Herpesvirus Infection of Endothelial Cells as a Systemic Pathological Axis in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Understanding the pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is critical for advancing treatment options. This review explores the novel hypothesis that a herpesvirus infection of endothelial cells (ECs) may underlie ME/CFS symptomatology. We review evidence linking herpesviruses to persistent EC infection and the implications for endothelial dysfunction, encompassing blood flow regulation, coagulation, and cognitive impairment-symptoms consistent with ME/CFS and Long COVID. This paper provides a synthesis of current research on herpesvirus latency and reactivation, detailing the impact on ECs and subsequent systemic complications, including latent modulation and long-term maladaptation. We suggest that the chronicity of ME/CFS symptoms and the multisystemic nature of the disease may be partly attributable to herpesvirus-induced endothelial maladaptation. Our conclusions underscore the necessity for further investigation into the prevalence and load of herpesvirus infection within the ECs of ME/CFS patients. This review offers conceptual advances by proposing an endothelial infection model as a systemic mechanism contributing to ME/CFS, steering future research toward potentially unexplored avenues in understanding and treating this complex syndrome.

Cardiovascular and haematological pathology in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): A role for viruses.

ME/CFS is a debilitating chronic condition that often develops after viral or bacterial infection. Insight from the study of Long COVID/Post Acute Sequelae of COVID-19 (PASC), the post-viral syndrome associated with SARS-CoV-2 infection, might prove to be useful for understanding pathophysiological mechanisms of ME/CFS. Disease presentation is similar between the two conditions, and a subset of Long COVID patients meet the diagnostic criteria for ME/CFS. Since Long COVID is characterized by significant vascular pathology - including endothelial dysfunction, coagulopathy, and vascular dysregulation - the question of whether or not the same biological abnormalities are of significance in ME/CFS arises. Cardiac abnormalities have for a while now been documented in ME/CFS cohorts, with recent studies demonstrating major deficits in cerebral blood flow, and hence vascular dysregulation. A growing body of research is demonstrating that ME/CFS is accompanied by platelet hyperactivation, anomalous clotting, a procoagulant phenotype, and endothelial dysfunction. Endothelial damage and dysregulated clotting can impair substance exchange between blood and tissues, and result in hypoperfusion, which may contribute to the manifestation of certain ME/CFS symptoms. Here we review the ME/CFS literature to summarize cardiovascular and haematological findings documented in patients with the condition, and, in this context, briefly discuss the potential role of previously-implicated pathogens. Overall, cardiac and haematological abnormalities are present within ME/CFS cohorts. While atherosclerotic heart disease is not significantly associated with ME/CFS, suboptimal cardiovascular function defined by reduced cardiac output, impaired cerebral blood flow, and vascular dysregulation are, and these abnormalities do not appear to be influenced by deconditioning. Rather, these cardiac abnormalities may result from dysfunction in the (autonomic) nervous system. Plenty of recently published studies are demonstrating significant platelet hyperactivity and endothelial dysfunction in ME/CFS, as well as anomalous clotting processes. It is of particular importance to determine to what extent these cardiovascular and haematological abnormalities contribute to symptom severity, and if these two systems can be targeted for therapeutic purposes. Viral reservoirs of herpesviruses exist in ME/CFS, and most likely contribute to cardiovascular and haematological dysfunction directly or indirectly. This review highlights the potential of studying cardiac functioning, the vasculature, and coagulation system in ME/CFS.

Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system.

BACKGROUND: Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation. METHODS: Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician. RESULTS: Our long COVID cohort's symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots. CONCLUSION: Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

The potential role of ischaemia-reperfusion injury in chronic, relapsing diseases such as rheumatoid arthritis, Long COVID, and ME/CFS: evidence, mechanisms, and therapeutic implications.

Ischaemia-reperfusion (I-R) injury, initiated via bursts of reactive oxygen species produced during the reoxygenation phase following hypoxia, is well known in a variety of acute circumstances. We argue here that I-R injury also underpins elements of the pathology of a variety of chronic, inflammatory diseases, including rheumatoid arthritis, ME/CFS and, our chief focus and most proximally, Long COVID. Ischaemia may be initiated via fibrin amyloid microclot blockage of capillaries, for instance as exercise is started; reperfusion is a necessary corollary when it finishes. We rehearse the mechanistic evidence for these occurrences here, in terms of their manifestation as oxidative stress, hyperinflammation, mast cell activation, the production of marker metabolites and related activities. Such microclot-based phenomena can explain both the breathlessness/fatigue and the post-exertional malaise that may be observed in these conditions, as well as many other observables. The recognition of these processes implies, mechanistically, that therapeutic benefit is potentially to be had from antioxidants, from anti-inflammatories, from iron chelators, and via suitable, safe fibrinolytics, and/or anti-clotting agents. We review the considerable existing evidence that is consistent with this, and with the biochemical mechanisms involved.

Immuno-Thrombotic Complications of COVID-19: Implications for Timing of Surgery and Anticoagulation.

Early in the coronavirus disease 2019 (COVID-19) pandemic, global governing bodies prioritized transmissibility-based precautions and hospital capacity as the foundation for delay of elective procedures. As elective surgical volumes increased, convalescent COVID-19 patients faced increased postoperative morbidity and mortality and clinicians had limited evidence for stratifying individual risk in this population. Clear evidence now demonstrates that those recovering from COVID-19 have increased postoperative morbidity and mortality. These data-in conjunction with the recent American Society of Anesthesiologists guidelines-offer the evidence necessary to expand the early pandemic guidelines and guide the surgeon's preoperative risk assessment. Here, we argue elective surgeries should still be delayed on a personalized basis to maximize postoperative outcomes. We outline a framework for stratifying the individual COVID-19 patient's fitness for surgery based on the symptoms and severity of acute or convalescent COVID-19 illness, coagulopathy assessment, and acuity of the surgical procedure. Although the most common manifestation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is COVID-19 pneumonitis, every system in the body is potentially afflicted by an endotheliitis. This endothelial derangement most often manifests as a hypercoagulable state on admission with associated occult and symptomatic venous and arterial thromboembolisms. The delicate balance between hyper and hypocoagulable states is defined by the local immune-thrombotic crosstalk that results commonly in a hemostatic derangement known as fibrinolytic shutdown. In tandem, the hemostatic derangements that occur during acute COVID-19 infection affect not only the timing of surgical procedures, but also the incidence of postoperative hemostatic complications related to COVID-19-associated coagulopathy (CAC). Traditional methods of thromboprophylaxis and treatment of thromboses after surgery require a tailored approach guided by an understanding of the pathophysiologic underpinnings of the COVID-19 patient. Likewise, a prolonged period of risk for developing hemostatic complications following hospitalization due to COVID-19 has resulted in guidelines from differing societies that recommend varying periods of delay following SARS-CoV-2 infection. In conclusion, we propose the perioperative, personalized assessment of COVID-19 patients' CAC using viscoelastic hemostatic assays and fluorescent microclot analysis.

Membrane Transporters Involved in the Antimicrobial Activities of Pyrithione in Escherichia coli.

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.

A protet-based, protonic charge transfer model of energy coupling in oxidative and photosynthetic phosphorylation.

Textbooks of biochemistry will explain that the otherwise endergonic reactions of ATP synthesis can be driven by the exergonic reactions of respiratory electron transport, and that these two half-reactions are catalyzed by protein complexes embedded in the same, closed membrane. These views are correct. The textbooks also state that, according to the chemiosmotic coupling hypothesis, a (or the) kinetically and thermodynamically competent intermediate linking the two half-reactions is the electrochemical difference of protons that is in equilibrium with that between the two bulk phases that the coupling membrane serves to separate. This gradient consists of a membrane potential term Δψ and a pH gradient term ΔpH, and is known colloquially as the protonmotive force or pmf. Artificial imposition of a pmf can drive phosphorylation, but only if the pmf exceeds some 150-170mV; to achieve in vivo rates the imposed pmf must reach 200mV. The key question then is 'does the pmf generated by electron transport exceed 200mV, or even 170mV?' The possibly surprising answer, from a great many kinds of experiment and sources of evidence, including direct measurements with microelectrodes, indicates it that it does not. Observable pH changes driven by electron transport are real, and they control various processes; however, compensating ion movements restrict the Δψ component to low values. A protet-based model, that I outline here, can account for all the necessary observations, including all of those inconsistent with chemiosmotic coupling, and provides for a variety of testable hypotheses by which it might be refined.

An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines.

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.

Gingipain R1 and Lipopolysaccharide From Porphyromonas gingivalis Have Major Effects on Blood Clot Morphology and Mechanics.

Background:Porphyromonas gingivalis and its inflammagens are 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 the brains of Alzheimer's disease patients and in the 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 microscopy, scanning electron microscopy, and AFM-Raman imaging. 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.

The Biology of Lactoferrin, an Iron-Binding Protein That Can Help Defend Against Viruses and Bacteria.

Lactoferrin is a nutrient classically found in mammalian milk. It binds iron and is transferred via a variety of receptors into and between cells, serum, bile, and cerebrospinal fluid. It has important immunological properties, and is both antibacterial and antiviral. In particular, there is evidence that it can bind to at least some of the receptors used by coronaviruses and thereby block their entry. Of importance are Heparan Sulfate Proteoglycans (HSPGs) and the host receptor angiotensin-converting enzyme 2 (ACE2), as based on other activities lactoferrin might prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from attaching to the host cells. Lactoferrin (and more specifically enteric-coated LF because of increased bioavailability) may consequently be of preventive and therapeutic value during the present COVID-19 pandemic.

The biology of ergothioneine, an antioxidant nutraceutical.

Ergothioneine (ERG) is an unusual thio-histidine betaine amino acid that has potent antioxidant activities. It is synthesised by a variety of microbes, especially fungi (including in mushroom fruiting bodies) and actinobacteria, but is not synthesised by plants and animals who acquire it via the soil and their diet, respectively. Animals have evolved a highly selective transporter for it, known as solute carrier family 22, member 4 (SLC22A4) in humans, signifying its importance, and ERG may even have the status of a vitamin. ERG accumulates differentially in various tissues, according to their expression of SLC22A4, favouring those such as erythrocytes that may be subject to oxidative stress. Mushroom or ERG consumption seems to provide significant prevention against oxidative stress in a large variety of systems. ERG seems to have strong cytoprotective status, and its concentration is lowered in a number of chronic inflammatory diseases. It has been passed as safe by regulatory agencies, and may have value as a nutraceutical and antioxidant more generally.

Iron Dysregulation and Inflammagens Related to Oral and Gut Health Are Central to the Development of Parkinson's Disease.

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.

L-(+)-Ergothioneine Significantly Improves the Clinical Characteristics of Preeclampsia in the Reduced Uterine Perfusion Pressure Rat Model.

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 H2O2 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 H2O2 (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.

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

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.

Iron Dysregulation and Dormant Microbes as Causative Agents for Impaired Blood Rheology and Pathological Clotting in Alzheimer's Type Dementia.

Alzheimer's disease and other similar dementias are debilitating neurodegenerative disorders whose etiology and pathogenesis remain largely unknown, even after decades of research. With the anticipated increase in prevalence of Alzheimer's type dementias among the more susceptible aging population, the need for disease-modifying treatments is urgent. While various hypotheses have been put forward over the last few decades, we suggest that Alzheimer's type dementias are triggered by external environmental factors, co-expressing in individuals with specific genetic susceptibilities. These external stressors are defined in the Iron Dysregulation and Dormant Microbes (IDDM) hypothesis, previously put forward. This hypothesis is consistent with current literature in which serum ferritin levels of individuals diagnosed with Alzheimer's disease are significantly higher compared those of age- and gender-matched controls. While iron dysregulation contributes to oxidative stress, it also causes microbial reactivation and virulence of the so-called dormant blood (and tissue) microbiome. Dysbiosis (changes in the microbiome) or previous infections can contribute to the dormant blood microbiome (atopobiosis), and also directly promotes systemic inflammation via the amyloidogenic formation and shedding of potent inflammagens such as lipopolysaccharides. The simultaneous iron dysregulation and microbial aberrations affect the hematological system, promoting fibrin amylodiogenesis, and pathological clotting. Systemic inflammation and oxidative stress can contribute to blood brain barrier permeability and the ensuing neuro-inflammation, characteristic of Alzheimer's type dementias. While large inter-individual variability exists, especially concerning disease pathogenesis, the IDDM hypothesis acknowledges primary causative factors which can be targeted for early diagnosis and/or for prevention of disease progression.

No effects without causes: the Iron Dysregulation and Dormant Microbes hypothesis for chronic, inflammatory diseases.

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.

Enhancing Drug Efficacy and Therapeutic Index through Cheminformatics-Based Selection of Small Molecule Binary Weapons That Improve Transporter-Mediated Targeting: A Cytotoxicity System Based on Gemcitabine.

The transport of drug molecules is mainly determined by the distribution of influx and efflux transporters for which they are substrates. To enable tissue targeting, we sought to develop the idea that we might affect the transporter-mediated disposition of small-molecule drugs via the addition of a second small molecule that of itself had no inhibitory pharmacological effect but that influenced the expression of transporters for the primary drug. We refer to this as a "binary weapon" strategy. The experimental system tested the ability of a molecule that on its own had no cytotoxic effect to increase the toxicity of the nucleoside analog gemcitabine to Panc1 pancreatic cancer cells. An initial phenotypic screen of a 500-member polar drug (fragment) library yielded three "hits." The structures of 20 of the other 2,000 members of this library suite had a Tanimoto similarity greater than 0.7 to those of the initial hits, and each was itself a hit (the cheminformatics thus providing for a massive enrichment). We chose the top six representatives for further study. They fell into three clusters whose members bore reasonable structural similarities to each other (two were in fact isomers), lending strength to the self-consistency of both our conceptual and experimental strategies. Existing literature had suggested that indole-3-carbinol might play a similar role to that of our fragments, but in our hands it was without effect; nor was it structurally similar to any of our hits. As there was no evidence that the fragments could affect toxicity directly, we looked for effects on transporter transcript levels. In our hands, only the ENT1-3 uptake and ABCC2,3,4,5, and 10 efflux transporters displayed measurable transcripts in Panc1 cultures, along with a ribonucleoside reductase RRM1 known to affect gemcitabine toxicity. Very strikingly, the addition of gemcitabine alone increased the expression of the transcript for ABCC2 (MRP2) by more than 12-fold, and that of RRM1 by more than fourfold, and each of the fragment "hits" served to reverse this. However, an inhibitor of ABCC2 was without significant effect, implying that RRM1 was possibly the more significant player. These effects were somewhat selective for Panc cells. It seems, therefore, that while the effects we measured were here mediated more by efflux than influx transporters, and potentially by other means, the binary weapon idea is hereby fully confirmed: it is indeed possible to find molecules that manipulate the expression of transporters that are involved in the bioactivity of a pharmaceutical drug. This opens up an entirely new area, that of chemical genomics-based drug targeting.

A Dormant Microbial Component in the Development of Preeclampsia.

Preeclampsia (PE) is a complex, multisystem disorder that remains a leading cause of morbidity and mortality in pregnancy. Four main classes of dysregulation accompany PE and are widely considered to contribute to its severity. These are abnormal trophoblast invasion of the placenta, anti-angiogenic responses, oxidative stress, and inflammation. What is lacking, however, is an explanation of how these themselves are caused. We here develop the unifying idea, and the considerable evidence for it, that the originating cause of PE (and of the four classes of dysregulation) is, in fact, microbial infection, that most such microbes are dormant and hence resist detection by conventional (replication-dependent) microbiology, and that by occasional resuscitation and growth it is they that are responsible for all the observable sequelae, including the continuing, chronic inflammation. In particular, bacterial products such as lipopolysaccharide (LPS), also known as endotoxin, are well known as highly inflammagenic and stimulate an innate (and possibly trained) immune response that exacerbates the inflammation further. The known need of microbes for free iron can explain the iron dysregulation that accompanies PE. We describe the main routes of infection (gut, oral, and urinary tract infection) and the regularly observed presence of microbes in placental and other tissues in PE. Every known proteomic biomarker of "preeclampsia" that we assessed has, in fact, also been shown to be raised in response to infection. An infectious component to PE fulfills the Bradford Hill criteria for ascribing a disease to an environmental cause and suggests a number of treatments, some of which have, in fact, been shown to be successful. PE was classically referred to as endotoxemia or toxemia of pregnancy, and it is ironic that it seems that LPS and other microbial endotoxins really are involved. Overall, the recognition of an infectious component in the etiology of PE mirrors that for ulcers and other diseases that were previously considered to lack one.

A Bacterial Component to Alzheimer's-Type Dementia Seen via a Systems Biology Approach that Links Iron Dysregulation and Inflammagen Shedding to Disease.

The progression of Alzheimer's disease (AD) is accompanied by a great many observable changes, both molecular and physiological. These include oxidative stress, neuroinflammation, and (more proximal to cognitive decline) the death of neuronal and other cells. A systems biology approach seeks to organize these observed variables into pathways that discriminate those that are highly involved (i.e., causative) from those that are more usefully recognized as bystander effects. We review the evidence that iron dysregulation is one of the central causative pathway elements here, as this can cause each of the above effects. In addition, we review the evidence that dormant, non-growing bacteria are a crucial feature of AD, that their growth in vivo is normally limited by a lack of free iron, and that it is this iron dysregulation that is an important factor in their resuscitation. Indeed, bacterial cells can be observed by ultrastructural microscopy in the blood of AD patients. A consequence of this is that the growing cells can shed highly inflammatory components such as lipopolysaccharides (LPS). These too are known to be able to induce (apoptotic and pyroptotic) neuronal cell death. There is also evidence that these systems interact with elements of vitamin D metabolism. This integrative systems approach has strong predictive power, indicating (as has indeed been shown) that both natural and pharmaceutical iron chelators might have useful protective roles in arresting cognitive decline, and that a further assessment of the role of microbes in AD development is more than highly warranted.

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

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