related conditions of the disease.

Background Type 2 Diabetes Mellitus (T2DM) is a well-known comorbidity to COVID-19 and coagulopathies are a common accompaniment to both T2DM and COVID-19. In addition, patients with COVID-19 are known to develop micro-clots within the lungs. The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated clotting severity and the extent of clotting pathologies in the individual who was infected or not would be highly desirable. Methods We show here that microclots can be detected in the native plasma of COVID-19, as well as T2DM patients, without the addition of any clotting agent, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. Results In COVID-19 plasma these microclots are significantly increased when compared to the levels in T2DM. Conclusions This fluorogenic test may provide a rapid and convenient test with 100% sensitivity (P

Molecular similarity is an elusive but core &ldquo, unsupervised&rdquo, cheminformatics concept, yet different &ldquo, fingerprint&rdquo, encodings of molecular structures return very different similarity values, even when using the same similarity metric. Each encoding may be of value when applied to other problems with objective or target functions, implying that a priori none are &ldquo, better&rdquo, than the others, nor than encoding-free metrics such as maximum common substructure (MCSS). We here introduce a novel approach to molecular similarity, in the form of a variational autoencoder (VAE). This learns the joint distribution p(z|x) where z is a latent vector and x are the (same) input/output data. It takes the form of a &ldquo, bowtie&rdquo, shaped artificial neural network. In the middle is a &ldquo, bottleneck layer&rdquo, or latent vector in which inputs are transformed into, and represented as, a vector of numbers (encoding), with a reverse process (decoding) seeking to return the SMILES string that was the input. We train a VAE on over six million druglike molecules and natural products (including over one million in the final holdout set). The VAE vector distances provide a rapid and novel metric for molecular similarity that is both easily and rapidly calculated. We describe the method and its application to a typical similarity problem in cheminformatics.

The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated whether the individual was infected or not would be highly desirable. Coagulaopathies are a common accompaniment to COVID-19, especially micro-clots within the lungs. We show here that microclots can be detected in the native plasma of COVID-19 patient, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. This provides a rapid and convenient test (P

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), coronavirus disease 2019 (COVID-19)-induced infection is strongly associated with various coagulopathies that may result in either bleeding and thrombocytopenia or hypercoagulation and thrombosis. Thrombotic and bleeding or thrombotic pathologies are significant accompaniments to acute respiratory syndrome and lung complications in COVID-19. Thrombotic events and bleeding, often occurs in subjects with weak multiple risk factors and co-morbidities. Of particular interest are the various circulating inflammatory coagulation biomarkers involved directly in clotting, with specific focus on fibrin(ogen), D-dimer, P-selectin and von Willebrand Factor (vWF). Central to activity of these biomarkers are their receptors and signaling pathways on endothelial cells, platelets and erythrocytes. In this review, we discuss vascular implications of COVID-19, and relate this to circulating biomarker, endothelial, erythrocyte and platelet dysfunction. During the progression of the disease, these markers may either be within healthy levels, upregulated or eventually depleted. Most significant is that patients need to be treated early in the disease progression, when high levels of vWF, P-selectin and fibrinogen are present with still low levels of D-dimer. Progression to vWF and fibrinogen depletion with high D-dimer levels and even higher P-selectin levels, followed by the cytokine storm, will be indicative of a poor prognosis. We conclude by looking at point-of-care devises and methodologies in COVID-19 management and suggest that a personalized medicine approach should be considered in the treatment of patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), also known as coronavirus disease 2019 (COVID-19)-induced infection, is strongly associated with various coagulopathies that may result in either bleeding and thrombocytopenia or hypercoagulation and thrombosis. Thrombotic and bleeding or thrombotic pathologies are significant accompaniments to acute respiratory syndrome and lung complications in COVID-19. Thrombotic events and bleeding often occur in subjects with weak constitutions, multiple risk factors and comorbidities. Of particular interest are the various circulating inflammatory coagulation biomarkers involved directly in clotting, with specific focus on fibrin(ogen), D-dimer, P-selectin and von Willebrand Factor (VWF). Central to the activity of these biomarkers are their receptors and signalling pathways on endothelial cells, platelets and erythrocytes. In this review, we discuss vascular implications of COVID-19 and relate this to circulating biomarker, endothelial, erythrocyte and platelet dysfunction. During the progression of the disease, these markers may either be within healthy levels, upregulated or eventually depleted. Most significant is that patients need to be treated early in the disease progression, when high levels of VWF, P-selectin and fibrinogen are present, with normal or slightly increased levels of D-dimer (however, D-dimer levels will rapidly increase as the disease progresses). Progression to VWF and fibrinogen depletion with high D-dimer levels and even higher P-selectin levels, followed by the cytokine storm, will be indicative of a poor prognosis. We conclude by looking at point-of-care devices and methodologies in COVID-19 management and suggest that a personalized medicine approach should be considered in the treatment of patients.

Introduction It is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites. Objectives Blood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to detect and identify metabolites present in serum, but to also establish a method capable of measure their uptake and secretion by different cell lines. Methods We develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the ‘exometabolome’ or metabolic footprint). Results Our method measures some 4000–5000 metabolic features in both positive and negative electrospray ionisation modes. We show that the metabolic footprints of different cell lines differ greatly from each other. Conclusion Our new, 15-min untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum. This will enable future research to study these differences in multiple cell lines that will relate this to transporter expression, thereby advancing our knowledge of transporter substrates, both natural and xenobiotic compounds.

ObjectiveThe risk of cardiovascular events in patients with RA is disproportionately heightened as a result of systemic inflammation. The relative effect of autoimmune-associated citrullination on the structure and thrombotic potential of fibrin(ogen) remains unknown. We therefore compared indices of vascular function, inflammation, coagulation and fibrin clot composition in RA patients with healthy controls and evaluated inter-parameter relationships.MethodsBlood samples were collected from 30 RA patients and 25 age- and gender-matched healthy volunteers. Levels of SAA, CRP, ICAM-1 and VCAM-1 was measured using a sandwich immunoassay. Whole blood coagulation was assessed using Thromboelastography. Fibrin clot networks and fiber structure was investigated using Scanning Electron Microscopy. The detection and quantification of citrullination in formed fibrin clots were performed using a fluorescently labeled Citrulline monoclonal antibody with Confocal Microscopy.ResultsConcentrations of SAA, CRP and ICAM-1 were significantly elevated in RA patients compared to controls. TEG parameters relating to coagulation initiation (R and K), rate of fibrin cross-linking (α-Angle), and time to reach maximum thrombus generation (TMRTG) were attenuated in RA patients. Parameters relating to clot strength (MA, MRTG, TGG) did not statistically differ between RA and controls. Logistic regression modelling revealed stronger association between acute phase reactants (CRP, SAA) with TEG parameters than endothelial function markers. Microscopic analysis revealed denser networks of thicker fibrin fibers in RA patients compared to controls [median (interquartile range) 214 (170-285)vs120 (100-144) nm respectively, pConclusionPatients with active RA display a coagulation profile that is dissimilar to general findings associated with other inflammatory conditions. The alteration of protein structures by autoimmune linked citrullination could play a role in determining the structure of fibrin and the potential of conferring a heightened thrombotic risk in RA patients.

Background: Porphyromonas gingivalis and its inflammagens is associated with a number of systemic diseases, such as cardiovascular disease and type 2 diabetes (T2DM). The proteases, gingipains have also recently been identified in brains of Alzheimer’s disease patients and in blood of Parkinson’s disease patients. Bacterial inflammagens, including lipopolysaccharides (LPSs) and various proteases in circulation may drive systemic inflammation. Methods: Here, we investigate the effects of the bacterial products LPS from Escherichia coli and Porphyromonas gingivalis, and also the P. gingivalis gingipain (recombinant P. gingivalis gingipain R1 (RgpA)), on clot architecture and clot formation in whole blood and plasma from healthy individuals, as well as in purified fibrinogen models. Structural analysis of clots was performed using confocal, scanning electron microscopy and AFM-Raman. We use thromboelastography (TEG) and rheometry to compare the static and dynamic mechanical properties of clots. Results: We found that these inflammagens may interact with fibrin(ogen) and this interaction causes anomalous blood clotting. Conclusions: These techniques, in combination provide insight into the effects of these bacterial products on cardiovascular health, and particularly clot structure and mechanics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genotypic microbial resistance to antibiotics with intracellular targets commonly arises from mutations that increase the activities of transporters (pumps) that cause the efflux of intracellular antibiotics.A prioriit is not obvious why this is so much more common than are mutations that simply inhibit the activity of uptake transporters for the antibiotics. We analyse quantitatively a mathematical model consisting of one generic equilibrative transporter and one generic concentrative uptake transporter (representing any number of each), together with one generic efflux transporter. The initial conditions are designed to give an internal concentration of the antibiotic that is three times the minimum inhibitory concentration (MIC). The effect of varying the activity of each transporter type 100-fold is dramatically asymmetric, in that lowering the activities of individual uptake transporters has comparatively little effect on internal concentrations of the antibiotic. By contrast, increasing the activity of the efflux transporter lowers the internal antibiotic concentration to levels far below the MIC. Essentially, these phenomena occur because inhibiting individual influx transporters allows others to ‘take up the slack’, whereas increasing the activity of the generic efflux transporter cannot easily be compensated. The findings imply strongly that inhibiting efflux transporters is a much better approach for fighting antimicrobial resistance than is stimulating import transporters. This has obvious implications for the development of strategies to combat the development of microbial resistance to antibiotics and possibly also cancer therapeutics in human.

BackgroundType 2 Diabetes Mellitus (T2DM) is a well-known comorbidity to COVID-19 and coagulopathies are a common accompaniment to both T2DM and COVID-19. In addition, patients with COVID-19 are known to develop micro-clots within the lungs. The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated clotting severity and the extent of clotting pathologies in the individual who was infected or not would be highly desirable.MethodsPlatelet poor plasma (PPP) was collected and frozen. On the day of analysis, PPP samples were thawed and analysed. We show here that microclots can be detected in the native plasma of twenty COVID-19, as well as ten T2DM patients, without the addition of any clotting agent, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. Results were compared to ten healthy age-matched individuals.ResultsIn COVID-19 plasma these microclots are significantly increased when compared to the levels in T2DM.ConclusionsThis fluorogenic test may provide a rapid and convenient test with 100% sensitivity (P

The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated whether the individual was infected or not would be highly desirable. Coagulaopathies are a common accompaniment to COVID-19, especially micro-clots within the lungs. We show here that microclots can be detected in the native plasma of COVID-19 patient, without the addition of any clotting agent, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. This provides a rapid and convenient test with 100% sensitivity (P

IntroductionPre-eclampsia is a major cause of maternal and fetal mortality and morbidity worldwide. Its pathophysiology remains unclear, but mitochondrial dysfunction and oxidative stress have been implicated. L-Ergothioneine is a naturally occurring, water-soluble betaine, that has demonstrated antioxidant properties. Using the reduced uterine perfusion pressure (RUPP) rat model of pre-eclampsia, this study aimed to define the plasma metabolic profile following treatment with L-Ergothioneine.MethodsThe effect of L-Ergothioneine (ET) treatment was explored using in vivo treatment in rats: Sham control (SC, n = 5), RUPP control (RC, n = 5), Sham +ET (ST, n = 5), RUPP +ET (RT, n = 5). Differential expression of plasma metabolites were obtained using untargeted liquid chromatography coupled to mass spectrometry. Statistical analysis was performed on normalised data comparing RC to SC, RT to RC, and RT to ST. Metabolites significantly altered (FDR < 0.05) were identified through database search.ResultsWe report significantly lower levels of L-palmitoylcarnitine in RC compared to SC, a fatty acyl substrate involved in beta-oxidation in the mitochondria. We report that a metabolite that has been associated with oxidative stress (Glutamylcysteine) was detected at significantly higher levels in RT vs RC and RT vs ST. Five metabolites associated with inflammation were significantly lower in RT vs RC and three metabolites in RT vs ST, demonstrating the anti-inflammatory effects of ET in the RUPP rat model of pre-eclampsia.ConclusionsL-Ergothioneine may help preserve mitochondrial function by increasing antioxidant levels, and reducing inflammatory responses associated with pre-eclampsia. This study shows the potential of L-Ergothioneine as a treatment for pre-eclampsia.

Systems biology has experienced dramatic growth in the number, size, and complexity of computational models. To reproduce simulation results and reuse models, researchers must exchange unambiguous model descriptions. We review the latest edition of the Systems Biology Markup Language (SBML), a format designed for this purpose. A community of modelers and software authors developed SBML Level 3 over the past decade. Its modular form consists of a core suited to representing reaction‐based models and packages that extend the core with features suited to other model types including constraint‐based models, reaction‐diffusion models, logical network models, and rule‐based models. The format leverages two decades of SBML and a rich software ecosystem that transformed how systems biologists build and interact with models. More recently, the rise of multiscale models of whole cells and organs, and new data sources such as single‐cell measurements and live imaging, has precipitated new ways of integrating data with models. We provide our perspectives on the challenges presented by these developments and how SBML Level 3 provides the foundation needed to support this evolution., Over the past two decades, scientists from different fields have been developing SBML, a standard format for encoding computational models in biology and medicine. This article summarizes recent progress and gives perspectives on emerging challenges.

Preeclampsia is a multifactorial hypertensive disorder of pregnancy founded on abnormal placentation, and the resultant placental ischemic microenvironment is thought to play a crucial role in its pathophysiology. Placental ischemia because of fluctuations in the delivery of oxygen results in oxidative stress, and recent evidence suggests that mitochondrial dysfunction may be a prime mediator. However, large clinical trials of therapeutic antioxidants such as vitamins C and E for the treatment of preeclampsia have been disappointing. L-(+)-ergothioneine (ERG)—an unusual amino acid betaine derived from histidine—has important cytoprotective and antioxidant properties under conditions of high oxidative stress. In this study, we investigated the potential therapeutic effects of administration of ERG in the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia. ERG (25 mg/kg per day) was administered to rats on gestational day 11. On gestational day 14, RUPP surgery was performed, and on gestational day 19, blood pressure (mean arterial pressure) and fetal growth were measured. Production of mitochondria-specific H 2 O 2 was analyzed in vivo in kidney samples. ERG ameliorated the hypertension (129±3 versus 115±4 mm Hg; P =0.01; n=8) and significantly increased pup weight in RUPP rats. ERG also significantly decreased circulating levels of antiangiogenic sFlt-1 (soluble fms-like tyrosine kinase-1) in RUPP rats (1367±245 pg/mL; P =0.04). Mitochondria-specific H 2 O 2 (0.022±0.003 versus 0.029±0.001; MitoP/B ratio, n=3; P =0.05) was also significantly decreased in kidney tissue in RUPP rats treated with ERG. These data support the potential use of ERG for the treatment of preeclampsia.

Since the successful conquest of many acute, communicable (infectious) diseases through the use of vaccines and antibiotics, the currently most prevalent diseases are chronic and progressive in nature, and are all accompanied by inflammation. These diseases include neurodegenerative (e.g. Alzheimer's, Parkinson's), vascular (e.g. atherosclerosis, pre-eclampsia, type 2 diabetes) and autoimmune (e.g. rheumatoid arthritis and multiple sclerosis) diseases that may appear to have little in common. In fact they all share significant features, in particular chronic inflammation and its attendant inflammatory cytokines. Such effects do not happen without underlying and initially 'external' causes, and it is of interest to seek these causes. Taking a systems approach, we argue that these causes include (i) stress-induced iron dysregulation, and (ii) its ability to awaken dormant, non-replicating microbes with which the host has become infected. Other external causes may be dietary. Such microbes are capable of shedding small, but functionally significant amounts of highly inflammagenic molecules such as lipopolysaccharide and lipoteichoic acid. Sequelae include significant coagulopathies, not least the recently discovered amyloidogenic clotting of blood, leading to cell death and the release of further inflammagens. The extensive evidence discussed here implies, as was found with ulcers, that almost all chronic, infectious diseases do in fact harbour a microbial component. What differs is simply the microbes and the anatomical location from and at which they exert damage. This analysis offers novel avenues for diagnosis and treatment.

Pyrithione (2-mercaptopyridine-N-oxide) is a metal binding modified pyridine, the antibacterial activity of which was described over 60 years ago. The formulation of zinc-pyrithione is commonly used in the topical treatment of certain dermatological conditions. However, the characterisation of the cellular uptake of pyrithione has not been elucidated, although an unsubstantiated assumption has persisted that pyrithione and/or its metal complexes undergo a passive diffusion through cell membranes. Here, we have profiled specific membrane transporters from an unbiased interrogation of 532 E. coli strains of knockouts of genes encoding membrane proteins from the Keio collection. Two membrane transporters, FepC and MetQ, seemed involved in the uptake of pyrithione and its cognate metal complexes with copper, iron, and zinc. Additionally, the phenotypes displayed by CopA and ZntA knockouts suggested that these two metal effluxers drive the extrusion from the bacterial cell of potentially toxic levels of copper, and perhaps zinc, which hyperaccumulate as a function of pyrithione. The involvement of these distinct membrane transporters contributes to the understanding of the mechanisms of action of pyrithione specifically and highlights, more generally, the important role that membrane transporters play in facilitating the uptake of drugs, including metal–drug compounds.

The UK Synthetic Biology Research Centre, SYNBIOCHEM, hosted by the Manchester Institute of Biotechnology at the University of Manchester is delivering innovative technology platforms to facilitate the predictable engineering of microbial bio-factories for fine and speciality chemicals production. We provide an overview of our foundry activities that are being applied to grand challenge projects to deliver innovation in bio-based chemicals production for industrial biotechnology.

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.

BackgroundIt 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.ResultsThe 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.

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.

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 diseasesin 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 yeastSaccharomyces cerevisiaeto 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 ofS. cerevisiaewas altered by knock-out ofTOR1 orYIH1. 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. BecauseS. cerevisiaeis 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.

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.

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.

Background. 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. Results. 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 - and -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). Conclusions. 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.Background. 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. Results. 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 - and -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). Conclusions. 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.

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.

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.

SummarySeveral 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.

Directed evolution (DE) is a powerful tool for optimizing an enzyme's properties toward a particular objective, such as broader substrate scope, greater thermostability, or increased k

Many chronic diseases, including those classified as cardiovascular, neurodegenerative, or autoimmune, are characterized by persistent inflammation. The origin of this inflammation is mostly unclear, but it is typically mediated by inflammatory biomarkers, such as cytokines, and affected by both environmental and genetic factors. Recently circulating bacterial inflammagens such as lipopolysaccharide (LPS) have been implicated. We used a highly selective mouse monoclonal antibody to detect bacterial LPS in whole blood and/or platelet poor plasma of individuals with Parkinson’s Disease, Alzheimer’s type dementia, or Type 2 Diabetes Mellitus. Our results showed that staining is significantly enhanced (P

In recent work, we discovered that the presence of highly substoichiometric amounts (10−8molar ratio) of lipopolysaccharide (LPS) from Gram-negative bacteria caused fibrinogen clotting to lead to the formation of an amyloid form of fibrin. We here show that the broadly equivalent lipoteichoic acids (LTAs) from two species of Gram-positive bacteria have similarly (if not more) potent effects. Using thioflavin T fluorescence to detect amyloid as before, the addition of low concentrations of free ferric ion is found to have similar effects. Luminescent conjugated oligothiophene dyes (LCOs), marketed under the trade name AmytrackerTM, also stain classical amyloid structures. We here show that they too give very large fluorescence enhancements when clotting is initiated in the presence of the four amyloidogens (LPS, ferric ions and two LTA types). The staining patterns differ significantly as a function of both the amyloidogens and the dyes used to assess them, indicating clearly that the nature of the clots formed is different. This is also the case when clotting is measured viscometrically using thromboelastography. Overall, the data provide further evidence for an important role of bacterial cell wall products in the various coagulopathies that are observable in chronic, inflammatory diseases. The assays may have potential in both diagnostics and therapeutics.

The thrombin-induced polymerisation of fibrinogen to form fibrin is well established as a late stage of blood clotting. It is known that Parkinson’s Disease (PD) is accompanied by dysregulation in blood clotting, but it is less widely known as a coagulopathy. In recent work, we showed that the presence of tiny amounts of bacterial lipopolysaccharide (LPS) in healthy individuals could cause clots to adopt an amyloid form, and this could be observed via scanning electron microscopy (SEM) or via the fluorescence of thioflavin-T. This could be prevented by the prior addition of lipopolysaccharide-binding protein (LBP). We had also observed by SEM this unusual clotting in the blood of patients with Parkinson’s Disease. We hypothesised, and here show, that this too can be prevented by LBP in the context of PD. This adds further evidence implicating inflammatory microbial cell wall products as an accompaniment to the disease, and May be part of its aetiology. This May lead to novel treatment strategies in PD designed to target microbes and their products.

Directed evolution (DE) is a powerful tool for optimizing an enzyme's properties toward a particular objective, such as broader substrate scope, greater thermostability, or increased kcat. A successful DE project requires the generation of genetic diversity and subsequent screening or selection to identify variants with improved fitness. In contrast to random methods (error-prone PCR or DNA shuffling), site-directed mutagenesis enables the rational design of variant libraries and provides control over the nature and frequency of the encoded mutations. Knowledge of protein structure, dynamics, enzyme mechanisms, and natural evolution demonstrates that multiple (combinatorial) mutations are required to discover the most improved variants. To this end, we describe an experimentally straightforward and low-cost method for the preparation of combinatorial variant libraries. Our approach employs a two-step PCR protocol, first producing mutagenic megaprimers, which can then be combined in a "mix-and-match" fashion to generate diverse sets of combinatorial variant libraries both quickly and accurately.

// 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.

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.

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.

Improving enzymes by directed evolution requires the navigation of very large search spaces; we survey how to do this intelligently., 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 (K d) and catalytic (k cat) 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 k cat (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.

Background Transporter proteins mediate the translocation of substances across the membranes of living cells. Many transport processes are energetically expensive and the cells use 20 to 60% of their energy to power the transportomes. We hypothesized that there may be an evolutionary selection pressure for lower energy transporters. Results We performed a genome-wide analysis of the compositional reshaping of the transportomes across the kingdoms of bacteria, archaea, and eukarya. We found that the share of ABC transporters is much higher in bacteria and archaea (ca. 27% of the transportome) than in primitive eukaryotes (13%), algae and plants (10%) and in fungi and animals (5–6%). This decrease is compensated by an increased occurrence of secondary transporters and ion channels. The share of ion channels is particularly high in animals (ca. 30% of the transportome) and algae and plants with (ca. 13%), when compared to bacteria and archaea with only 6–7%. Therefore, our results show a move to a preference for the low-energy-demanding transporters (ion channels and carriers) over the more energy-costly transporter classes (ATP-dependent families, and ABCs in particular) as part of the transition from prokaryotes to eukaryotes. The transportome analysis also indicated seven bacterial species, including Neorickettsia risticii and Neorickettsia sennetsu, as likely origins of the mitochondrion in eukaryotes, based on the phylogenetically restricted presence therein of clear homologues of modern mitochondrial solute carriers. Conclusions The results indicate that the transportomes of eukaryotes evolved strongly towards a higher energetic efficiency, as ATP-dependent transporters diminished and secondary transporters and ion channels proliferated. These changes have likely been important in the development of tissues performing energetically costly cellular functions. Electronic supplementary material The online version of this article (10.1186/s12864-018-4816-5) contains supplementary material, which is available to authorized users.

Although it is widely recognised as involving two stages (poor placentation followed by oxidative stress/inflammation), the precise originating causes of pre-eclampsia (PE) remain elusive. We have previously brought together some of the considerable evidence that a (dormant) microbial component is commonly a significant part of its aetiology. However, apart from recognising, consistent with this view, that the many inflammatory markers of PE are also increased in infection, we had little to say about immunity, whether innate or adaptive. In addition, we focussed on the gut, oral and female urinary tract microbiomes as the main sources of the infection. We here marshall further evidence for an infectious component in PE, focussing on the immunological tolerance characteristic of pregnancy, and the well-established fact that increased exposure to the father’s semen assists this immunological tolerance. As well as these benefits, however, semen is not sterile, microbial tolerance mechanisms may exist, and we also review the evidence that semen may be responsible for inoculating the developing conceptus with microbes, not all of which are benign. It is suggested that when they are not, this may be a significant cause of preeclampsia. A variety of epidemiological and other evidence is entirely consistent with this, not least correlations between semen infection, infertility and PE. Our view also leads to a series of other, testable predictions. Overall, we argue for a significant paternal role in the development of PE through microbial infection of the mother via insemination.“In one of the last articles which he wrote, the late Professor F J Browne (1958) expressed the opinion that all the essential facts about pregnancy toxaemia are now available and that all that is required to solve the problem is to fit them together in the right order, like the pieces of a jigsaw puzzle” [1]“It appears astonishing how little attention has been given in reproductive medicine to the maternal immune system over the last few decades.” [2]

Motivation Synthetic biology is typified by developing novel genetic constructs from the assembly of reusable synthetic DNA parts, which contain one or more features such as promoters, ribosome binding sites, coding sequences and terminators. PartsGenie is introduced to facilitate the computational design of such synthetic biology parts, bridging the gap between optimization tools for the design of novel parts, the representation of such parts in community-developed data standards such as Synthetic Biology Open Language, and their sharing in journal-recommended data repositories. Consisting of a drag-and-drop web interface, a number of DNA optimization algorithms, and an interface to the well-used data repository JBEI ICE, PartsGenie facilitates the design, optimization and dissemination of reusable synthetic biology parts through an integrated application. Availability and implementation PartsGenie is freely available at https://parts.synbiochem.co.uk.

Armed with the digital availability of two natural products libraries, amounting to some 195 885 molecular entities, we ask the question of how we can best sample from them to maximize their "representativeness" in smaller and more usable libraries of 96, 384, 1152, and 1920 molecules. The term "representativeness" is intended to include diversity, but for numerical reasons (and the likelihood of being able to perform a QSAR) it is necessary to focus on areas of chemical space that are more highly populated. Encoding chemical structures as fingerprints using the RDKit "patterned" algorithm, we first assess the granularity of the natural products space using a simple clustering algorithm, showing that there are major regions of "denseness" but also a great many very sparsely populated areas. We then apply a "hybrid" hierarchical K-means clustering algorithm to the data to produce more statistically robust clusters from which representative and appropriate numbers of samples may be chosen. There is necessarily again a trade-off between cluster size and cluster number, but within these constraints, libraries containing 384 or 1152 molecules can be found that come from clusters that represent some 18 and 30% of the whole chemical space, with cluster sizes of, respectively, 50 and 27 or above, just about sufficient to perform a QSAR. By using the online availability of molecules via the Molport system (www.molport.com), we are also able to construct (and, for the first time, provide the contents of) a small virtual library of available molecules that provided effective coverage of the chemical space described. Consistent with this, the average molecular similarities of the contents of the libraries developed is considerably smaller than is that of the original libraries. The suggested libraries may have use in molecular or phenotypic screening, including for determining possible transporter substrates.

Synthetic biology is typified by developing novel genetic constructs from the assembly of reusable synthetic DNA parts, which contain one or more features such as promoters, ribosome binding sites, coding sequences and terminators. While repositories of such parts exist to promote their reuse, there is still a need to design novel parts from scratch.PartsGenie, freely available at http://parts.synbiochem.co.uk, is introduced to facilitate the computational design of such synthetic biology parts. PartsGenie has been designed to bridge the gap between optimisation tools for the design of novel parts, the representation of such parts in community-developed data standards such as Synthetic Biology Open Language (SBOL), and their sharing in journal-recommended data repositories.Consisting of a drag-and-drop web interface, a number of DNA optimisation algorithms, and an interface to the well-used data repository JBEI ICE, PartsGenie facilitates the design, optimisation and dissemination of reusable synthetic biology parts through a single, integrated application. PartsGenie can therefore be used as a single, stand-alone tool, or integrated into larger synthetic biology pipelines that are being developed in the SYNBIOCHEM centre and elsewhere.