653 research outputs found

    The Difference Between the Accuracy of Real and the Corresponding Random Model is a Useful Parameter for Validation of Two-State Classification Model Quality

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    The simplest and the most commonly used measure for assess the classification model quality is parameter Q2 = 100 (p + n) / N (%) named the classification accuracy, p, n and N are the total numbers of correctly predicted compounds in the first and in the second class, and the total number of elements of classes (compounds) in data set, respectively. Moreover, the most probable accuracy that can be obtained by a random model is calculated for two-state model by the formulae Q2,rnd = 100 [(p + u) (p + o) + (n + u) (n + o)] / N2 (%), where u and o are the total number of under-predictions (when class 1 is predicted by the model as class 2) and over-predictions (when class 2 is predicted by the model as class 1) in data set, respectively. Finally, the difference between these two parameter ΔQ2 = Q2 – Q2,rnd is introduced, and it is suggested to compute and give ΔQ2 for each two-state classification model to assess its contribution over the accuracy of the corresponding random model. When data set is ideally balanced having the same numbers of elements in both classes, the two-state classification problem is the most difficult with maximal Q2 = 100 % and Q2,rnd = 50 %, giving the maximal ΔQ2 = 50 %. The usefulness of ΔQ2 parameter is illustrated in comparative analysis on two-class classification models from literature for prediction of secondary structure of membrane proteins and on several quanti¬tative structure-property models. Real contributions of these models over the random level of accuracy is calculated, and their ΔQ2 values are compared mutually and with the value of ΔQ2 (= 50 %) for the most difficult two-state classification model

    Structural Requirements of N-Substituted Spiropiperidine Analogues as Agonists of Nociceptin/Orphanin FQ Receptor

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    The nociceptin/orphanin FQ (NOP) receptor is involved in a wide range of biological functions, including pain, anxiety, depression and drug abuse. Especially, its agonists have great potential to be developed into anxiolytics. In this work, both the ligand- and receptor-based three-dimensional quantitative structure–activity relationship (3D-QSAR) studies were carried out using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques on 103 N-substituted spiropiperidine analogues as NOP agonists. The resultant optimal ligand-based CoMSIA model exhibited Q2 of 0.501, R2ncv of 0.912 and its predictive ability was validated by using an independent test set of 26 compounds which gave R2pred value of 0.818. In addition, docking analysis and molecular dynamics simulation (MD) were also applied to elucidate the probable binding modes of these agonists. Interpretation of the 3D contour maps, in the context of the topology of the active site of NOP, provided insight into the NOP-agonist interactions. The information obtained from this work can be used to accurately predict the binding affinity of related agonists and also facilitate the future rational design of novel agonists with improved activity

    Structural and Functional Insights into Ghrelin Acylation by Ghrelin O-Acyltransferase

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    Ghrelin is a peptide hormone primarily secreted by the stomach that acts as the endogenous ligand for the GHSR1a G-protein-coupled receptor. This 28 amino acid peptide stimulates appetite and has been associated with the cardiovascular, gastrointestinal, neuroendocrine, and immune systems. Ghrelin requires a unique posttranslational modification to be biological active. Ghrelin O-acyltransferase (GOAT) is responsible for catalyzing octanoylation of the 3rd serine side chain of ghrelin to generate the active form of this hormone. Thus, GOAT is considered as a potential therapeutic target for treatment of obesity and other disorders linked to the peptide. Yet, the active site architecture and mechanism for GOAT catalyzed ghrelin acylation are presently undetermined. Purification of an active form of GOAT has been challenging, impeding structural studies of this integral membrane protein. To improve on these challenges, we have synergistically combined computational modeling and biochemical validation to construct the first structural model of the eukaryotic membrane-bound human GOAT protein. Our structure revealed an unforeseen strategy for transmembrane protein acylation with catalysis occurring in an internal channel connecting the lumen and cytoplasm. Structure-guided studies used in this work, have revealed essential amino acid responsible for important substrate-enzyme interactions. Moreover, by using a tight-binding fluorescent ghrelin derived peptide, we demonstrate GOAT’s interaction with extracellular ghrelin and facilitation of ligand cell internalization. Our work provides a new understanding of GOAT’s catalytic mechanism, establishes key substrate-enzyme interactions, and advances structure-guided inhibitor design to target therapeutically important but experimentally intractable membrane proteins

    Nutritional Systems Biology Modeling: From Molecular Mechanisms to Physiology

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    The use of computational modeling and simulation has increased in many biological fields, but despite their potential these techniques are only marginally applied in nutritional sciences. Nevertheless, recent applications of modeling have been instrumental in answering important nutritional questions from the cellular up to the physiological levels. Capturing the complexity of today's important nutritional research questions poses a challenge for modeling to become truly integrative in the consideration and interpretation of experimental data at widely differing scales of space and time. In this review, we discuss a selection of available modeling approaches and applications relevant for nutrition. We then put these models into perspective by categorizing them according to their space and time domain. Through this categorization process, we identified a dearth of models that consider processes occurring between the microscopic and macroscopic scale. We propose a “middle-out” strategy to develop the required full-scale, multilevel computational models. Exhaustive and accurate phenotyping, the use of the virtual patient concept, and the development of biomarkers from “-omics” signatures are identified as key elements of a successful systems biology modeling approach in nutrition research—one that integrates physiological mechanisms and data at multiple space and time scales

    Systems chronotherapeutics

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    Chronotherapeutics aim at treating illnesses according to the endogenous biologic rhythms, which moderate xenobiotic metabolism and cellular drug response. The molecular clocks present in individual cells involve approximately fifteen clock genes interconnected in regulatory feedback loops. They are coordinated by the suprachiasmatic nuclei, a hypothalamic pacemaker, which also adjusts the circadian rhythms to environmental cycles. As a result, many mechanisms of diseases and drug effects are controlled by the circadian timing system. Thus, the tolerability of nearly 500 medications varies by up to fivefold according to circadian scheduling, both in experimental models and/or patients. Moreover, treatment itself disrupted, maintained, or improved the circadian timing system as a function of drug timing. Improved patient outcomes on circadian-based treatments (chronotherapy) have been demonstrated in randomized clinical trials, especially for cancer and inflammatory diseases. However, recent technological advances have highlighted large interpatient differences in circadian functions resulting in significant variability in chronotherapy response. Such findings advocate for the advancement of personalized chronotherapeutics through interdisciplinary systems approaches. Thus, the combination of mathematical, statistical, technological, experimental, and clinical expertise is now shaping the development of dedicated devices and diagnostic and delivery algorithms enabling treatment individualization. In particular, multiscale systems chronopharmacology approaches currently combine mathematical modeling based on cellular and whole-body physiology to preclinical and clinical investigations toward the design of patient-tailored chronotherapies. We review recent systems research works aiming to the individualization of disease treatment, with emphasis on both cancer management and circadian timing system–resetting strategies for improving chronic disease control and patient outcomes

    Brain serotonergic circuitries

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    Brain serotonergic circuitries interact with other neurotransmitter systems on a multitude of different molecular levels. In humans, as in other mammalian species, serotonin (5-HT) plays a modulatory role in almost every physiological function. Furthermore, serotonergic dysfunction is thought to be implicated in several psychiatric and neurodegenerative disorders. We describe the neuroanatomy and neurochemistry of brain serotonergic circuitries. The contribution of emergent in vivo imaging methods to the regional localization of binding site receptors and certain aspects of their functional connectivity in correlation to behavior is also discussed. 5-HT cell bodies, mainly localized in the raphe nuclei, send axons to almost every brain region. It is argued that the specificity of the local chemocommunication between 5-HT and other neuronal elements mainly depends on mechanisms regulating the extracellular concentration of 5-HT, the diversity of high-affinity membrane receptors, and their specific transduction modalities

    “ Obesity serum factors affect human platelets SERT: we need a cellular model to investigate “

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    Amongst molecules active in modulating 5-HT transmission, SERT exerts a main function by promoting 5-HT clearance and re-uptake into the pre-synaptic terminal, therefore controlling either the duration and extent of the transmitter action at specific targets after release or its “reserve” inside pre-synaptic vesicles. For this reason, SERT expression and function is under the control of several intracellular phosphorylation systems (Ramamoorthy et al. 2007). More recently, a relationship between obesity and inflammatory-immune response has been suggested (Matarese and La Cava, 2004), indicating a cross-talk between CNS and periphery in the control of body weight. In a preliminary attempt to elucidate these networks, the present study was thus designed to investigate the influence of 5-HT on body weight by measuring the equilibrium binding parameters (maximal binding capacity, Bmax and dissociation constant, Kd) of the high affinity SERT ligand [3H]-paroxetine in platelets from 174 subjects recruited on the basis of their body mass index (BMI), starting from 18 to > 40 kg/m2. The high affinity ligand [3H]-paroxetine was used as a selective tracer of SERT and subjects recruited to obtain five groups as regards to BMI: 1. normal weight (18-25 kg/ m2), 2. overweight (26-30 kg/ m2), 3. obese, I grade, (31-35 kg/ m2), 4. obese, II grade, (36-40 kg/ m2); 5. obese, III grade, (>40 kg/ m2). All subjects were also accurately monitored for clinical-biochemical parameters, including insulinemia and leptinemia blood glucose, insulin, leptin cytokines and other clinical-chemical parameters. We have chosen to study platelets as these non nucleate cells display an identical SERT to the brain one as well as several 5-HT receptor subtypes. For such reasons, platelets are considered “indicators” of 5-HT-regulated CNS-periphery connections and have been extensively studied in psychiatric-affective disorders since many years (Mellerup et al, 1983; Martini C. et al., 2004). Finally, platelets respond to agonist-induced activation by rapid phosphorilation and represent, therefore, a good model to study neuronal signal transduction events in periphery (Aharanovitz and Granot, 1996 ). Our study is the first that showed a significant reduced SERT density in platelets of obese subjects (p<0.05), providing new insights in the role of 5-HT system and obesity. Moreover, no BMI-dependent changes in [3H]-paroxetine Kd (nM) values, but a significant inverse correlation (p<0,0001) between [3H]-paroxetine Bmax values (fmol/mg protein) and BMI. Consequently, a negative significant correlation was observed between [3H]-paroxetine Bmax values and: blood leptin, TNF-α, insulin, glicemia, PAS, aptoblobin and triglycerides. In conclusion, these findings show that platelet SERT number is reduced in obese subjects and such a decrease could be linked, in part, to the efficiency of the insulin/adipokine-related downstream signaling cascade in periphery. These results could explain that adipose tissue entity are able to modulate SERT expression, but not his functionality, in platelets membrane. Identification of isolated cellular model, capable to express SERT and having biochemical characteristics of platelet, could be useful to study the modulation of serotonin transporter by different factors related with obesity. We identified this model in MEG-01, a human megakaryoblastic leukaemia cell line, which specifically retains the morphological and functional properties of bone marrow megakaryocytes. This cell line is considered to be the most suitable one for evaluating human megakaryocytic maturation and differentiation into platelet-like cells (Isakari Y. et al 2007). The evidence that SERT is present in megakaryoblasts suggests the physiological relevance of a balanced control of 5-HT levels during the differentiation processes leading to platelet formation (Liu YS et al 2006; Yang M et al 2007). After 8 days of MEG-01 cell culture with -TPA, significant cell morphological variations were observed, accompanied by changes in SERT immunostaining, from a perinuclear (undifferentiated cells) to a cytoplasm-diffuse (differentiated cells) fluorescence pattern. In -TPA activated MEG-01 cells a maximal increase of SERT mRNA was observed after 3 day of stimulation, reaching the steady state plateau; a moderate increase in protein expression was noticeable after 8 days of culture in-TPA, as shown by densitometric analysis of SERT immunoblot band and, even if not significantly, by 5-HT uptake results. 5-HT re-uptake experiments have shown that SERT is present in the plasma membrane of MEG-01 cells. Western blot revealed a single SERT band in control and treated cells, with an apparent size of 71 KDa, a M.W. comparable to that commonly reported for SERT in human brain and platelets (67-75 KDa). These findings demonstrate that megakaryoblastic cells increase SERT expression during megakaryocytopoiesis and that differentiation into megakaryocyte and proplatelet-like formation ensures an adequate SERT reserve and 5-HT storage in circulating platelets. The enhancement of SERT expression in -TPA treated cells seems in apparent contradiction with -TPA early events observed in platelets (Marazziti D et al 1999c; Jayanthi LD et al.2005; Carneiro AM et al. 2006). Nevertheless, some authors have reported that 1 M -TPA is able either to reduce (after 2h) or to stimulate (after 16 h) SERT uptake velocity in JAR placental cells (Ramamoorthy JD et al 1995). Jiang and coauthors (2002) have observed that both fibronectin and protein kinase C-dependent ERK1/2 MAPK activities are essential to promote megakaryoblastic differentiation. These authors have demonstrated that activation of protein kinase C alone (serum free -TPA stimulation) is not able to promote a full megakaryocytopoiesis process. Thus, the observed SERT increase might depend from -TPA-protein kinase C pathways and other signaling cascades. The protein kinase network and downstream signal convergent MAPK pathways could play a key role in SERT modulation. Proteomic analysis and gene expression studies together the use of selective protein kinase (also protein kinase C) or MAPK inhibitors will clarify the signaling pathways involved. On the basis of results we hypothesize that our findings derived from an equilibrium between different SERT regulatory effects: differentiation events, inducing morphological changes and leading to the formation of cytoskeleton-organized cells with 5-HT storage vesicles (Ogura M et al 1998), increase total SERT protein density (necessity to accumulate 5-HT in dense granules); phosphorylation, down-regulation and trafficking mechanisms (linked to cytoskeleton formation) can start having influence on SERT protein expression and function, especially in late events of megakaryocytopoiesis. This could explain the observed discrepancy between SERT mRNA and protein expression after 8 days of treatment with -TPA. We cannot, in fact, exclude that our Western blot protein extracts were enriched fractions of cytoplasm and plasma membrane components. These data seem to be in accordance with binding results obtained on cell membranes. Confocal microscopy of MEG-01 cells activated by TPA will verify SERT localization during early and late differentiation events. Since diverse protein regulatory patterns are present in immature megakaryoblasts cells as regards to pro-platelets and platelets (Tytgat GAM et al. 2002), the MEG-01 model should be primarily applied as a developmental model, for investigations regarding molecular differentiation events. For instance, it should be mentioned that a SERT regulatory endoproteolytic cleavage has been observed in human platelets, producing fragments of different size after immunoblot analysis, whereas a single SERT band was resolved in MEG-01 cell extracts, using the same primary antibodies, as here reported. Megakaryoblastic MEG-01 differentiation is a complex phenomenon leading to up or down-regulation of a variety of genes (Isakari Y et al 2009). Nevertheless, taking into account all limits, the use of MEG-01 cells can be the starting point for many investigations as the evaluation of hormone and transmitter effects on megakaryoblastic differentiation. Moreover, this cell line could be a cellular model to screen those of obesity serum factors are mostly important for long-term SERT down or up-regulation

    Molecular mechanism of orlistat hydrolysis by the thioesterase of human fatty acid synthase for targeted drug discovery

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    Indiana University-Purdue University Indianapolis (IUPUI)Fatty acid synthase (FASN) is over-expressed in many cancers, and novel inhibitors that target FASN may find use in the treatment of cancers. It has been shown that orlistat, an FDA approved drug for weight loss, inhibits the thioesterase (TE) of FASN, but can be hydrolyzed by TE. To understand the mechanisms of TE action and for designing better FASN inhibitors, I examined the mechanism of orlistat hydrolysis by TE using molecular dynamics simulations. I found that the hexyl tail of orlistat undergoes a conformational transition, destabilizing a hydrogen bond that forms between orlistat and the active site histidine. A water molecule can then hydrogen bond with histidine and become activated to hydrolyze orlistat. These findings suggest that rational design of inhibitors that block hexyl tail transition may lead to a more potent TE inhibitor. To search for novel inhibitors of TE, I performed virtual DOCK screening of FDA approved drugs followed by a fluorogenic assay using recombinant TE protein and found that proton pump inhibitors (PPIs) can competitively inhibit TE. PPIs, which are used for the treatment of gastroesophageal reflux and peptic ulcers, work to decrease gastric acid production by binding irreversibly with gastric hydrogen potassium ATPase in the stomach. Recently, PPIs have been reported to reduce drug resistance in cancer cells when used in combination with chemotherapeutics, although the mechanism of resistance reduction is unknown. Further investigation showed that PPIs are able to decrease FASN activity and cancer cell proliferation in a dose-dependent manner. These findings provide new evidence that FDA approved PPIs may synergistically suppress cancer cells by inhibiting TE of FASN and suggests that the use of PPIs in combinational therapies for the treatment of many types of cancer, including pancreatic cancer, warrants further investigation
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