Internal jugular vein thrombosis is a serious event with potentially fatal outcome, where the clinical symptoms may be vague or absent. This paper refers to a rare case where routine carotid Doppler ultrasound prior to coronary artery bypass grafting (CABG) and aortic valve replacement (AVR) in a 76-year-old man, incidentally revealed thrombosis of the right internal jugular vein. Thoracic CT demonstrated an underlying, large, benign substernal multinodular goiter, mainly involving the right lobe, causing compression and displacement of the great vessels. A successful, one-stage operation including ligation of the internal jugular vein to avoid pulmonary embolism and hemithyroidectomy, combined with the scheduled CABG and AVR, was performed. This case illustrates that benign substernal goiter may be associated with asymptomatic internal jugular vein thrombosis. Carotid Doppler ultrasound should involve evaluation of the internal jugular vein concerning thrombosis as its presence may reveal space-occupying lesions in the thorax.  Internal jugular vein thrombosis (IJVT) is a serious event with potentially fatale outcome, including pulmonary embolism as well as intracranial propagation of the thrombus . IJVT isA 76-year-old man with a medical history of hypertension, lipid disorder, and stable angina presented with sustained ventricular tachycardia and non-ST elevation myocardial infarction. He was without dilatation of the neck veins or any other signs of SVC syndrome. Further examination revealed coronary triple-vessel disease and hemodynamic significant aortic valve stenosis. Routine preoperative carotid Doppler ultrasound was performed. This examination revealed non-compressible intraluminal echoes inside the right internal jugular vein consistent with an intravenous thrombosis, partly adherent to the vessel wall  . CABG and biological AVR, combined with right-sided hemithyroidectomy and ligation of the right internal jugular vein were performed in one stage. Surgery started with isolation and ligation of the internal jugular vein in order to prevent dislodgment of thrombotic material from the large vein during and after surgery, which could have caused pulmonary artery embolism. Histopathological examination revealed a benign multinodular goiter with a large, dominantly cystic nodule in the right lobe.The postoperative course was somewhat prolonged, but without serious complications. He was given acetylsalicylic acid 75 mg once per day and enoksaparin 60 mg once per day as prophylaxis against thrombotic complications. Vocal cord function was normal at laryngoscopy. He was transferred to a local hospital on the 7th postoperative day. On clinical follow-up, two and eight months postoperatively, he was in good health without clinical symptoms or signs of heart- or endocrinological dysfunction. Retrospectively, the patient appears to have had an episode of transient swelling of neck and face, and development of superficial varicose veins on the neck some years earlier, which, however, did not lead to any further examination at that time.The present case shows how routine carotid Doppler ultrasound performed prior to CABG and AVR incidentally detected an IJVT on the right side, leading to CT diagnosis of a huge, undetected, normal functioning, multinodular goiter, mainly involving the right lobe. These lesions were subjected to one-stage surgery, combining CABG, AVR, right-sided hemi-thyroidectomy and ligation of the jugular vein, all performed without serious complications.In the majority of cases, IJVT is caused by fast-growing infiltrating malignant disease presenting as superior vena cava syndrome due to compression of several mediastinal structures or as a complication to central venous catheterization , 3, 8. SThe cause of the IJVT in this case, substernal goiter mainly involving the right lobe containing a large cyst compressing the great vessels, however, may not be as straightforward as it seems due to (1) prior episode of transient swelling of the neck and face possibly caused by jugular vein thrombosis and (2) current presence of antiphospholipid antibodies, which thus may be part of an antiphospholipid syndrome. Lack of prior episodes of venous thrombosis in other locations than the neck and the rather low predictive value of antiphospholipid antibodies as a single finding, point towards the giant intrathoracic goiter as the most important cause of IJVT in this case . HoweverBecause of the simultaneous presentation of serious coronary artery disease with non-ST elevation myocardial infarction and aortic valve stenosis combined with a giant intrathoracic goiter and IJVT, surgery was performed in one stage, starting with ligation of the thrombosed internal jugular vein in order to prevent per- and postoperative lung embolism . Based uThis case illustrates that benign substernal goiter may be associated with IJVT. Although the combination is extremely rare, the silent presentation suggests routine evaluation of the internal jugular vein by ultrasound prior to thyroidectomy in patients with large substernal goiter to prevent per-and postoperative lung embolism. In addition, detection of an IJVT during carotid Doppler ultrasound may be the first sign of a space-occupying lesion in the thorax. If an IJVT is detected by ultrasound, ligation of the thrombosed internal jugular vein should be considered.No competing interests exist.Written informed consent was obtained from the patient for publication of this paper and accompanying images.


F test, the r2 test,and the profile plot, we have assessed the effectiveness of kernel density clusteringfor recovering clusters, and its robustness against noise on clustering both simulatedand real GEP data. Our results show that the kernel density clustering method hasexcellent performance in recovering clusters from simulated data and in groupinglarge real expression profile data sets into compact and well-isolated clusters, andthat it is the most robust clustering method for analysing noisy expression profiledata compared to the other three methods assessed.Kernel density smoothing techniques have been used in classification or supervised learning of gene expression profile (GEP) data, but their applications to clusteringor unsupervised learning of those data have not been explored and assessed. Herewe report a kernel density clustering method for analysing GEP data and compareits performance with the three most widely-used clustering methods: hierarchicalclustering, K-means clustering, and multivariate mixture model-based clustering.Using several methods to measure agreement, between-cluster isolation, and withinclustercoherence, such as the Adjusted Rand Index, the Pseudo


The important role of equilibrium of environmental factors during the embryo-fetal period is undisputable. Women of reproductive age are increasingly exposed to various environmental risk factors such as hypoxia, prenatal viral infections, use of drugs, smoking, complications of birth or stressful life events. These early hazards represent an important risk for structural and/or functional maldevelopment of the fetus and neonates. Impairment of oxygen/energy supply during the pre- and perinatal period may affect neuronal functions and induce cell death. Thus when death of the newborn is not occurring following intrauterine hypoxia, various neurological deficits, including hyperactivity, learning disabilities, mental retardation, epilepsy, cerebral palsy, dystonia etc., may develop both in humans and in experimental animals. In our animal studies we used several approaches for modeling hypoxia in rats during pregnancy and shortly after delivery, i.e. chronic intrauterine hypoxia induced by the antiepileptic drug phenytoin, neonatal anoxia by decreased oxygen saturation in 2-day-old pups. Using these models we were able to test potential protective properties of natural  and synthetic (stobadine) compounds. Based on our results, stobadine was also able to reduce hypoxia-induced hyperactivity and the antioxidant capacity of stobadine exceeded that of vitamin E and melatonin, and contrary to vitamin E, stobadine had no adverse effects on developing fetus and offspring. Many studies have been investigating neurological deficits following perinatal hypoxia, including seizures, cerebral palsy, mental retardation, attention deficit-hyperactivity disorder, anxiety as well as other mental diseases.  have been shown to induce limb and central nervous system (CNS) defects in developing rats when the exposure occurs during fetal stages. Although it is a chemically and physically diverse group, exposure to each of the chemicals or events studied has been found to have vasoactive or cardioactive consequences that result in transient uteroplacental hypoperfusion  and provokes anoxia in nitrogen atmosphere of tight sealed glass chamber. Adapting the Dell'Anna et al.  model to et al., . These eet al., Above mentioned approaches represent acute models hypoxia/ischemia. However, hypoxia/ischemia is often caused also with chronic hypoxia-reoxygenation during maturation of organs. For this sake the model of chronic intrauterine hypoxia induced pharmacologically (phenytoin – PHT) was introduced by our lab. The proposed teratogenicity mechanism of antiseizure medication phenytoin is due to embryonic hypoxia/ischemia and production of free radicals  daily in sensitive stages of organ development or during whole pregnancy  could by make only after insult and therefore the data represents changes not mechanisms. In this matter animals are used for elucidation of mechanisms leading to origins of eclampsia, placental transfer disturbances, maternal-fetal complex communications etc. For extrapolation to humans, threshold effect and intra-/interspecies differences in the timing of developmental events must also be taken into account  were used for assessment not only structural disturbances (teratology studies) but also functional disorders . Neonatal anoxia showed that 1- and 2-day old rat pups were extremely resistant to anoxic insult  was shown to exert an overwhelming effect on embryo-fetal and neuromotor development (Ujházy The aim of our experiments is to widen our knowledge on the possible protective effects of antioxidants in a hypoxia/ischemia models. Based on our results, the antioxidant capacity of STO exceeded that of VitE, and contrary to VitE, STO had no adverse effects on offspring. Even though STO did not fully alleviate the PHT teratogenicity, it was able to reduce PHT-induced hyperactivity and had beneficial effects on some reproductive variables. These results are indicative of a prospective use of STO as a potential protectant and a supportive therapeutic agent in pregnancies with high risk of pre-eclampsia, perinatal asphyxia or pre-term delivery, in which oxidative injury may play a crucial role.et al., The experimental results also demonstrating that ethological approaches in pharmacology and toxicology are an integral part of relative safety drug assessment (Dubovický


Anaemia is a common occurrence in patients with cancer and contributes to the clinical symptomatology and reduced quality of life (QOL) seen in cancer patients. Many aspects of reduced QOL, including fatigue, are known to be associated with suboptimally low levels of haemoglobin. Even mild-to-moderate anaemia adversely affects patient-reported QOL parameters. Red blood cell transfusions are associated with many real and perceived risks, inconveniences, costs, and only temporary benefits. Recombinant human erythropoietin (rHuEPO) is an effective therapy to increase haemoglobin values in over half of anaemic cancer patients receiving concurrent chemotherapy. These increased haemoglobin values are closely correlated with improvements in QOL. Despite these objectively defined benefits, less than 50% of anaemic patients undergoing cytotoxic chemotherapy receive rHuEPO, in contrast to patients with chronic renal failure on dialysis, where anaemia is universally and aggressively treated to more optimal haemoglobin values. However, there are several barriers that may limit more widespread use of rHuEPO. These include inconvenience associated with frequent dosing; failure of a large proportion (40 to 50%) of patients to respond; relatively slow time to response; absence of reliable early indicators of response; and current lack of rigorous pharmacoeconomic data demonstrating cost-effectiveness. Darbepoetin alfa is a novel erythropoiesis stimulating protein (NESP) that is biochemically distinct from rHuEPO, and which has been proven to stimulate red blood cell production. The molecule has a 3-fold longer half-life and increased biological activity that will allow less frequent dosing, facilitating improved management of the anaemia of cancer. With this new option for therapy, further avenues of investigation should lead to renewed interest in the clinical benefits of optimal haemoglobin levels for patients with cancer. © 2001 Cance Cancer Research Campaign


Some developmental anomalies, such as NTDs, are to a large extent prevented by supplementation of folic acid before conception, but supplementation does not seem to prevent cardiovascular disease or cognitive decline. These diverging results can be elucidated by introduction of the PrOO concept, as MTHFR polymorphisms and inherent low folate levels induce both non-optimal maturation of the oocyte and unsuccessful DNA methylation and demethylation, i.e. epigenetic mutations. The PrOO concept is testable and predicts in a random population the following: (1) female carriers of specific genetic MTHFR variants exhibit more ovulatory disturbances and inherent subfecundity traits, (2) descendents from a carrier mother, when compared with those from a wild-type mother, are more frequently conceived in PrOO high-risk conditions and, thus, (3) disadvantaged in life expectancy. If so, some MTHFR polymorphisms represent a novel, genetically determined, PrOO high-risk conception category comparable to those which are environmentally and behaviorly influenced. These high-risk conditions may cause developmental anomalies and defective epigenetic reprogramming in progeny. The interaction between genetic and environmental factors is a plausible mechanism of multifactorial inheritance.Several conditions apparent at birth, e.g., neural tube defects (NTDs) and cardiac anomalies, are associated with polymorphisms in folate-related genes, such as the 677C → T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene. Similar associations have been established for several constitutional chronic diseases in adulthood, such as schizophrenia, cardiovascular diseases, dementia, and even neoplasias in different organ systems. This spectrum of developmental anomalies and constitutional diseases may be linked to high-risk conceptions related to  Most theories related to the origin of adult diseases focus on genetic causes and direct environmental effects preceding disease onset by several years at most. The view that diseases in adulthood can partly be explained by conditions earlier in life or even before birth is gaining scientific support. Previously, we proposed non-optimal oocyte ripening or impaired oocyte maturation can be an important cause of developmental anomaly and disease later in adult life -4. The bab ovo for some congenital anomalies and constitutional diseases. Indeed, a genetic PrOO determinant emerges as an explanation for the diverging preventive effects of folate in NTDs versus adult diseases. We also discuss the relationship between low folate levels and unsuccessful DNA methylation patterns in the context of epigenetic mutations. Furthermore, testing strategies are proposed to establish the causality of the relation between MTHFR polymorphisms and PrOO conceptions in random populations.In this paper, we review current knowledge on MTHFR polymorphisms and folate levels as they relate to developmental anomalies at birth and selected constitutional disease in adulthood. We also recapitulate the ovopathy concept and posit that the 677C → T variant and inherent low folate levels are accompanied by low estrogenisation, and that this condition induces preovulatory overripeness ovopathy (PrOO). This leads to a high-risk conception mediated by genetic factors, analogous to the environmentally and behaviorally conditioned high-risk conceptions, and an origin Folate is an important B vitamin that plays a pivotal role in remethylation of homocysteine to methionine, which is essential for DNA-synthesis, DNA-repair, and DNA-imprinting processes . ReductiIt has become evident that the 677C → T variant as well as low-folate intake by the mother contribute to increased risks of NTDs and cardiac anomalies. The underlying pathogenic mechanism which causes this detrimental effect is not fully understood ,10. HoweAn additional enigma is the marginal association between MTHFR polymorphisms and several neoplasias with diverging incidences according to age ,28. A meth and 20th century as well as observations in human reproduction [optimally ripened oocytes (OptRO) are apparent during optimal conditions for reproduction. This coincides with optimal maternal age, adequate nutritional state, and post-pregnancy restoration of a regulatory ovulatory pattern, and with the seasonally-bound ovulation peaks, guaranteeing optimal embryo quality and favourable downstream effects on subsequent events, i.e., on life expectancy [This concept was initially derived from animal research in the 19oduction . Meioticpectancy . In factpectancy .preovulatory overripeness ovopathy (PrOO), and leads to fertilization of non-optimally matured oocytes [In contrast, abnormal estrogen concentrations cause deficient oocyte maturation which is the basis of  oocytes -4. The i oocytes -3.dose-response fallacy in reproductive studies described by Selevan and Lemasters [Excessive dysfunctional oocyte maturation results in poor quality oocytes and, after surpassing a certain threshold, in disproportionately increasing numbers of pathological conceptuses. Excess vanishing of pathological outcome will turn into excess preterm loss, and eventually in deficits of pathological births. This is called the emasters . In our The spectrum of developmental anomalies associated with MTHFR polymorphisms and/or folate deficiency appears analogous to the spectrum of anomalies related to PrOO induced by endocrine disturbances during the transitional stages of reproductive life and/or by divergent reproductive behavior, e.g. unusual maternal age or pregnancy interval  see Fig. Low foldose-response fallacy being inherent to the PrOO concept. Other findings are also in accordance with disproportionate levels of oocyte deterioration and a dose-response fallacy: a dose-specific reduced risk of progeny with cleft lip with or without cleft palate(CL/P) from mothers carrying either one or two copies of the 677C → T variant , a negative association for children with CL/P (RRs: 1.05 and 0.74) versus a positive one for cleft palate only , and a fourfold increased risk of orofacial clefts in mothers using folic acid [This genetically conditioned high-risk for PrOO explains several unexplained phenomena related to MTHFR variants as well as to low dietary folate. Both conditions are associated with increased fetal loss, intrauterine growth retardation, and heart defects in female mice , as welllic acid .The finding of MTHFR 677C → T mutations in the mother   is in geab ovo. In other words, preventive measures are effective only before conception but not afterward, and that threatened oocytes may be the culprit of specific constitutional diseases encountered long after birth.It should be noted that periconceptional multivitamin use including folic acid reduces the dose-response risk of preeclampsia and ovulatory infertility ,18. In Iab ovo for both developmental anomalies and constitutional disease is consistent with the environmental determinants related to non-optimally maturing oocytes [The conjecture of a common origin  oocytes -4. This  oocytes ,3,48,49  oocytes -54. AnalAs mentioned previously, adequate folate levels are a prerequisite not only for optimal oocyte maturation but also for remethylation of homocysteine to methionine – the key epigenetic contributor to gene activation and/or reprogramming . These eIncorrect DNA reprogamming results in epigenetic mutation, which does not follow the classic rules of Mendelian inheritance. Active transport of methionine happens in all animal and human oocytes and in early preimplantation embryos. DNA-methylation is concentrated at specific stages when developmental potency of cells changes, i.e., during the final phase of oocyte maturation. In contrast, DNA-demethylation occurs immediately after fertilization ,55,56. I12 and folate restricted diet during the periconceptional period, resulted in obesity, insulin resistance, hypertension, and altered immune responses when full grown [The combined effect of PrOO and epigenetic mutations evoked by MTHFR variants (and low folate levels) can explain many developmental puzzles in animals and humans. For example, epigenetic modifications in lambs derived from oocytes retrieved from ewes given a vitamin Bll grown . This wall grown . Additioll grown . Furtherll grown . A numbell grown , schizopll grown , and cerll grown ,65,66.MTHFR polymorphism status in females is hypothesized to entail a genetically determined propensity to PrOO, which may be likened to the high-risk conceptions elicited by environmental and behavioral conditions. The variant alleles operate either independently or in concert and may strengthen each other. Therefore, we predict that:1) Heterozygous and homozygous 677C → T carrier females suffer from subfecundity traits in a dose-response manner when compared to females with no mutation (CC). Apart from giving birth to affected progeny, they also experience more menstrual disorders, longer time to achieve pregnancy, longer interpregnancy intervals, and more pronounced reproductive seasonal variation.2) Offspring from homozygous (and to a lesser extent from heterozygous 677C → T carrier mothers), being genetically more susceptible to environmental triggers than mothers without this mutation, are more frequently conceived in high-risk conditions characterized by ovulatory disturbances. Typical PrOO characteristics are depicted in Figure 3) Descendents from homozygous (and to a lesser extent from heterozygous 677 → T carrier women) have reduced life expectancy compared to offspring from mothers without this polymorphism.dose-response fallacy will in particular occur at the extremes of maternal age, birth interval or at the seasonal transitions (Figure These effects may be obscured by early loss before birth as a consequence of preterm premature rupture of membranes and eclampsia associated with MTHFR polymorphisms ,38 or dus Figure  and 2c.conclusion, MTHFR polymorphisms and resulting low folate levels warrant consideration as factors inducing non-optimally matured oocytes before conception. They represent a novel, genetically determined, high-risk PrOO condition comparable to the endocrine disturbances elicited by environmental and behavioral conditions. Further study of the interaction between genetic and environmental factors may indentify mechanisms of multifactorial inheritance and explain many commonly associated enigmata in chronic constitutional diseases.In The authors declare that they have no competing interests.th of June 2007 at the Department of Epidemiology, Biostatistics, and Health Technology Assessment at the Radboud University Nijmegen, the Netherlands. The co-authors AV, MdH, and NR participated in the design of the study and the coordination for the manuscript preparation. They revised it critically for important intellectual content and interpretation of the arguments. All authors read and approved the final manuscript.The content of this manuscript was subject of the fare-well lecture of PHJ on 12


Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age-related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro-inflammatory, tissue-remodeling, senescent-like state. Other mesenchymal progenitors also can acquire a pro-inflammatory, adipocyte-like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro-inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress-responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent-like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro-inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role. Major changes in fat distribution and function occur throughout life. In old age, these changes are associated with diabetes, hypertension, cancers, cognitive dysfunction, and atherosclerosis leading to heart attacks and strokes . ConversExciting new data are beginning to point to the cell biological and molecular mechanisms that determine how aging impacts fat tissue function and how this, in turn, leads to age-related disease. Lessons from what happens in obesity are especially illuminating. In particular, inflammatory processes linked to cellular senescence in fat tissue could be pivotal. Fat tissue is important in host defense, immunity, injury responses, and production of inflammatory cytokines and chemokines. It is rich in progenitors that can produce pro-inflammatory factors and that are susceptible to cellular senescence. We suggest the possibility that cellular injury responses, activation of innate immunity, accumulation of dysfunctional, dysdifferentiated progenitors [mesenchymal macrophage- and adipocyte-like default (MAD) cells , and activates hormones, for example glucocorticoids and sex steroids. It releases paracrine factors in an endocrine-like fashion by developing in target organs and releasing factors that impact their function. For example, fat in muscle regulates muscle glucose homeostasis and insulin responses  deficiency. While short-term growth hormone exposure decreases visceral fat by enhancing lipolysis, in animals with lifelong excess growth hormone, fat redistribution begins earlier, advances faster, and is associated with decreased life span . ConversSubstantial changes in fat tissue metabolic function occur during aging, with declines in insulin, lipolytic, and fatty acid responsiveness  and interleukin (IL-6), can increase with aging  may be the main source of inflammatory cytokines and chemokines produced by fat  are reduced in obese women , in obesity adipogenesis, C/EBPα, PPARγ, and their downstream targets are decreased in preadipocytes and fat tissue , IL-6, transforming growth factor-β, and other signals eventually resulting in focal accumulation of heterochromatin  senescent cells restricted to a single tissue can have widespread systemic effects  Does failure of the immune system to remove senescent cells contribute to their accumulation in old age? and (vi) Is cellular senescence really at the root of age- and obesity-related fat tissue inflammation and metabolic dysfunction? As discussed later, suggestive evidence supports affirmative answers to some of these questions, but more work is required to address them definitively.This speculative model and recent findings about fat tissue cellular senescence and inflammation prompt several questions about cellular senescence (Table S1). Among these are the following: (i) Is cellular senescence effectively an alternative form of differentiation? (ii) Can a senescent-like state develop in terminally differentiated cells? (iii) Can senescence occur at any stage during life? (iv) Does senescence spread from cell to cell in fat tissue Is cellular senescence effectively an alternative form of differentiation? Cellular senescence can be viewed as a response to cellular stress . In preaCan a senescent-like state develop in terminally differentiated cells? Terminally differentiated fat cells might be able to acquire a senescent-like state in both aging and obesity (Can senescence occur at any stage during life? If our hypothetical model is correct, cellular senescence should be inducible at any phase in life. Even at early passage, some cells in cultured human skin fibroblast strains express markers of senescence . The ansin vivo that have DNA double-strand breaks and γH2AX induced by radiation can induce DNA damage responses and γH2AX foci in nearby cells (Does cellular senescence spread from cell to cell? Cultured human cells and mouse cells Does failure of the immune system to remove senescent cells contribute to their accumulation in old age? Macrophage function generally declines with aging , possiblIs cellular senescence at the root of age- and obesity-related fat tissue inflammation and metabolic dysfunction? The potentially central role of preadipocytes in genesis of fat tissue inflammation and metabolic dysfunction has not received much attention. Preadipocyte cellular stress can result from repeated replication, hypoxia, ROS, chronic effects of free fatty acids or other lipids such as ceramide, hyperglycemia, or other metabolic signals. These appear to activate innate immune responses in preadipocytes, causing further cytokine and chemokine generation, potentially inducing spread of activation of pro-inflammatory responses to nearby preadipocytes and other cell types and attraction of immune cells. The preadipocyte pro-inflammatory phenotype might impede protection against lipotoxicity, contributing to systemic consequences in aging similar to those in lipodystrophies and obesity. The pathways and processes culminating in this generation of activated or senescent preadipocytes and fat cells represent potential new targets for intervention.It seems a senescent-like, pro-inflammatory fate can be acquired in response to intra- or extracellular danger signals, inflammation, infection, excessive replication, or toxic metabolites. This spectrum of cell fates may prevent differentiation along pathways that are not desirable in the context of damage. In the case of fat tissue, this could involve preventing development of new fat cells or enlargement of existing ones in favor of acquiring cells capable of facilitating repair. Perhaps terminally differentiated cells are able to enter a senescent secretory state related to the molecular pathways that initiate senescence in dividing cell types. Studies in fat tissue are beginning to suggest that cellular senescence could be an alternative cell fate involving activation of pro-inflammatory responses owing to intra- or extracellular injury signals at any stage of life.


There are many biological activities attributed to isoflavonoids. The majority of them could be beneficial and some of them may be detrimental, depending on specific circumstances. Isoflavonoids play an important role in human nutrition as health promoting natural chemicals. They belong to plant secondary metabolites that mediate diverse biological functions through numerous pathways. They are structurally similar to estrogens, exerting both estrogenic and antiestrogenic properties in various tissues. The results of epidemiologic studies exploring the role of isoflavonoids in human health have been inconclusive. Some studies support the notion of a protective effect of their consumption in immunomodulation, cognition, risk reduction of certain cancers, cardiovascular and skin diseases, osteoporosis and obesity, as well as relief of menopausal symptoms. Other studies failed to demonstrate any effects. However, primarily they are naturally present in their β-D-glycoside form as genistin, daidzin, glycitin. The aglycone form of isoflavones is biologically active. In addition to soy, isoflavone daidzein and genistein, their precursors formononetin, biochanin A, their glycosides, glycosides malonates and acetyl glycosides were determined in red clover (Trifolium pratense) extracts using chromatographic and spectrometric methods  two new isoflavonoids, millewanin-F and furowanin-A, together with previously known five isoflavonoids. The bioavailability of isoflavones has been shown to be influenced by their chemical form in foods , susceptibility to degradation, the microbial flora of the consumer, and by the food matrix. Bioavailability of isoflavonoids may depend to some extent upon interaction with other dietary components , CYP19 (generated by a G → T substitution in intron 6), involved in the sex hormone biosynthesis pathway, and Pvull (lipoprotein lipase) polymorphism (generated by a C → T substitution in intron 1) in the estrogen receptor ESR1 gene and the risk of breast cancer was demonstrated , rs1008805 (A/G) and rs730154 (C/T) revealed that premenopausal women carrying at least one G alelle at Cyp 19 locus was associated with an increased breast cancer risk  and a high body mass index (BMI) in postmenopausal women  polymorphism is a potential marker for identifying gastric cancer individuals at a high-risk, and useful in defining who may benefit from specific chemopreventive interventions.Additionally, results obtained by Boccia et al. , proved Isoflavonoids have been shown to possess many biological properties that may account for cancer prevention. Isoflavones exert their effects through numerous pathways and with respect to cancer prevention, they use mechanisms of action which appear to be various, complementary and/or overlapping.at al., et al., Some mechanisms of action have been identified for isoflavone/flavone prevention of cancer, including estrogenic/antiestrogenic activity, antiproliferation, induction of cell cycle arrest and apoptosis, prevention of oxidation, induction of detoxification enzymes, regulation of host immune system and changes in cellular signaling . They may inhibit also proliferation of hormone-independent breast cell lines via a number of mechanisms, including inhibition and down-regulation of protein tyrosine kinases, which are involved in growth signaling pathways. Genistein was shown to inhibit tyrosine kinase, particularly the autophosphorylation and activation of epidermal growth factor receptor, which is important in regulating apoptosis and cell proliferation. Genistein inhibited protein tyrosine kinase-dependent transcription of c-fos and subcellular proliferation in estrogen receptor negative human breast cancer cell lines. Key enzymes implicated in cancer invasion are also affected by genistein .Changes in cell signaling were demonstrated in the HC11 mouse mammary epithelial cell line. Soy isoflavone genistein upregulated epithelial adhesion molecule E-cadherin expression and attenuated β-catenin signaling in mammary epithelial cells. In addition, it diminished basal and Wnt-1-(wingless)-induced cell proliferation and attenuated Wnt-1 targets et al., Isoflavonoids have been increasingly advocated as potential natural alternatives to hormone replacement therapy. There are however only limited data on the effect of isoflavones on breast density, despite the fact that isoflavonoids were proved to be present in breast fluids and could directly act on breast tissue  cholesterol oxidation and to the development of cancer through DNA strand breaks induction. The antioxidative efficiency of dietary isoflavonoids is associated not only with their reductive capacity but also with their protein-binding properties. In humans isoflavones may prevent low-density lipoprotein LDL cholesterol oxidation and lower markers of DNA oxidative stress The postmenopausal status increases cardiovascular risk due to accelerated atherosclerosis progression. Prevention of intracellular lipid deposition may inhibit the formation of atherosclerotic lesions at the primary stage of atherogenesis at the level of the arterial wall. Although isoflavonoids may prevent atherosclerosis development in postmenopausal women, little is known about their direct effects on atherogenesis. Myasoedova and Sobenin  showed tResearch on the effect of genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium-dependent vasodilatation in postmenopausal women revealed that genistein therapy improved flow-mediated endothelium-dependent vasodilatation in healthy postmenopausal women. This improvement was mediated by a direct effect of genistein on vascular function and could be the result of an increased ratio of nitric oxide to endothelin . Adipogenesis is then regulated by the hormonally induced cooperative interaction between members of the enhancer binding protein and peroxisome proliferator-activated receptor (PPAR) families and the primary adipogenic transcription factors. They act by synergistically transactivating the expression of several adipogenic effector genes. Unlike the highly specific ERs, PPARs bind a wide number of ligands and directly affect lipid metabolism by enhancing transcription of PPAR-regulated genes , osteoprotegerin (an inhibitor of osteoclast activity) and IGF (a promoter of osteoblast activity) resulting in reduced bone resorption and increased bone formation  secretion from G-209 osteosarcoma cell lines (Chrzan and Bradford,


Cyclosporin A (Cy A), a novel immunosuppressive agent with apparently selective inhibitory effects on T lymphocytes and little myelotoxicity, was tested for its effects on a variety of syngeneic animal tumours including sarcomas, carcinomas and a T-cell lymphoma. Cy A, given orally or parenterally in repeated doses, had no effect on the growth rates of any of the tumours tested, but a highly significant effect on metastasis was seen in many cases. All the sarcomas examined in both rats and mice, and also the lymphoma, showed a marked increase in their metastases, in some cases even when administration of Cy A was delayed until after excision of the "primary" tumour implants. In contrast no effect of Cy A on metastasis was observed in animals bearing poorly immunogenic mammary or squamous-cell carcinomas. The metastases developing in Cy A-treated animals, when transplanted into normal syngeneic animals, showed no evidence of enhanced metastatic potential compared with their "parent tumours.


The coincidence of autoantibodies against the acetylcholine receptor (AChR) and musclestriational antigens (SA) is a characteristic finding in thymoma-associated myastheniagravis (MG), but their origins are still unresolved. Some common muscle antigens thatwere shown to be targets of anti-SA autoantibodies in thymoma-associated MG havealso been detected in normal or neoplastic thymic epithelial cells, suggesting that therelease of  antigens from the thymic tumors could elicit SAautoimmunity. In contrast to this model, we report here that titin, which is a recentlyreported target of SA autoimmunity, is not expressed in thymomas. In addition, weshow that skeletal muscle type-II fibers exhibit a striational immunoreactivity withmonoclonal antibody mAb155, which was previously identified to label a veryimmunogenic cytoplasmic epitope of the AChR and neoplastic epithelial cells of MGassociatedthymomas. We conclude from these findings that titin autoimmunity inthymoma-associated MG is either due to a molecular mimicry mechanism involvingtumor antigens (other than titin) or is a secondary phenomenon following release of titinfrom muscle. Based on the common immunoreactivity of the AChR, a striational antigenand thymoma, we suggest as the pathogenetic mechanism of thymoma-associated MGa"circulus vitiosus" in which SA autoimmunity could help maintain the AChRautoimmunity that is primarily elicited by the thymomas.


Genome signatures are used to identify and cluster sequences de novo from an acid biofilm microbial community metagenomic dataset, revealing information about the low-abundance community members. Analyses of DNA sequences from cultivated microorganisms have revealed genome-wide, taxa-specific nucleotide compositional characteristics, referred to as genome signatures. These signatures have far-reaching implications for understanding genome evolution and potential application in classification of metagenomic sequence fragments. However, little is known regarding the distribution of genome signatures in natural microbial communities or the extent to which environmental factors shape them.We analyzed metagenomic sequence data from two acidophilic biofilm communities, including composite genomes reconstructed for nine archaea, three bacteria, and numerous associated viruses, as well as thousands of unassigned fragments from strain variants and low-abundance organisms. Genome signatures, in the form of tetranucleotide frequencies analyzed by emergent self-organizing maps, segregated sequences from all known populations sharing < 50 to 60% average amino acid identity and revealed previously unknown genomic clusters corresponding to low-abundance organisms and a putative plasmid. Signatures were pervasive genome-wide. Clusters were resolved because intra-genome differences resulting from translational selection or protein adaptation to the intracellular (pH ~5) versus extracellular (pH ~1) environment were small relative to inter-genome differences. We found that these genome signatures stem from multiple influences but are primarily manifested through codon composition, which we propose is the result of genome-specific mutational biases.An important conclusion is that shared environmental pressures and interactions among coevolving organisms do not obscure genome signatures in acid mine drainage communities. Thus, genome signatures can be used to assign sequence fragments to populations, an essential prerequisite if metagenomics is to provide ecological and biochemical insights into the functioning of microbial communities. The prevalence of horizontal gene transfer, recombination, and population-level genomic diversity underscores the dynamic nature of bacterial and archaeal genomes and demands reconsideration of fundamental issues such as microbial taxonomy ,2 and thfunction -7. To dafunction -11, elucfunction , redefinfunction -15, uncofunction -19 and tfunction , and shofunction .An important approach to study evolutionary and ecological processes, pioneered by Karlin and others , is the Oligonucleotide frequencies capture species-specific characteristics of nucleotide composition more effectively than %GC . AnalyseGenome signatures also offer a compelling means of assigning metagenomic sequence fragments to microbial taxa, a procedure termed 'binning' . This isGenome signatures are a promising approach for sequence classification. However, it is important to understand the source of the signal and how environmental effects and evolutionary distance will compromise it. To date, sequence signatures have been explored using genomes from cultivated microbes ,30-34, a2) dissolution [et al., submitted). These assembled composite genomes provide the organism affiliation of sequences with which binning accuracy can be evaluated. Therefore, the dataset allows assessment of binning performance while capturing sequence heterogeneity that is an intrinsic feature of natural microbial populations. We find that AMD biofilm microorganisms are indeed distinguished by population-specific genome signatures and show that sequence signatures can be used to identify and cluster sequences from low-abundance community members de novo, without reference genomes or reliance on databases. Our results have implications for metagenomic binning and provide new insights into the sources of genome signatures that distinguish coexisting populations.Here we present a comprehensive analysis of genome signatures in sequences derived from natural biofilms inhabiting a subsurface chemolithoautotrophic acid mine drainage (AMD) ecosystem in the Richmond Mine at Iron Mountain, CA . The biosolution . Due to solution ,55,56 (BLeptospirillum spp. group II and group III (Nitrospirae) populations, for which near-complete genomes have been reconstructed [An overview of our methodology is shown in Figure structed ,56. The structed , and A-pstructed ,55.From the combined dataset, near-complete genomes were reconstructed for ARMAN-2, I-plasma, E-plasma, G-plasma, and A-plasma Table . In addiFerroplasma acidarmanus fer1, which was cultivated from AMD solutions in the Richmond Mine [We constructed a dataset that contained all sequences from the combined assembly (assigned and unassigned), previously assembled composite genome sequences, and the genome sequence from ond Mine ,57  [Leptospirillum group II, which share 95% average nucleotide identity [Ferroplasma types I and II, which share 83% average nucleotide identity and are known to participate in homologous recombination [Leptospirillum groups II and III was achieved, except for certain genomic regions containing mobile elements, as described further below. Among members of the Thermoplasmatales, populations were distinguished by genome signatures but borders were variably well-defined   to resolve sequences from coexisting populations of the community. To quantify the degree of segregation of fragments from genomes at various evolutionary distances, we adapted a method using fixed point kernel densities  ) mapped identity   sequence fragments were analyzed, but precision remained remarkably high even for 1,400-bp fragments . Lower sensitivity is due to sequence fragments that fall between clusters, beyond the borders of any bin. Notably, the tetra-ESOM correctly assigned sequence fragments as short as 500 bp, provided that some larger fragments were included in the analysis . To address the question of how genome completeness influences performance, genomes randomly subsampled at different levels were analyzed by tetra-ESOM. Binning accuracy was maintained even at 20% genome sequence; only at 10% subsampling was a notable decline observed, and even then only for certain genomes .To quantitatively evaluate binning performance on sequence fragments of different lengths, tetra-SOMs were run on the same dataset (including unassigned sequences and reconstructed composite genomes) but with sequences broken into various fragment sizes. Binning accuracy was calculated for a subset of genomes for which deeply sampled and manually curated assemblies are available . For sequence fragments 5 kb or larger, sensitivity (percentage of fragments from each genome correctly identified) and precision (percentage of fragments in each bin belonging to the correct genome) rates of > 90% were achieved . Sensitivity was somewhat lower for Leptospirillum groups II and III  of incorrectly binned fragments from all five reference genomes show evidence of transposons, prophage, or integrated plasmids. Other frequently unresolved genomic regions contain CRISPR elements  and rRNAI Figure  was domisequence ) and strFerroplasma in our binning analysis.Interestingly, many strain-unique regions were correctly binned with their host genomes. There are 197 strain-unique genes between the fer1 and fer1(env) genomes, the majority of which occur in distinct genomic blocks of up to 24 genes with atypical %GC content inferred to be the result of prophage insertion . Ninety-Leptospirillum most closely related to Leptospirillum ferrodiazotrophum (group III); several members of the Thermoplasmatales for which genomic sequence had not been previously obtained ; several Actinobacteria; and multiple more shallowly sampled populations, including a gammaproteobacterium and several Sulfobacillus-like organisms .The tetra-ESOM revealed large regions of the map that were devoid of sequence fragments of known organism affiliation , a trend that has been observed previously for cultivated viruses and hosts [We calculated tetranucleotide frequencies for viral genomes that were recently reconstructed from the same genomic datasets and linked to their hosts via CRISPR viral resistance system sequences  . Three ond hosts ,63. InteAs expected, the frequency at which each tetranucleotide occurs is related to overall %GC: GC-rich tetranucleotides are abundant in high-GC genomes and uncommon in low-GC genomes. However, patterns of tetranucleotide usage extend beyond trends in %GC  and genomes with near-identical %GC were effectively segregated by tetra-SOM. Because tetranucleotide frequencies are calculated with a 1-bp sliding window and reverse complementary pairs of tetranucleotides are summed together, all possible reading frames on both strands are sampled. In addition to spanning complete single codons, adjacent pairs of partial codons are also sampled Figure . TherefoFerroplasma versus other Thermoplasmatales. SOMs based on codon composition were notably more accurate than amino acid composition and comparable to those based on tetranucleotide frequency .To assess the contributions of these potential sources of genome signature signal, we compared SOMs based on amino acid composition, codon composition, and tetranucleotide frequency. Amino acid composition alone distinguished certain genomes . This was especially true for phylogenetically distant organisms , but some separation was also apparent among groups within some lineages such as Ferroplasma types I and II, G-plasma, E-plasma). However, SOMs run excluding all 16 palindromic tetranucleotides distinguished populations with accuracy comparable to that achieved using all tetranucleotides, indicating that palindrome avoidance is not a primary component of the genome signature.Additional features of the relationship between codon composition and tetranucleotide frequency were revealed by comparing the observed frequency of tetranucleotides to the frequency predicted from genome-wide codon usage . Observed and predicted tetranucleotide frequency correlated strongly Figure , and difThe correlation of genome signatures with codon usage raises the question of whether they persist in intergenic regions. Thus, we extracted intergenic regions from assembled and annotated genomes and analyzed them with coding regions by tetra-ESOM . Intergenic regions from each genome formed discrete, cohesive clusters that mapped adjacent to coding regions from the same genome but were separated by U-Matrix boundaries . Intergenic sequences from each genome were grouped based on length, concatenated, and analyzed by ESOM; all size classes of intergenic regions from the same genome clustered together regardless of length, from the shortest (4 to 20 bp) to longest  (data not shown). The noncoding complement of each Thermoplasmatales genome formed a distinct cluster adjacent to noncoding regions of the other Thermoplasmatales. The only outlier to this trend was A-plasma, which has the highest %GC among these organisms. Based on U-Matrix background, the distance between noncoding sequences of different genomes is comparable to the distance between noncoding and coding sequences of the same genome. To determine if the presence of noncoding sequence influences binning accuracy in the initial experiments, we calculated the percentage of coding sequence on incorrectly binned fragments from the five reference genomes (5 kb and 1 kb window sizes). For many genomes, the incorrectly binned fragments do indeed have a smaller average percentage of coding sequence. However, this percentage varied widely on incorrectly binned fragments. Only a small fraction of such fragments had a percentage of coding sequence smaller than one standard deviation below the genome-wide average .Leptospirillum group II-UBA proteins that are consistently enriched in the extracellular fraction according to environmental shotgun proteomics data [Leptospirillum group II genes. No systematic differences were detected via tetra-ESOM, suggesting that genome signatures persist even when gene sequences are influenced by considerable protein-coding constraints .For sequence signatures to differentiate populations in a genome-wide manner, it is necessary that within-genome differences resulting from atypical regions of amino acid and/or synonymous codon usage are smaller than between-genome differences. This issue is especially relevant in AMD, where proteins are under diverse constraints depending on whether they function in the extracellular (around pH 1) or intracellular (around pH 5) environment . Indeed,ics data ,66 and cLeptospirillum group II proteins most abundantly detected via environmental shotgun proteomics [Leptospirillum groups II and III), highly expressed genes clustered with the rest of the genome .Selection for codons that optimize translation rate may also influence codon usage. We analyzed genome signatures for the 50 oteomics ,66. WithThrough analysis of a deeply sampled and extensively curated community genomic dataset, we have demonstrated that genome signatures can be used to differentiate coexisting microbial populations despite functional and environmental constraints, processes such as lateral gene transfer, and pressures imposed by viral predation that might have diminished them to the point that they are no longer diagnostic. The genome-wide nature of the signatures makes them potentially useful for classification of sequence fragments. Results from our AMD dataset show that the signal can be detected on fragments as small as 500 bp, genome clusters can be defined using fragments as short as 1,400 bp  and a small fraction of the genome . These findings suggest broad applicability of the tetra-ESOM approach for metagenomic studies. However, in order to understand and predict its utility for binning, it is important to identify sources of genome signatures as well as processes that are likely to diminish the signal.It has been suggested that environmental constraints strongly shape nucleotide composition ,49-51. IOur results show that genome signatures are related to several traits, including %GC, amino acid composition, synonymous codon usage, and palindrome avoidance. These characteristics are interrelated and further connected to a host of biochemical, ecological, and evolutionary processes . Large differences in %GC and/or amino acid composition guarantee distinctive genome signatures but are not required to differentiate genomes. At finer evolutionary scales, where %GC and amino acid composition are not informative, populations can be readily distinguished through subtle differences in tetranucleotide frequency, which correlate with genome-specific synonymous codon usage. Tetra-ESOM analyses based on codon usage and tetranucleotide frequency displayed similar clustering resolution, indicating that little signal derives from longer-range characteristics such as codon pair bias. It should be noted, however, that using tetranucleotide frequency rather than codon composition has practical advantages for binning because it is independent of coding strand and reading frame and thus insensitive to errors in gene-calling or frame shifts due to poor quality sequence. These issues are particularly important for short, low-coverage sequence fragments.Although genome signatures are largely manifested through codon composition, the observation that population-specific signatures also occur in non-coding regions  suggests a mechanism of generation that is independent of protein coding. We hypothesize this underlying process is mutational bias associated with DNA replication and repair, which exerts directional pressure on nucleotide composition . In factFerroplasma, that have only minor differences in tRNA gene content, and these differences do not correspond to observed differences in codon usage. In addition to tRNA gene complement, there may be changes in tRNA gene regulation, which can significantly impact cellular tRNA concentrations and have been correlated with changes in codon usage [An alternative to the mutation bias hypothesis, at least for coding sequences, is that genome signatures are shaped by factors related to translation. Changes in codon usage can be driven by changes in the tRNA gene complement ,70 that on usage . Thus, aLeptospirillum group II genome fragment encoding nine ribosomal proteins and two translation elongation factors had distinctive tetranucleotide composition, indicating that this mode of codon bias occurs in AMD organisms. However, as commonly construed, translational selection would influence within-genome codon bias, not the genome-wide codon biases that differentiate populations as observed in our study. It is tempting to speculate that differences in ecological strategy  could hFinally, restriction avoidance places another selective genome-wide constraint on DNA composition that may contribute to genome signatures. Under-representation of palindromic tetranucleotides Figure  has beenDue to the high levels of diversity in most natural systems, random sequencing approaches yield fragmentary data, often comprising genomic sequences no more than a few kilobases in length. While more comprehensive coverage of individual organisms can be achieved by single cell genomics -80 or taet al., in preparation), a putative plasmid, and many discrete but less well-sampled populations.A key challenge for virtually all shotgun metagenomics investigations is the assignment of genome fragments to the organism they derive from. This step links organism to metabolism and function and is essential if we are to understand microbial community dynamics and predict ecosystem level impacts of changes in community membership and structure. Binning is particularly challenging for lower-abundance organisms, which may play keystone roles that are critical to ecosystem function. Thus, our finding that tetra-ESOM can resolve the phylogenetic affiliation of genome fragments on the scale of two mate-paired reads is of great significance. This approach has clear applicability to low-complexity datasets such as those derived from our AMD biofilms, bioreactors , and enrTetra-ESOM may also provide a powerful method for analysis of unassembled data from complex samples such as soil, seawater, and the human microbiome if representative isolate genomes are available. The feasibility of binning metagenomic sequences from complex samples using reference genomes will increase with current initiatives to fill in the phylogenetic tree with genome sequences from cultivated microorganisms.An important advantage of unsupervised, compositional-based approaches such as tetra-ESOM is that gene sequences need not be represented in databases to be identified; only representation of the genome signature is required. This is in contrast to fragment recruitment  and BLASBecause the reach of composition-based approaches to binning extends beyond gene content of reference genomes, they hold great promise for identifying and classifying genes from the variable fraction of the pan-genome (present in only a subset of strains or species), an important determinant of pathogenicity and niche differentiation -88. In A222 ways to code for a typical protein in our samples (based on an average protein size of 467 amino acids and assuming an average of 3 possible ways to code for any amino acid). This richness of protein coding space suggests ample capacity for numerous genome signatures. To date, SOMs have shown promising results in resolving up to 81 complete genomes, in successfully classifying fragments of 1,502 genomes into phylogenetic groups, and in visualizing phylogenetic clustering of sequences in complex environmental samples [Although the tetra-ESOM method works well to separate sequence fragments from organisms distinct at the genus or higher level, it has some limitations. Tetra-ESOM is generally unable to distinguish closely related species or strains. An important question, especially for more diverse samples, is whether limitations in genome sequence signature space will impose an inherent constraint on the number of populations that can be resolved. There are a staggering 6 × 10 samples  and grow samples .Bacterial, archaeal, and viral populations in the AMD biofilm community have genome-wide signatures of nucleotide composition that are effectively captured and visualized through self-organizing maps of tetranucleotide frequency. We conclude that even under extremely acidic conditions, shared environmental pressure does not obscure genome signatures of nucleotide composition. Our data point to pervasive mechanisms of generating and maintaining genome signatures; although a variety of factors and processes contribute, we propose that mutational bias is the primary underlying mechanism driving the divergence of genome signature between closely related organisms. The resulting signal, evident through synonymous codon usage, is genome-wide and sufficiently diagnostic to classify fragmentary metagenomic data from coexisting populations of a natural microbial community at approximately the genus level. However, distinguishing features of genome signatures may be subtle, being masked by within-genome heterogeneity and the multidimensional nature of tetranucleotide frequency patterns. Tetra-ESOM is a key method for visualizing and exposing these potentially weak signals. Being unsupervised, it requires no database representation of the organisms present. Visualization of the data structure highlights differences between populations and reveals atypical regions corresponding to biologically meaningful genomic features such as mobile elements or previously unrecognized genotypes present at low abundance in the community. When employed in conjunction with complementary methods such as genomic assembly and analysis of phylogenetic marker genes, genome signatures offer powerful perspectives on metagenomic data.Leptospirillum spp. removed). This included 229,082 reads and approximately 210 Mb of total sequence, which assembled into 15,929 contigs and 36.6 Mb of composite sequence.An overview of the samples and methodology used in this study is provided in Figure The phylogenetic tree of 16S rRNA genes was constructed by neighbor joining (default parameters) with the ARB software package  and 'SILTetranucleotide frequencies were determined for each assembled contig using a custom Perl script. Frequencies were calculated with a 1-bp sliding window and pairs of reverse complementary tetranucleotides were summed in order to avoid strand bias. Longer contigs and assembled genomes were split into 5-kb windows and only contigs longer than 2 kb were considered unless noted otherwise. To assess binning accuracy, data points (representing contigs/windows) are colored according to their genome of origin (when known), but this information is not available to the clustering process.Contigs were clustered by tetranucleotide frequency utilizing Databionics ESOM Tools . The inpTo quantify the degree of clustering between closely related genomes, we analyzed SOM maps using fixed point kernel densities . SpatialThe predicted frequency of each unique pair of reverse complementary tetranucleotides was calculated based on genome-wide frequencies of potentially contributing codons. As shown in Figure Sensitivity (percentage of fragments from each genome correctly identified) and precision (percentage of fragments in each bin belonging to the correct genome) of binning were calculated for a subset of assembled genomes that are deeply sampled and manually curated  to monitor variance in sequence signatures from intergenic regions of differing lengths. All coding sequences were similarly concatenated with interleaving 'N's. Concatenated coding and non-coding regions were then broken into 5-kb windows and run against the same background dataset of assembled genomes and unassigned sequences as usual.Leptospirillum group II extracellular and whole cell fractions from the ABend, ABfront, and UBA locations of the Richmond mine [Shotgun proteomics data were obtained for ond mine ,66. ProtThis Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the project accessions ACXJ00000000 (unassigned contigs), ACXK00000000 (A-plasma), ACXL00000000 (E-plasma), ACXM00000000, (I-plasma), and ACVJ00000000 . The versions described in this paper are the first versions, ACXJ01000000, ACXK01000000, ACXL01000000, ACXM01000000, and ACVJ01000000.AMD: acid mine drainage; ESOM: emergent self-organizing map; %GC: percentage content of guanine plus cytosine; SOM: self-organizing map.GJD, AFA, SLS, and JFB conceived and designed the experiments. GJD, BJB, SLS, APY, and BCT performed the experiments. GJD, AFA, SLS, BCT, BJB, APY, and JFB analyzed the data. GJD and JFB wrote the paper.Leptospirillum group II genes coding for highly expressed proteins or proteins enriched in the extracellular fraction analyzed as separate fractions from the rest of the genome .Click here for fileHeat map of average genome-wide frequency of each tetranucleotide for each genome, including bacteria, archaea, viruses, and a putative plasmid.Click here for fileComparison of tetra-ESOMs of assembled genomes based on (a) amino acid composition, (b) codon composition, and (c) tetranucleotide frequencyClick here for fileThe observed difference in frequency of each tetranucleotide between pairs of genomes correlates with the difference predicted based on codon composition.Click here for fileTetra-ESOM of deeply sampled genomes for which coding and noncoding regions were separated.Click here for filePercentage of sequence coding for genes in comparison with the genome-wide coding percentage for incorrectly binned fragments.Click here for fileLeptospirillum group II genes coding for highly expressed proteins or proteins enriched in the extracellular fraction analyzed as separate fractions from the rest of the genome.Tetra-ESOM of Click here for fileProcesses and factors influencing genome signature.Click here for file