Like cancer cells, virally infected cells have dramatically altered metabolic requirements. We analyzed global metabolic changes induced by latent infection with an oncogenic virus, Kaposi's Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposi's Sarcoma (KS), the most common tumor of AIDS patients. Approximately one-third of the nearly 200 measured metabolites were altered following latent infection of endothelial cells by KSHV, including many metabolites of anabolic pathways common to most cancer cells. KSHV induced pathways that are commonly altered in cancer cells including glycolysis, the pentose phosphate pathway, amino acid production and fatty acid synthesis. Interestingly, over half of the detectable long chain fatty acids detected in our screen were significantly increased by latent KSHV infection. KSHV infection leads to the elevation of metabolites involved in the synthesis of fatty acids, not degradation from phospholipids, and leads to increased lipid droplet organelle formation in the infected cells. Fatty acid synthesis is required for the survival of latently infected endothelial cells, as inhibition of key enzymes in this pathway led to apoptosis of infected cells. Addition of palmitic acid to latently infected cells treated with a fatty acid synthesis inhibitor protected the cells from death indicating that the products of this pathway are essential. Our metabolomic analysis of KSHV-infected cells provides insight as to how oncogenic viruses can induce metabolic alterations common to cancer cells. Furthermore, this analysis raises the possibility that metabolic pathways may provide novel therapeutic targets for the inhibition of latent KSHV infection and ultimately KS tumors. In recent years there has been a resurgence in the study of metabolic changes in tumor cells. To determine if an oncogenic virus alters similar metabolic pathways as cancer cells, we measured the levels of a large number of metabolites in endothelial cells infected with Kaposi?s Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposi's Sarcoma (KS), the most common tumor of AIDS patients world wide. Latent KSHV infection of endothelial cells altered a significant proportion of the host cell metabolites. Many metabolic pathways that are altered in most tumor cells were also altered by KSHV. In particular, KSHV upregulated fatty acid synthesis, a pathway that provides membrane material and metabolites critical for cell proliferation. Inhibitors of fatty acid synthesis kill many types of tumor cells and we found that these inhibitors led to death of cells latently infected with KSHV. In summary, we found that a directly oncogenic virus alters the same host metabolic pathways that are dysregulated in many cancer cells and that inhibition of these pathways can be used to kill off infected cells, thereby providing novel therapeutic targets for KSHV and ultimately KS tumors. Many metabolic pathways are dramatically altered in cancer cells. These alterations are thought to provide cancer cells with the necessary energy and substrates for rapid cell division. Otto Warburg first demonstrated that most cancer cells have increased levels of glycolysis, even in the presence of oxygen, indicating that cancer cells dramatically alter their metabolism Recently, global changes in cellular metabolism have been studied using metabolomic approaches As non-living entities, viruses do not inherently have their own metabolism. However, upon infection, viruses dramatically alter the metabolism of the host cell. Viral alteration of host cell metabolism can provide the substrates necessary for viral replication. For example, alteration of host cell metabolism can provide the increased nucleotides necessary for genome replication or increased free amino acids needed for rapid viral protein synthesis. Virally-induced alterations of host metabolic pathways are likely to also be important for viral pathogenesis. Viral metabolomic studies were first used to identify changes in host cellular metabolism induced by human cytomegalovirus (HCMV) lytic infection Kaposi's Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS). KS is the most common tumor in parts of central Africa as well as in AIDS patients worldwide. KS lesions are predominantly populated by spindle cells of endothelial origin Our previous studies found that KSHV infection of cultured endothelial cells directly induces the Warburg effect de novo fatty acid synthesis (FAS or lipogenesis), and was concurrent with increased lipid droplets in latently infected endothelial cells. Lipid droplets are lipid-rich cytoplasmic organelles (diameter <1–100 µm) that store cellular energy in the form of triacylglycerols and also store phospholipids and sterols, the building blocks of membranes To determine if KSHV induces other metabolic changes similar to cancer cells, we performed a global screen to detect metabolites in mock- and KSHV-infected cells at 48 and 96 hours post infection (hpi). At 48 and 96 hpi, greater than 90% of the cells expressed a latent marker of infection, while only 1–5% of the cells expressed a marker of lytic replication. By 48 hpi, KSHV altered greater than one-quarter of the approximately 200 detectable metabolites and nearly one-third were altered by 96 hpi. Metabolites of several pathways commonly dysregulated in tumor cells were altered by KSHV, including glycolysis, amino acid metabolism, the pentose phosphate pathway and lipogenesis. KSHV infection leads to elevated levels of over half of the detectable metabolite products of de novo lipogenesis. Normal cells predominantly acquire fatty acids from dietary sources rather than de novo lipogenesis. However, the majority of fatty acids from cancer cells are derived by de novo lipogenesis even when exogenous fatty acids are available Fatty acids are essential substrates for energy metabolism and are the major components of all biological lipid membranes To determine global changes in host cell metabolism induced by latent KSHV infection, we utilized a metabolomic approach. Tert-immortalized human dermal microvascular endothelial cells (TIME cells) A variety of curation procedures were carried out by Metabolon to ensure the quality of the data set presented. System artifacts, mis-assignments, and background noise were limited by Metabolon data analysts through the use of proprietary visualization and interpretation software to confirm the consistency of peak identification among the various samples. Bradford assays were used to normalize each sample by protein concentration and each biochemical in the mock samples was re-scaled to yield a median equal to 1. KSHV-infected cells are shown as fold change compared to mock-infected cells. 194 named biochemical were unambiguously identified in our samples. Following log transformation and imputation with minimum observed values for each compound, Welch's two–sample t-tests were used to determine which biochemicals are significantly altered by KSHV infection at 48 and 96 hpi across the 6 biological replicates. An estimate of the false discovery rate  was calculated to take into account the multiple comparisons that normally occur in metabolomic-based studies. Low q-values were calculated for significantly different biochemical (p≤0.05) for both comparisons, indicating a low estimated false discovery rate. The q-values for the significantly altered compounds are 0.06 and 0.09 at 48 hours and 96 hpi respectively.de novo fatty acid synthesis (FAS) as described more extensively below. Of note, many of the metabolites that are increased by infection, are increased to higher levels at 96 hours as compared to 48 hours post infection. For example, increased glucose is only increased 1.38 fold at 48 hours with a p-value of greater than 0.05, while it is increased more than 10 fold at 96 hours with a p-value below 0.05  , the buiLipid droplets are small cytoplasmic organelles that contain triacyglycerols for cellular energy storage, as well as, phospholipids and sterols for membrane production. Excess cellular LCFA can lead to the formation of lipid droplets Inhibitors of FAS have been shown to selectively kill cancer cells and inhibit tumor cell growth Fatty acid inhibitor C75 specifically inhibits fatty acid synthase (FASN), thereby preventing the catalysis of acetyl-CoA and malonyl-CoA into palmitic acid . Cancer The FAS inhibitor results were confirmed in TIME cells  and 1°hDTo determine the mechanism of cell death induced by TOFA and C75 in latently infected endothelial cells, classic markers of apoptosis were examined. Caspase 3 cleavage is an early event in many forms of programmed cell death and leads to the induction of PARP cleavage Inhibition of ACC1 by TOFA could lead to apoptosis through inhibition of the synthesis of necessary downstream metabolites or through increased accumulation of precursors that could potentially be harmful to the cell. To determine if downstream pathways are necessary for latent infected cell survival we added palmitic acid, a metabolite that is downstream of the enzyme ACC1. Palmitic acid is the fundamental fatty acid precursor that iscreated by the conversion of acetyl-CoA and malonyl-CoA by the enzyme FASN. Addition of palmitic acid to tumor cell culture media has been shown to overcome blocks in fatty acid synthesis in tumor cells It has long been known that viruses can alter specific metabolic processes. However, global changes in metabolism have only recently been examined for a few lytic viruses. The first landmark study of viral metabolism was performed on HCMV infected cells A number of enveloped lytic viruses have been shown to be inhibited by a block in FAS Interestingly, KSHV induces and requires lipogenesis during latent infection of endothelial cells. The increase in lipogenesis and lipid droplet accumulation was seen in the bulk of the population where greater than 85% of the cells expressed latent markers and less than 5% of the cells express lytic markers. This indicates that increased lipid droplets occurred in most of the latently infected cells. However, this does not rule out the possibility that lipid droplet formation requires a paracrine factor produced by lytic cells. Increased lipogenesis may predispose infected cells to oncogenesis as lipogenesis is upregulated in most cancer cells and is required for their survival. Inhibition of FAS led to high levels of apoptosis in KSHV latently infected cells, while the same concentration of inhibitors had little effect on the mock-infected cells. These effects were overcome by the addition of exogenous palmitic acid, a key FAS metabolite downstream of the early FAS inhibitor blockage. This indicates that downstream fatty acid metabolic processes are necessary for KSHV latency and that FAS inhibitors are not causing cell death simply due to build up of toxic pre-cursors. Importantly, apoptosis is also induced in many cancer cells following inhibition of FAS All current drugs for herpesviruses specifically target only lytic replication steps. FAS inhibitors may provide novel targets for the inhibition of KSHV as they inhibit KSHV latency. Therefore, it is possible that FAS inhibitors could be used to treat KS tumors as they have the capacity to kill latently infected cells in the tumor. Overall, our metabolomics studies have identified a number of metabolic pathways altered by KSHV that are also commonly altered in cancer cells including glycolysis, amino acid synthesis, the pentose phosphate pathway, polyamine metabolism and fatty acid synthesis. These alterations in metabolism provide novel targets for inhibition of KSHV and possibly KS tumors. Experiments to identify the role of other metabolic pathways identified in our screen on KSHV pathogenesis are ongoing.Tert-immortalized microvascular endothelial (TIME) cells or primary human dermal microvascular endothelial cells (1°hDMVECs) were maintained as monolayer cultures in EBM-2 media (Lonza or PromoCell) supplemented with a bullet kit containing 5%fetal bovine serum (FBS), vascular endothelial growth factor, basicfibroblast growth factor, insulin-like growth factor 3, epidermalgrowth factor, and hydrocortisone.Staurosporin (Sigma) was diluted in DMSO and were used at a final concentration of 1 µM. 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA) (Sigma) and C75  were diluted in DMSO and used at the indicated final concentrations. Palmitic Acid (Sigma) was diluted in DMSO to 100 mM and used at a final concentration of 24 µM. Image-It Dead Green Viability Stain (Invitrogen) was diluted in DMSO and was used at a final concentration of 100 nM. Antibodies recognizing cleaved Caspase-3 , Poly-A Ribose Polymerase  ORF 59 (Advanced Biotechnology inc.) were used at 1∶1000 dilutions as specified by the manufacturer and antibodies to β-Actin (Sigma) was used at 1∶10,000. Antibodies that recognize KSHV protein, LANA (kind gift from Polson and Ganem) was used at 1∶1000 dilutions.KSHV inoculum from induced BCBL-1 cells was concentrated, titered and used to infect TIME and primary hDMVECs as previously described TIME cells were mock- or KSHV-infected, harvested using a cell scraper and pelleted. An aliquot of thecells was seeded onto chamber slides for immunofluorescenceanalysis as described above. Cell pellets were washed once in cold phosphate-buffered saline and then resuspended in RIPA lysis buffer . Proteinconcentrations were determined by the bicinchoninic acid assay(Pierce). 15–50 µg of protein was fractionated on a sodiumdodecyl sulfate-polyacrylamide gel, transferred to a polyvinyldifluoride membrane, blotted with the indicated primary antibody diluted as described above, and subsequently with horseradish peroxidase-conjugated donkeyanti-goat , goat anti-rabbit , goat anti-mouse .Immunoreactive proteins were visualized by chemiluminescence using the Amersham ECL Plus Western blotting detection reagents.48 hpi, mock- or KSHV-infected TIME cells or 1°hDMVECs were seeded sub-confluently  into 12 well plates and treated with TOFA  for 48 hours or treated with C75  for 24 hours. Cells were trypsinized and pelleted with cellular supernatant for 5 minutes and cell pellet was resuspended in ∼50 uL media. Cell death rates were determined by counting cells using a hemocytometer after addition of trypan blue, which is actively exported from the cytoplasm of live cells but remains in dead cells. Cell death rate (%) = number of dead cells/number of total cells (*100%). For the Dead Green viability assay, TIME cells or 1°hDMVECs were mock- or KSHV-infected for 48 hours, seeded sub-confluently in 12 well plates and treated 0 µg/mL or 4 µg/mL C75 for 24 hours, or 0 µg/mL or 20 µg/mL TOFA for 24 hours. Cells were then treated with Image-It Dead Green Viability stain for 15 min and pictures were taken with a fluorescent microscope. For Palmitic Acid rescue experiments, 48 hpi, mock- and KSHV infected TIME cells were seeded sub-confluently  in 12 well plates and treated with TOFA at 20 µg/ml in the presence or absence of 24 µM Palmitic Acid for an additional 48 hours. Both trypan blue and Dead Green viability assays were completed as described above.48 hpi, mock- and KSHV-infected TIME cells were seeded in a chamber slide, fixed with 10% Formalin in PBS for 40–60 minutes and washed with 60% Isopropanol for 5 minutes. The cells were then exposed to fresh Oil-Red-O working solution for 10 minutes to stain the lipid droplets. Cells were then washed with dH2O, counterstained with hematoxylin for 1 minute to stain the nuclei and then washed and mounted in Aqua Poly/Mount (Polysciences Inc.). Pictures were taken under bright field microscopy. Images represent frames with high levels of lipid droplets for each condition.Mock- and KSHV-infected cells were trypsinized to remove cells from plates, counted and processed for flow cytometry. For detection of lipid droplets, the Lipid Droplets Florescence Assay Kit  was used as recommended by the manufacturer. Briefly, 48 hpi TIME cells were washed in 1 mL Assay Buffer, fixed for 10 minutes at room temperature, washed with 1 mL Assay buffer, resuspended in Nile Red Staining Solution for 15 minutes at room temperature, washed and resuspended in Assay Buffer. Samples were analyzed by FACScan caliber flow cytometer  or by FACS Canto flow cytometer. All Samples were analyzed by FloJo flow cytometry analysis software .Table S1Relative levels of allthe metabolites identified in KSHV infected cells compared to mock infected cells at 48 and 96 hours post infection. Values shown are normalized levels from 6 distinct KSHV and mock infections of TIME cells. The major metabolic pathways are indicated in the right columns and the biochemical name and the mass spectrometry platforms used are indicated in the middle columns. Metabolites that are significantly increased (p<0.05) are shaded in red and those significantly decreased (p<0.05) are shaded in green. Numbers in blue are altered in KSHV infected cells but with lower statistical significance (0.05<p<0.01).(DOCX)Click here for additional data file.


Identification of effective prognostic biomarkers and targets are of crucial importance to the management of estrogen receptor positive (ER+) breast cancer. CCNA2  belongs to the highly conserved cyclin family and is significantly overexpressed in various cancer types. In this study, we demonstrated that CCNA2 had significant predictive power in distant metastasis free survival, disease free survival, recurrence free survival and overall survival of ER+ breast cancer patients. We also found that CCNA2 was closely associated with tamoxifen resistance. In addition, gene set enrichment analysis (GSEA) revealed that its expression was positively associated with genes overexpressed in endocrine therapy resistant samples. Finally, though CCNA2-Drug interaction network, we demonstrated the interactions between CCNA2 and several available cancer drugs. Overall, we suggest that CCNA2 is a biomarker for the prognosis of ER+ breast cancer and monitoring of tamoxifen efficacy. It's also a promising target for developing new strategies to prevent or even reverse tamoxifen resistance. Moreover, CCNA2 expression may help monitoring tamoxifen efficacy and directing personalized therapies. Nevertheless, in vivo and in vitro experiments and multi-center randomized controlled clinical trials are still needed before its application in clinical settings. Breast cancer is the most common malignancy among women in the United States, among which 70% of them are ER+. The selective ER modulator tamoxifen has shown great success in the treatment of ER+ breast cancerCCNA2  belongs to the highly conserved cyclin family and is expressed in almost all tissues in human bodyNevertheless, the prognostic power of CCNA2 in ER+ breast cancer and its relation with tamoxifen resistance have never been reported before.We have the right to use datasets from Gene Expression Omnibus (GEO) by complying with all requirements according to each dataset. The Research Ethics Committee of Peking University Cancer Hospital & Institute waived the requirement for ethical approval of this analysis because the registry is a de-identified database.Several publicly available datasets were downloaded and analyzed to explore the prognostic value of CCNA2 in ER+ breast cancer patients and its potential role in tamoxifen resistance.GSE47561 contains expression data from 1570 breast cancer samples, of them, 514 ER+ patients with distant metastasis free survival (DMFS) data (114 of them are known to be treated with tamoxifen), 513 ER+ patients with recurrence free survival (RFS) data, 125 ER+ patients with disease free survival (DFS) and overall survival (OS) data, 167 ER- patients with DMFS data. Van cohort contains expression data from 226 ER+ breast cancer samples that with DMFS, DFS and OS data. Gyorffy dataset (256 samples) and GSE3494 (83 samples) are expression data from ER+ breast cancer samples that with DMFS data and are known to be treated with tamoxifen. ISDB3008 dataset contains expression data from 2795 breast cancer samples and is obtained through InsilicoMerging packageAmong all the datasets used in this study, GSE47561, GSE3494, GSE33366 and GSE26459 are obtained from Gene Expression Omnibus (GEO) dataset; van dataset and Gyorffy dataset were obtained from supplementary data of previous publications. For all the study subjects in this research, ER positivity was defined as greater than 10 fmol/mg tumor tissue and greater than 1% nuclear staining or immunohistochemistry (IHC) score of at least 3 for the biochemical and immunohistochemical assays, respectively.The mRNA expression profiling of all the samples in this study were performed on the Human U133A Gene Chip or Human genome U133 plus 2.0 platforms . GSE47561GSE33366 and GSE26459NULL computed with randomly assigned phenotypes. The nominal p value estimates the statistical significance of the enrichment score for a single gene set. A P value of zero (0.0) indicates an actual P value of less than 1/number-of-permutations. In the present study, we performed 1000 permutations. Significance was defined as a P value of less than 0.05. Analyses were performed using R 3.0.1 , GraphPad Prism 5.01  and SPSS version 21 .Clinical and gene expression profiling data were analyzed using standard statistical tests including the logrank test and unpaired t-test. In GSEA, we assess the significance of an observed enrichment score (ES) by comparing it with the set of scores ESBy analyzing gene expression profiles and corresponding clinical information of breast cancer patients from publicly available datasets, we found that high expression of CCNA2 confers poor distant metastasis free survival (DMFS), disease free survival (DFS), recurrence free survival (RFS) and overall survival (OS) in ER+ breast cancer patients , Fig. S1Specifically, CCNA2 overexpression is associated with poor distant metastasis survival in ER+ breast cancer cohorts GSE47561  = 0.3.All these data indicates that CCNA2 confers poor prognosis in ER+ breast cancer.Since high expression of CCNA2 confers poor DMFS, DFS, RFS and OS in ER+ breast cancer patients and tamoxifen is one of the most widely used drugs for the management of those ER+ patients, CCNA2 may play a potential role in tamoxifen resistance. To validate this hypothesis, several datasets were downloaded from publicly available datasets and analyzed. Results showed that high expression of CCNA2 was significantly associated with poor tamoxifen efficacy in ER+ breast cancer within GSE47561, Gyorffy et al. and GSE3494  indicated that CCNA2 expression was positively correlated with genes overexpressed in endocrine therapy resistant samples  = 0.006.All these data suggests that CCNA2 expression is correlated tamoxifen resistance.Next, we sought to explore how CCNA2 and available cancer drugs could influence each other. CCNA2-Drug interaction network was constructed in Cytoscape based on data from The Comparative Toxicogenomics Database (CTD) . Please The therapy of ER+ breast cancer, which represents more than 70% of breast tumors, is based on anti-hormonal compoundsNumerous studies have been performed, which combined endocrine therapy with agents that could modulate these mechanisms, so as to prevent the occurrence of tamoxifen resistanceCCNA2, a key regulator of cell cycle, is overexpressed in many human cancers, including breast cancer. However, the association between CCNA2 overexpression and tamoxifen resistance in ER+ breast cancer remains unclear. Our results show the significant prognostic power of CCNA2 in ER+ breast cancer progression and tamoxifen resistance. Importantly, high level of CCNA2 is correlated with tamoxifen treatment failure and poor DMFS. We might be able to stratify ER+ breast cancer patients by testing the mRNA expression level of CCNA2and decide when and how to use tamoxifen treatment in combination with appropriate therapeutic drug in the future. Moreover, we demonstrated that CCNA2 is down-regulated in MCF-7 tumor xenografts treated with tamoxifen compared with control in vivo. However, tamoxifen has no significant influence on CCNA2 expression in tamoxifen resistant subclones (P = 0.9783), which indicates CCNA2's potential role in tamoxifen resistance. Nevertheless, whether CCNA2 is a driver gene in tamoxifen resistance still requires experimental validation.Provided the high expression of CCNA2 is involved in the development of tamoxifen resistance, how to manage cancer patients with CCNA2 overexpression remains a great challenge. Here we show several drugs that could influence the expression of CCNA2 . For insAlthough much information about ER and cancer has been provided in the past three decades since the arrival of tamoxifen in the clinic, a lot more needs to be elucidated for favorable therapeutic outcomes. More concrete research outcomes will warrant the translational research that may lead to more efficient and safer treatment for breast cancer patients as well as women at high risk of advanced breast cancer.Taken together, this study indicates that CCNA2 expression may help monitoring tamoxifen efficacy. In addition, it suggests the relevance of CCNA2 in the development of tamoxifen resistance. Furthermore, it could provide guidance personalized therapies. Nevertheless, multi-center randomized controlled clinical trials and in vivo/in vitro experiments are still needed before its application in clinical settings.Figure S1Kaplan-Meier plot for DMFS (A), DFS (B and C), RFS(D) of ER+ breast cancer patients classified according to the tertile of CCNA2 expression levels. (E and F) Kaplan-Meier plot for OS of breast cancer pateints classified according to the tertile of CCNA2 expression level. Significance was assessed by logrank test.(TIF)Click here for additional data file.Figure S2(A and B) Kaplan-Meier plot for DMFS of tamoxifen treated breast cancer patients classified according to the tertile analysis of CCNA2 expression level, significance was assessed by logrank test. Gene set enrichment analysis of CCNA2 mRNA expression in relation to gene set up-regulated in endocrine resistant patients (C) and gene set down-regulated in tamoxifen resistant MCF-7 cells (D) using breast cancer expression profiles GSE47561.(TIF)Click here for additional data file.


ATP in the neuron. Neuronal ATP is used to drive the Na+/K+ATPase pumps, which maintain ionic gradients necessary for neuronal firing. When placed in the metabolic loop, the neuron exhibits sustained firing only when the stimulation current is more than a minimum threshold. For various combinations of initial neuronal [ATP] and external current, the neuron exhibits a variety of firing patterns including sustained firing, firing after an initial pause, burst firing etc. Neurovascular interactions under conditions of constricted vessels are also studied. Most models of cerebral circulation describe neurovascular interactions exclusively in the “forward” neuron→vessel direction. The proposed model indicates possibility of “reverse” influence also, with vasomotion rhythms influencing neural firing patterns. Another idea that emerges out of the proposed work is that brain's computations may be more comprehensively understood in terms of neuro-glial-vascular dynamics and not in terms of neural firing alone.We present a computational, biophysical model of neuron-astrocyte-vessel interaction. Unlike other cells, neurons convey “hunger” signals to the vascular network via an intervening layer of glial cells (astrocytes); vessels dilate and release glucose which fuels neuronal firing. Existing computational models focus on only parts of this loop (neuron→astrocyte→vessel→neuron), whereas the proposed model describes the entire loop. Neuronal firing causes release of a neurotransmitter like glutamate which triggers release of vasodilator by astrocytes via a cascade of biochemical events. Vasodilators released from astrocytic endfeet cause blood vessels to dilate and release glucose into the interstitium, part of which is taken up by the astrocyticendfeet. Glucose is converted into lactate in the astrocyte and transported into the neuron. Glucose from the interstitium and lactate (produced from glucose) influx from astrocyte are converted into  ATP) molecules ATP is used to fuel the pump activity necessary to restore ionic gradients necessary to sustain neuronal activity. In the present work, the above mentioned events in neuro-glial-vascular interaction have been modeled in an elaborate biophysical model  gradients required to sustain neuronal firing. The efflux of +K and influx of +Na during action potential propagation is reversed by Na+/K+ ATPase pump at the expense of ATP. Neuron conveys its “hunger signals”, both by direct action on the vessels by release of vasodilators like NO NMDA (N-methyl- D-aspartate) receptors leading to activation of neuronal nitric oxide synthase (nNOS) and subsequent catalysis of NO production NO diffuses across neuronal tissue and acts on smooth muscles to cause vasodilation GABA, acetylcholine, histamine, adenosine and ATP2+Ca oscillations in astrocytes which may be transmitted to neighboring astrocytes via gap junctions to elicit astrocyte-mediated vasodilation.Action potential propagation through neuron disturbs ionic (mGluR) on astrocyticprocesses by synaptic glutamate triggers a cascade of biochemical reactions leading to release of vasoactive molecules. Activation of mGluR increases cytosolic [2+Ca] 2A evoking production of arachidonic acid (AA) from membrane phospholipids. AA is precursor to various vasoactive molecules, including prostaglandins and epoxyeicosatrienoic acids (EETs) EAAT 1 and 2). Glutamate is co-transported with +Na resulting in activation of Na+/K+ ATPase pump. Elevation of [2+Ca]i leads to opening of large conductance 2+Ca-activated +K (BK) channels in astrocyte. BK channels allow release of +K ions from astrocyticendfeet which also compensates for +K uptake by Na+/K+ ATPase pump 2+-activated +K channels. Opening of Ca2+ activated +K channels makes the membrane potential more negative and consequently blocks the L-type voltage-gated 2+Ca channels 2+Ca]i and consequently suppresses phosphorylation of myosin light chain which is essential for maintaining contracted state of smooth muscle.Activation of metabotropic glutamate receptors (ATP by Na+/K+ ATPase pump stimulates glucose uptake and glycolysis in neuron. Hence release of glutamate in synapse triggers activity-dependent glucose uptake and glycolysis. Monocarboxylate transporters (MCT 1 and2) shuttle lactate to neuron  via interstitium which is converted to pyruvate by lactate dehydrogenase (LDH) enzyme. Pyruvate is metabolized via tricarboxylic acid (TCA) cycle to produce ATP both in neuron and astrocyte ATP produced in astrocyte is used for sustaining housekeeping functions and buffering of K+ ions. Na+/K+ ATPase pump is fueled by astrocytic ATP to buffer K+Na+ ions co-transported into the astrocyte along with glutamate molecules. Na+/K+ ATPase pump in neuron actively uptakes K+ ions and removes Na+ ions to restore the ionic gradients lost during action potential propagation.Consumption of From the above discussion it is clear that neurons, astrocytes and vessels form a closed loop information transfer system and astrocytes can be visualized as a hub, relaying biochemical signals between neurons and vessels in the forward direction, and conveying energy molecules from the vessels to the neurons in the reverse direction. To the best of our knowledge there is no existing model to simulate the entire information transfer system.fMRI) and Positron Emission Tomography (PET) measure hemodynamic response as an indicator of neuronal activity Although a model of neuron-astrocyte-vessel system has an obvious interest from basic science point of view, such a model also has an applied dimension, particularly in the area of functional neuroimaging 2+ oscillations were discovered in glial cells 2+ oscillations in myocytes of parenchymal arterioles in brain slices Glial function is often described as if it is under passive control of neural signals. However, low-frequency spontaneous CaK+ in the interstitium. Ion exchange between neuron and interstitium described in the proposed model is designed on the lines of 3IP-mediated release of EET which is a potent vasodilator. In the cerebrovascular network model of Boas et al. (2008) Various models have been proposed to describe various components of neuro-glial-vessel interactions. Kager et al. (2000) The outline of the paper is as follows. Section 2.0 describes the proposed model of neuron-astrocyte-vessel loop. Simulation results are described in Section 3.0 and discussed in the final section.Our model of mammalian system consists of four compartments - neuron, astrocyte, vessel and interstitium. Individual compartments were developed separately and results were matched with existing literature. To keep the presentation simple, model equations are given in the Appendix S.A; only a general description of mechanisms incorporated in each component is presented below. The integrated system consisting of 89 equations (Appendix S.A) is programmed and simulated in Matlab® 7.nτ) by a factor nτ, firing rates in range of 20 Hz to 80 Hz were obtained which corresponds to gamma range firing rates.The neuron model used is a single-compartment model closely resembling the Hodgkin-Huxley model, with the primary difference being that the Nernst potentials are not constant and vary as a function of intracellular and interstitial ion concentrationsat physiological temperature of 37°C. The shortcoming of model was that it could not be used to generate firing rate below 100 Hz since it is a resonate-and-fire type neuron model K+ buffering, Na+/K+ ATPase action based on ATP – these components of the proposed neuron model were modeled after Variation of Nernst potentials as a function of ion concentrations, Na,T and INa,P), potassium currents , and N-methyl-D-aspartate (NMDA) receptor-controlled currents (INMDA) at appropriate regions of the model cell; these currents were not included in the proposed model. The proposed minimal model of neuron is mere extension of Hodgkin-Huxley model which explains the difference in firing rates between The model of Kager et al. (2000) Furthermore, the model for quantal release of glutamate described by The simulated results were found to be consistent with the results published by Lee et al. (2009) The series of events that occur in the neuron starting from current injection to neurotransmitter release are described by equations in Appendix S.A.1.Glutamate concentration in synapse is detected by astrocytes, which in turn release vasodilators. The model of 3IPinside astrocyte 3IP concentration modulates 2Ca+ release from internal calcium stores 2Ca+ concentration crosses a threshold value, it produces EET which is released from astrocyte mGluR on astrocyte detects synaptic glutamate and facilitates release of This model was simulated with various concentrations of glutamate pulses for 2 s as performed by Bennett et al. (2008) EET profile in our model can be attributed to omission of diffusion kinetics. Instead a degradation term is introduced for removal of EET (S.A.2) which follows first order Michaelis–Menten kinetics. The parameters of this degradation term are fine-tuned to obtain approximate match with the control experiment. Furthermore, the effect of EET concentration on smooth muscle is controlled by passing it through a sigmoidal function.These results are matched with the simulation results from EET is a potent vasodilator that opens theK+ channels of vascular smooth muscle membrane releasing K+ ions from the smooth muscle cell which hyperpolarizes the smooth muscle cell and leads to muscle relaxation, vessel dilation and increased glucose flux from the vessel mGluR on astrocyte, culminating in the release of EET, are described by equations in Appendix S.A.2.SMV)has a linear relation with vessel radius. The maximum SMV of −30 mV (resting condition) corresponds to the minimum vessel radius of 8 µm (contracted state) and minimum VSM of −75 mV (hyperpolarized state) corresponds to maximum vessel radius of 30 µm (dilated state). Vessel dilation enhances glucose flux into the astrocytes and initiates metabolic feedback. Mangia et al., (2009) GLUTs) and monocarboxylate transporters (MCT) for glucose and lactate respectively. In the model of ATP in neuron, are described by equations in Appendix S.A.3.We assume that smooth muscle cell membrane voltage  cycle to generate ATP . The neuronal firing continues as the metabolic feedback diminishes. In this case, the ATP consumption is substantially more than the production rate of ATP. Once the ATP concentration becomes too low to sustain pump activity, the neuron tends to depolarize as the ionic gradients across the membrane are exhausted.A steep fall in neuronal 2 current which is greater than the threshold current of 0.07 mA/cm2, however, the initial [ATP] is 20 µM. Once the stimulus is removed the membrane potential stabilized at resting potential , even though 2Ca+ build up is roughly half in comparison to the case of continuous firing, the EET released  follows slow channel activation dynamics due to which there is a delay in release of cytosolic 2Ca+. Action of EET on smooth muscle cells causes their hyperpolarization and as a consequence the vessel dilates  are constant. But in the present model, since the ionic concentrations vary, even constant application of sub-threshold currents tend to depolarize the membrane, which causes reduction in NaE  the neuron maintains resting potential, whereas when the initial ATP reserve is low, for instance 5 µM, the neuron switches to bursting behavior signaling demand for energy.When the neuron is stimulated with a stimulation current greater than threshold current of 0.07 mA/cmATP reserve essential for maintaining resting potential is exhausted. If the initial ATP is adequate, the neuron will continued to remain in resting condition whereas if the initial ATP is low, the neuron resorted to bursting. While bursting, the neuron must fire for a duration sufficient to cause elevation of cytosolic [2Ca+] beyond a threshold in astrocyte, initiating release of EET as signal for vasodilation and subsequent metabolic feedback. However if neuronal bursting fails to initiate metabolic feedback, the ionic gradients will be destroyed, rendering neuronal firing impossible.For a sub-threshold stimulation pulse of duration 1 s, the neuron remains in polarized state until neuronal vasomotionIn Section 3.5, effects of vessel oscillations on neuronal firing pattern are studied. Small vessels are known to produce low-frequency, spontaneous oscillations known as ATP concentration and stimulation current determines the duration of initial pause before the onset of neuronal firing. Under metabolic stress, the neuron observes a relatively longer delay before the onset of firing for lower value of sub-threshold stimulation current. With increase in initial [ATP] the neuron exhibits a transition from bursting to firing with initial pause  and inserted it in the complete neuron-astrocyte-vessel loop (Supplement S). The astrocyte and vessel components of the model are not altered. Simulations show that the new model shares many features with the model described in the previous sections. Neural firing patterns depend on the input stimulation current and initial [ATP]. In the case of firing with an initial pause, the pause duration is found to depend on initial [ATP]. Under conditions of metabolic deficiency (low [ATP]), and constricted vessel, vasomotion rhythms are seen to influence amplitude of neural action potentials. However, one key difference between the current model and the CA1-neuron-based model is that the neuron does not show bursting behavior; only continuous firing and firing with initial pause are observed. (See Supplement section S2 for more details).NO is not incorporated. Action of vasoactive molecules released by astrocytes upon activation which are essentially arachidonic acid derivatives (except EET) has been neglected but can be implemented to fit experimental data. Furthermore, to simulate vessel dynamics we used a lumped model and there is no explicit representation of endothelium and smooth muscle dynamics. Metabolic feedback due to vessel dynamics does not take into account diffusive and convective transport of glucose and lactate through various compartments. Also, this model does not incorporate oxygen delivery to various tissues and assumes that sufficient oxygen is available for oxidation of glucose and lactate in neuron and astrocyte.This model captures the essential behavior of each compartment even though all the biophysical pathways are not considered. Neurons uptake neurotransmitters via astrocyte vascular computation. These intriguing possibilities can be the subject matter of future computational and experimental studies on networks underlying cerebral circulation.Furthermore, compartmental modeling allows creation of a network level model which may provide deeper insight into how brain functions. The network level model will further encourage developing models for simulating neurovascular disorders and investigating metabolic basis of neural disorders. Such network models would suggest that brain's computation as being performed, not solely by a network of neurons, but by three networks – neural, astrocytic and vascular - working in tandem. Recent work shows that astrocytes also exhibit “tuned responses” to visual stimuli, and therefore may be thought to be involved in information processing Supplement S1Model with hippocampal CA1 neuron in neuron-astrocyte-vessel loop.(DOCX)Click here for additional data file.Appendix S1List of equations and constants.(DOCX)Click here for additional data file.Figure S1(TIF)Click here for additional data file.Figure S2(TIF)Click here for additional data file.Figure S3(TIF)Click here for additional data file.Figure S4(TIF)Click here for additional data file.Figure S5(TIF)Click here for additional data file.Figure S6(TIF)Click here for additional data file.


Protective antibodies play a critical role in an effective HIV vaccine; however, eliciting antibodies to block infection by viruses from diverse genetic subtypes remains a major challenge. As the world’s most populous country, China has been under the threat of at least three major subtypes of circulating HIV-1 viruses. Understanding the cross reactivity and specificities of serum antibody responses that mediate broad neutralization of the virus in HIV-1 infected Chinese patients will provide valuable information for the design of vaccines to prevent HIV-1 transmission in China. Sera from a cohort of homosexual men, who have been managed by a major HIV clinical center in Beijing, China, were analyzed for cross-sectional neutralizing activities against pseudotyped viruses expressing Env antigens of the major subtype viruses  circulating in China. Neutralizing activities in infected patients’ blood were most capable of neutralizing viruses in the homologous subtype; however, a subset of blood samples was able to achieve broad neutralizing activities across different subtypes. Such cross neutralizing activity took 1–2 years to develop and CD4 binding site antibodies were critical components in these blood samples. Our study confirmed the presence of broadly neutralizing sera in China’s HIV-1 patient population. Understanding the specificity and breadth of these neutralizing activities can guide efforts for the development of HIV vaccines against major HIV-1 viruses in China. Infection with HIV-1 in humans leads to the production of serum antibodies against HIV’s envelope glycoprotein (Env). Several studies have systematically analyzed HIV-1 positive sera and discovered that not all HIV-1 infected patient sera achieve the same potency or breadth of neutralizing activities against primary HIV-1 viruses and only a fraction of infected sera can achieve broad neutralization against viruses from different genetic subtypes However, HIV-1 isolates circulating throughout the world demonstrate great diversity. In China’s AIDS endemic, there are three major HIV-1 subtypes: Thai-B, CRF07/08_BC, and CRF01_AE In the current study, sera from a cohort of homosexual men were analyzed for neutralizing activities against pseudotyped viruses expressing Env antigens representing the major subtype viruses circulating in China. Key specificities responsible for the neutralizing activities have also been mapped in this exploratory study. Our study confirmed the presence of broadly neutralizing sera in this patient population and provided a scientific basis to develop HIV vaccines to achieve broad protection against major HIV-1 viruses in China. Such information is also useful for researchers to understand the global pattern of neutralizing antibodies (NAb) in HIV-1-infected patients, possibly leading to better control of the AIDS epidemic throughout the world.Blood samples were collected from 36 homosexual male patients from the AIDS Clinic at Youan Hospital, Beijing, China in the summer of 2009. These patients had various lengths of confirmed diagnosis of HIV-1 infection.Written informed consent was obtained from all study participants. Sera and plasma samples were collected from the study subjects. They were diagnosed with HIV-1 infection but had not been on regular antiviral treatment prior to the start of the study. This study was carried out in strict accordance with the requirements for clinical studies established by the Capital Medical University, China. The protocol was approved by Youan Hospital’s Ethics Review Committee.TCA ACT CAA -3′), and inner anti-sense primer TR2 (5′ - ATA TCT CCT CCT CCA ggT CTg AA -3′). The PCR products were sequenced by Invitrogen . The genotype of each envelope was determined using the PhyloPlace software from the US NIH HIV sequence database.RNA was extracted from patient plasma samples using a QIAmp Viral RNA kit . The envelope gene fragment covering C2∼V5 was amplified with the access RT-PCR system . Specific primers  used to The neutralization assay was performed using a single round of infection in TZM-bl cells in 96-well plates, as previously described To examine the potential contribution of V3-specific or membrane proximal external region (MPER)-specific antibodies to the neutralizing abilities of patient sera, V3 or MPER peptide absorption of HIV-1 patient sera was performed prior to neutralization, as described previously In order to analyze the antibody specificity of patient sera, HIV-1 viral capture competition assays were performed against known HIV-1 neutralizing monoclonal antibodies, including CD4bs-specific b12, VRC01 and VRC03 Fisher’s exact test was used to determine the statistical differences in NAb among patient sera collected at various time points and their subtype specificities. Correlations between neutralization breadth and V3 peptide absorption or between neutralization breadth and antibody titers competing with neutralizing mAb IgG1 b12 were analyzed using Spearman’s rank correlation.Blood samples were collected from 36 male homosexual patients with a well-documented history of HIV-1 infection and most have the dates of their first diagnosis of HIV infection. Samples were collected during one of the patients’ regular clinic visits as a part of routine monitoring of their disease condition. The average age of this group was 30 years with an average blood CD4+ T cell count of 502 . The perenv gene was amplified from patient plasma by reverse transcription (RT) and nested-polymerase chain reaction (PCR), using primers described in env gene sequences were analyzed for HIV-1 subtypes based on the NIH HIV sequence database. The majority of env gene sequences isolated from this group of patients belonged to subtypes B/ThaiB, BC, and AE, which are the three major circulating HIV-1 subtypes in China , three  could only neutralize one virus, and the remaining six samples  could not neutralize either virus. Among these nine samples that could not neutralize either sensitive virus, all, except one (NJ016), had a history of infection for less than 1-year, indicating that sera from patients with a shorter time period after infection were unable to generate effective neutralizing activities. In support of this view, the only two serum samples (NJ015 and NJ021) that could not neutralize any viruses expressing primary Env in our analysis were among these nine samples. The remaining seven samples had neutralizing activities against no more than 1–3 primary Env pseudotyped viruses.Among 34 serum samples where For the 25 patient sera samples that were able to neutralize both of the sensitive viruses, only two (NJ006 and NJ035), both from patients with less than 1-year infection, could not neutralize any other primary Env pseudotyped viruses, suggesting that once NAb are elicited, sera can neutralize not just the sensitive viruses, but also additional primary Env viruses.As reported in the past, these serum samples showed preferred neutralization against viruses from the autologous subtype . By usinWhen a more quantitative analysis was conducted by combining all positive neutralizing activities together (both red and yellow (50–74% inhibition) boxes, serum samples from subtype B patients had the highest percentage of neutralization against subtype B viruses, which was significantly higher than the subtypes BC (p = 0.002) and AE (p = 0.015) . SubtypeA subset of patient sera showed quite broad neutralizing activities . Among 1Since many serum samples included in the current study did not show broad NAb responses until later in infection, it was interesting to examine whether a longer period of infection in the host may be needed in order to elicit broad NAb responses. Using subtype B virus-infected patient sera as an example, In order to further understand the specificity of anti-Env antibody responses that may be responsible for the breadth of NAb against primary HIV-1 viruses in this cohort of HIV-1-infected patients, two types of assays were conducted. In the first assay, serum samples were treated with HIV-1 subtype B consensus V3 peptide before the same neutralization study was conducted against SF162, which can be neutralized by majority of the patient sera in this study. Decreases in NAb responses upon the treatment of this V3 peptide were observed although a significant number of sera only had a moderate drop (20–40%) with V3 peptide treatment . An overNine serum samples with relative broad NAb activities were further treated with HIV-1 Group M consensus MPER peptides (six peptide pool spanning both 2F5 and 4E10 epitopes) before the same neutralization study was conducted against SF162. Decreases in NAb activities upon treatment of MPER peptides were only observed in patient NJ040 and not in the other eight sera samples  . To contThe next study examined the antibody components in patients’ sera that can compete against b12, a well-known CD4 binding site (CD4bs) neutralizing monoclonal antibody. Here, a significant correlation was observed between titers of b12-competing antibody responses and the number of primary viruses being neutralized  .To further support this finding, four broadly neutralizing sera from the current patient cohort were analyzed for the presence of antibodies that may compete against two newly discovered broadly neutralizing human mAbs, VRC01 and VRC03, which also target the CD4bs . Two serTo further examine the specificity of patient sera with relative broad NAb activities, we conducted mAb competition assays against glycan-specific mAb 2G12 and quaternary epitope-specific mAb PG9 . Among nUsing the same virus capture competition assay, two mAbs recognizing glycosylated Env antigens and quarternary structure (2G12 and PG9) were tested to compete against HIV-infected sera . Except In this study, we reported neutralizing activities in a group of HIV-1-infected homosexual male patients from Beijing, China. Like many other countries, China has not escaped the AIDS endemic that has occurred over the past two decades. While initially HIV-1 infections have been mainly observed in injection drug users in certain border provinces, the latest data suggest an increase in HIV-1 infections in urban areas through sexual transmission It is well-documented in the literature that the genetic makeup of HIV-1 in China is quite complex due to the way various viral subtypes infect different risk groups within the population. Three main subtypes have dominated HIV-1 infections in China including CRF07/08_BC (BC), subtype B’ (B’), and CRF01_AE (AE). Previous studies have suggested that each of these subtypes was associated with a particular mode of transmission Co-circulation of multiple subtypes of HIV-1 is a challenge to vaccine development in China. Furthermore, China offers a unique geographic model to examine cross-neutralizing antibody activities against well-defined subtypes in the same region. While early studies did not reach a consensus on the presence of subtype-specific humoral responses in HIV-1-infected people However, our data also suggest that such subtype specificity is not absolute. Many sera showed various degrees of cross-neutralizing activities against viruses from other subtypes. Between 40–50% of the tested sera were able to neutralize viruses in more than three subtypes but the real broadly neutralizing sera, which could neutralize more than 50% of the virus panel, represented only 15% of the sera; results from this study also showed that it will take more than 1–2 years’ time to develop such breadth. Previous studies on HIV-infected patients in China either did not pay particular attention to identifying broadly neutralizing sera It is particularly critical to understand the specificity of antibody responses responsible for the induction of broadly NAb against viruses in all three subtypes. Our data suggest that there are antibodies against more than one neutralizing epitope present in these sera and CD4bs antibodies may play a critical role in the breadth of neutralizing activities. Our results did not identify antibodies similar to either 2G12 or PG9. Peptide absorption using a MPER peptide was not able to reduce the NAb identified in our HIV patient sera. However, the current study is only exploratory and detailed studies are needed to examine neutralizing activity against pseudotyped viruses with selected mutations on those existing Env to further map out critical sites involved in neutralizing viruses in both autologous and heterologous subtypes; such information will guide the design of Env vaccine formulations to elicit broadly neutralizing activities.Our data also point to a major challenge in eliciting NAb responses, i.e. the lengthy period required to detect such antibody responses in naturally-infected hosts. We realize the limitation of the cross-sectional nature of our data as we did not follow these patients over time with multiple sampling time points. It is not clear whether such a long process is needed to let the viruses evolve in order to allow for the development antibodies against a more diverse viral population, or whether a long B-cell maturation process is needed to produce high-quality antibody responses against the same key epitopes. In the former situation, future vaccines will need to be based on “polyvalent’ principles to expand the breadth of immunogens. In the latter scenario, more effective immunization approaches may be needed, such as the use of adjuvants to selectively accelerate the necessary steps for the production of high quality antibodies. Both approaches can be tested in future vaccine studies to determine if similar cross-subtype antibody responses can be reproduced.Figure S1Phylogenetic tree analysis of the C2–C4 region of HIV-1 Env genes included in the current study. Each sequence included in the analysis is labeled. Red-colored branches show representative reference HIV-1 env gene sequences from GenBank. HIV-1 subtype clusters are marked. A neighbor joining tree model was used for the analysis.(DOC)Click here for additional data file.Table S1Primers for RT-PCR.(DOC)Click here for additional data file.Table S2Cohort of HIV-1 infected patents.(DOC)Click here for additional data file.Table S3Env gene subtyping.(DOC)Click here for additional data file.


Gestating F0 generation females were exposed to dioxin during fetal day 8 to 14 and adult-onset disease was evaluated in F1 and F3 generation rats. The incidences of total disease and multiple disease increased in F1 and F3 generations. Prostate disease, ovarian primordial follicle loss and polycystic ovary disease were increased in F1 generation dioxin lineage. Kidney disease in males, pubertal abnormalities in females, ovarian primordial follicle loss and polycystic ovary disease were increased in F3 generation dioxin lineage animals. Analysis of the F3 generation sperm epigenome identified 50 differentially DNA methylated regions (DMR) in gene promoters. These DMR provide potential epigenetic biomarkers for transgenerational disease and ancestral environmental exposures. Observations demonstrate dioxin exposure of a gestating female promotes epigenetic transgenerational inheritance of adult onset disease and sperm epimutations.Environmental compounds can promote epigenetic transgenerational inheritance of adult-onset disease in subsequent generations following ancestral exposure during fetal gonadal sex determination. The current study examined the ability of dioxin  to promote epigenetic transgenerational inheritance of disease and DNA methylation epimutations in sperm Epigenetic transgenerational inheritance involves the germline transmission of an altered epigenome and phenotypes across generations in the absence of direct environmental exposures The current study was designed to investigate the potential that dioxin  promotes epigenetic transgenerational inheritance of adult onset disease. In rodents TCDD has a half-life of weeks and causes liver disease, weight loss, thymic atrophy and immune suppression. In humans direct dioxin exposure influences chronic diseases, lymphomas and leukemias Animal models have been used to study the toxicological effects of dioxin. Dioxin has been shown to produce cleft palates and kidney malformations in newborn mice Environmental chemicals (fungicide vinclozolin and pesticide methoxychlor) were initially found to promote epigenetic transgenerational inheritance of adult onset diseases following ancestral exposure The epigenetic mechanisms involved in the transgenerational inheritance of disease have been previously reviewed The epigenetic transgenerational action of dioxin administered to F0 generation female rats transiently during days 8 to 14 of gestation was investigated. The F1 generation animals were bred to generate the F2 generation and the F2 generation bred to generate the F3 generation as previously described One of the major diseases/abnormalities observed in dioxin lineage males was kidney disease. Kidney disease was characterized by the presence of an increased number of proteinaceous fluid filled cysts, reduction in size of glomeruli and thickening of Bowman’s capsules . PreviouAs previously reported As previously reported The F1 and F3 generation rats did not present any change in the incidence of tumor development . The DMR are on average 800 bp in size. The chromosomal locations of these DMR are presented in As previously described, the transgenerational effects of dioxin on the sperm epigenome are unique The current study demonstrates that dioxin (TCDD), an environmental toxicant and contaminant present in herbicides such as Agent Orange, can promote epigenetic transgenerational inheritance of diseases in unexposed progeny of the F0 generation females exposed during gestation. These observations extend previous studies in mice Due to the bioaccumulation of dioxin and up to decade long half-life in humans, any woman becoming pregnant even 20 years after dioxin exposure runs the risk of transmitting dioxin effects to her fetus and later generations. A generational study in the Seveso Italy exposed population supports this concept demonstrating health effects in progeny born 25 years following the exposure This study did not use toxic doses of dioxin, but used only pharmacological doses based on 0.1% of the oral LD50 dose for dioxin. Therefore, no major toxic effects of dioxin were observed. However, the dose and route of administration used in the current study does not allow risk assessment of dioxin exposure. The objective of the study was to investigate if exposure to TCDD could promote epigenetic transgenerational inheritance of disease/abnormality phenotypes, and not to assess environmental risk of exposure to dioxin. These observations can now be used in future studies with appropriate modes of administration and doses to design more effective risk assessment analysis. However, the current study demonstrates the potential of dioxin to promote epigenetic transgenerational inheritance of disease.In the current study, the transgenerational diseases/abnormalities observed includes kidney disease, ovary disease/abnormality, and pubertal abnormalities. Kidney disease incidence was higher in the transgenerational F3 generation dioxin lineage males. Chronic kidney disease in humans is correlated with high dioxin levels As previously observed Pubertal abnormalities were increased only in female F3 generation animals of dioxin lineage. In an earlier study The molecular mechanism of epigenetic transgenerational inheritance of phenotypes involves the reprogramming of the germline epigenome during male gonadal sex determination Transgenerational diseases are promoted by many environmental compounds All experimental protocols for the procedures with rats were pre-approved by the Washington State University Animal Care and Use Committee . Female and male rats of an outbred strain Hsd:Sprague Dawley®™SD®™ (Harlan) at about 70 and 100 days of age were fed ad lib with a standard rat diet and ad lib tap water for drinking. To obtain time-pregnant females, the female rats in proestrus were pair-mated with male rats. The sperm-positive (day 0) rats were monitored for diestrus and body weight. On days 8 through 14 of gestation 2 inhalation for tissue harvest. Body and organ weights were measured at dissection time. Testis, epididymis, prostate, seminal vesicle, ovaries, uterus and kidney were fixed in Bouin’s solution (Sigma) and 70% ethanol, then processed for paraffin embedding by standard procedures for histopathology examination. Five-micrometer tissue sections were made and were either unstained and used for TUNEL analysis or stained with H & E stain and examined for histopathology. Blood samples were collected at the time of dissection, allowed to clot, centrifuged and serum samples stored for steroid hormone assays.Rats at 1-year of age were euthanized by COTestis sections were examined by Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay . Sections were deparaffinized and rehydrated through an alcohol series. They were deproteinized by Proteinase K , washed with PBS and then 25 µl of the enzyme-label solution mix was applied and incubated at 37°C for 90 min. After PBS washes, slides were mounted and kept at 4°C until examination in a fluorescent microscope in dark field. Both testis sections of each slide were microscopically examined to identify and to count apoptotic germ cells by the bright fluorescence.All histopathology was examined in randomly selected animals by three independent observers. Testis histopathology criteria included the presence of a vacuole, azoospermic atretic seminiferous tubule and ‘other’ abnormalities including sloughed spermatogenic cells in center of the tubule and a lack of a tubule lumen. Prostate histopathology criteria included the presence of vacuoles, atrophic epithelial layer of ducts and hyperplasia of prostatic duct epithelium. Kidney histopathology criteria included reduced size of glomerulus, thickened Bowman’s capsule and the presence of proteinaceous fluid-filled cysts. A cut-off was established to declare a tissue ‘diseased’ based on the mean number of histopathological abnormalities plus two standard deviations from the mean of control tissues by each of the three individual observers. This number was used to classify rats into those with and without testis, prostate or kidney disease/abnormality in each lineage. A rat tissue section was finally declared ‘diseased’ only when at least two of the three observers marked the same tissue section ‘diseased’. The proportion of rats with obesity or tumor development was obtained by accounting those that had these conditions out of all the animals evaluated. The number of animals per litter (litter representation) mean ± SEM used for the control versus dioxin lineage comparisons for each specific disease/abnormality was found not to be statistically different (p>0.05). Therefore, no litter representation differences or litter bias was detected for any of the specific disease/abnormality assessed.th section of each pair of ovaries was stained with hematoxylin and eosin and three stained sections (150 µm apart) through the central portion of the ovary with the largest cross sections being evaluated. Ovary sections were assessed for two histopathologies, primordial follicle loss and polycystic ovary disease. Primordial follicle loss was determined by counting the number of primordial follicles per ovary section and averaging across three sections. An animal was scored as having primordial follicle loss if the primordial follicle number was less than that of the control mean minus two standard deviations. Primordial follicles had an oocyte surrounded by a single layer of either squamous or both squamous and cuboidal granulosa cells Every 30Onset of puberty was assessed in females by daily examination for vaginal opening from 30 days of age and in males by balano-preputial separation from 35 days of age. For identifying a rat with a pubertal abnormality the mean from all the rats in control lineage evaluated for pubertal onset was computed and its standard deviation calculated. A range of normal pubertal onset was chosen based on the mean ±2 standard deviations. Any rat with a pubertal onset below this range was considered to have had an early pubertal onset and any rat with a pubertal onset above this range was considered to have had a delayed pubertal onset. The proportion of rats with pubertal abnormalities was computed from the total number of rats evaluated.A table of the incidence of individual diseases/abnormalities in rats from each lineage was created and the proportion of individual disease, total disease and multiple disease incidences was computed. For the individual diseases, only those rats that showed a presence of histopathology (plus) or absence of disease (minus) are included in the computation, Supplemental g and stored in fresh buffer at −20°C until processed further. Sperm heads were separated from tails through sonication following previously described protocol (without protease inhibitors) The epididymis was dissected free of connective tissue, a small cut made to the cauda and placed in 5 ml of F12 culture medium containing 0.1% bovine serum albumin for 10 minutes at 37°C and then kept at 4°C to immobilize the sperm. The epididymal tissue was minced and the released sperm centrifuged at 13, 000×The comparative MeDIP-Chip was performed with Roche Nimblegen’s Rat DNA Methylation 3×720 K CpG Island Plus RefSeq Promoter Array, which contains three identical sub-arrays, with 720,000 probes per sub-array, scanning a total of 15,287 promoters . Probe sizes range from 50–75 bp in length with the median probe spacing of 100 bp. Three different comparative (MeDIP vs. MeDIP) hybridization experiments were performed (3 sub-arrays) for dioxin lineage versus control, with each subarray encompassing DNA samples from 6 animals (3 each from dioxin and control). MeDIP DNA samples from experimental lineages were labeled with Cy3 and MeDIP DNA samples from the control lineage were labeled with Cy5.The MeDIP = PCR was used to confirm the MeDIP-Chip analysis observations using two genes. The MeDIP genomic DNA was used for a semiquantitative PCR involving 30 cycles and primers specific to the DMR sites. The genes and primers used were:Hdac3, 3′TGGCGTATTTCTACGACCCC, 5′GGAATGTTTCCGGTGCCTTC and.Npc2, 3′AGAATGCTTCCACTTGCCGA, 5′CTCACCGCAGTCCTTGAAGT.The PCR density for control versus dioxin lineage F3 generation sperm MeDIP samples from three different experiments were determined and normalized for fold change. A mean ± SEM was determined and statistical differences assessed with a U-Mann Whitney analysis.−5. The statistically significant differential DNA methylated regions were identified and P-value associated with each region presented. Each region of interest was then annotated for gene and CpG content. This list was further reduced to those regions with an average intensity value exceeding 9.5  and a CpG density ≥1 CpG/100 bp.The bioinformatic analysis was performed as previously described http://www.genome.jp/kegg/pathway.html).Associations between genes containing DMR and particular physiologic cellular processes were determined by an automated, unbiased survey of published literature using Pathway Studio™ software . Signaling pathway enrichment with genes containing DMR was determined by querying the library of KEGG pathways . All treatment differences were considered significant if P value was less than 0.05.Figure S1A. Control and dioxin F3 generation lineage serum testosterone concentrations. Testosterone concentrations (ng/dl) in F3 generation control and dioxin lineage male rats. B. Serum estradiol concentrations in proestrus-estrus in F3 generation control and dioxin lineage females. C. Serum estradiol concentrations in diestrus in F3 generation control and dioxin lineage females. D. Testicular spermatogenic cell apoptosis. Number of apoptotic germ cells normalized to control means in control (open bars) and dioxin (black bars) lineage (**P<0.01).Dioxin and transgenerational endocrine effects. (PDF)Click here for additional data file.Table S1(PDF)Click here for additional data file.Table S2(PDF)Click here for additional data file.Table S3(PDF)Click here for additional data file.Table S4(PDF)Click here for additional data file.


High protein (HP) diets are suggested to positively modulate obesity and associated increased prevalence of non-alcoholic fatty liver (NAFLD) disease in humans and rodents. The aim of our study was to detect mechanisms by which a HP diet affects hepatic lipid accumulation.To investigate the acute and long term effect of high protein ingestion on hepatic lipid accumulation under both low and high fat (HF) conditions, mice were fed combinations of high (35 energy%) or low (10 energy%) fat and high (50 energy%) or normal (15 energy%) protein diets for 1 or 12 weeks. Effects on body composition, liver fat, VLDL production rate and the hepatic transcriptome were investigated.Mice fed the HP diets displayed a lower body weight, developed less adiposity and decreased hepatic lipid accumulation, which could be attributed to a combination of several processes. Next to an increased hepatic VLDL production rate, increased energy utilisation due to enhanced protein catabolic processes, such as transamination, TCA cycle and oxidative phosphorylation was found upon high protein ingestion.Feeding a HP diet prevented the development of NAFLD by enhancing lipid secretion into VLDL particles and a less efficient use of ingested calories. Within the context of the metabolic syndrome clear links are demonstrated between non-alcoholic fatty liver disease (NAFLD) and insulin resistance, type 2 diabetes mellitus and obesity The amount of hepatic lipids can be modified by the macronutrient composition of the diet by modulating hepatic fatty acid uptake, lipogenesis, fatty acid oxidation and triglyceride secretion de novo lipogenesis, nutrient oxidation and VLDL secretion.The present study in mice aims at analysing mechanisms reducing hepatic lipid content in response to a HP diet. Mice were fed different combinations of high fat and high protein diets for 1 or 12 weeks to investigate the acute and long term effect of such a diet on body adiposity, liver fat, VLDL production rate and hepatic gene expression. We hypothesise that augmenting the amount of dietary protein affects liver lipid uptake, All animal experiments were approved by the Animal Experimentation Board at Wageningen University (record #2010017) and carried out according to the guidelines of the European Convention of Vertebrate Animals Used for Experimentation, under European Council Directive 86/609/EEC dated November, 1986.ad libitum access to their habitual diet. The day of sacrifice, mice received the calibrated meal at the habitual time and were anaesthetised with Isoflurane 2 hours later. Blood was taken by orbital puncture and plasma was stored. Liver and epididymal adipose tissue were removed, weighed and snap frozen in liquid nitrogen. Tissue was stored at 80°C until further analysis.Male C57BL/6J mice aged 8 weeks were purchased from Charles River  and housed in pairs in the light and temperature-controlled animal facility of Wageningen University . At 10 weeks of age, mice were stratified according to their bodyweight into 9 diet groups (n = 10 per group). Diets were provided as powder in specifically designed food cups to minimise spillage. All groups received a low fat-normal protein diet  as run-in of two weeks prior to the dietary intervention. One group was sacrificed after the run-in period to serve as reference group. The other groups either continued on the LF-NP diet or were switched to a high fat-normal protein , a low fat-high protein  or a high fat-high protein  diet which they received for 1 or 12 weeks. For diet compositions see Liver dry weight was determined by lyophilisation of samples at −40/−50°C and 10 Torr. Water content was calculated subsequently. Liver protein and triglycerides were determined in 5% liver homogenates prepared in buffer containing 2 mM EDTA and 10 mM Tris at pH 7.5 using enzymatic colorimetric assays . For oil red O/haematoxylin staining frozen livers were sectioned at 5 µM in a Cryostat , mounted on glass slides and air-dried for 30 minutes at room temperature. Sections were fixed in 4% buffered formaldehyde for 10 minutes prior to staining which was performed using standard protocols. Pictures of liver sections were taken with a light microscope  at 20x magnification.VLDL production was determined on an additional set of mice (n = 9/group) after feeding the 4 diets for 1 week. Diets, treatment, food intake and body weight development were highly similar to the first set (data not shown). The experiment started at the beginning of the dark phase. After 12 hours of fasting mice were anaesthetised by an intra-peritoneal injection of hypnorm and dormicum . Subsequently the LPL-inhibitor tyloxapol  in saline) was injected intra-orbitally. Plasma was sampled from tail bleedings and triglyceride and glycerol concentrations were measured every 30 minutes for 180 minutes to determine VLDL production. VLDL-TG production rate was determined from the slope of the curve from all individual mice.Plasma triglyceride concentration was determined using an enzymatic colorimetric test . Concentrations were corrected for free plasma glycerol . For determination of plasma β-hydroxybutyrate, the enzymatic assay β-Hydroxybutyrate LiquiColor  was used. Plasma leptin was measured using the Leptin (Mouse/Rat) ELISA .All data are expressed as means ± SD. Effects of increased dietary protein and fat within one time point were tested for statistical significance using general linear model with HP and HF as fixed factors in PASW Statistics 18.0 software . Mean values were compared to the reference group using one way ANOVA and Dunnett as post hoc test. Body weight was tested using a linear mixed model with diet as main and time as repeated factor. Least significant difference (LSD) test was used as post hoc test. Effects of the diets on food intake and VLDL production rate were tested using one way ANOVA. For all tests, p-values ≤0.05 were considered to be statistically significant.Total RNA was extracted from liver tissue with TRIzol reagent  and purified using SV total RNA isolation system . Concentrations of RNA samples were determined on a NanoDrop ND-1000 UV-Vis spectrophotometer . Total purified liver RNA of 5 mice per group was subsequently pooled at a concentration of 500 ng/mouse. Quality of the pooled RNA was assessed on an Agilent 2100 bioanalyzer with the 6000 Nano Kit using the Eukaryote Total RNA Nano assay . RNA was judged as being suitable for array hybridization only if samples showed intact bands corresponding to the 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products and had a RNA integrity number (RIN) above 8.0. The Ambion WT Expression kit  in conjunction with the Affymetrix GeneChip WT Terminal Labelling kit  was used for the preparation of labelled cDNA from 100 ng of total RNA without rRNA reduction. Samples were hybridized on Affymetrix GeneChip Mouse Gene 1.0 ST arrays. Quality control and normalisation were performed using Bioconductor packages integrated in an on-line pipeline Changes in gene expression due to dietary protein independent of intervention duration and background diet were related to functional changes using gene set enrichment analysis (GSEA) Cumulative food intake did not differ between the dietary groups (p = 0.3235)  A. HowevAugmenting dietary protein leads to an increase in liver weight compared to the reference group after both 1 and 12 weeks of diet . The higHepatic VLDL production was determined in fasted mice after 1 week of dietary intervention  A. VLDL de novo lipogenesis and fatty acid oxidation were not significantly enriched. All genes contained in gene sets describing fatty acid oxidation are expressed in Hepatic gene expression was determined by microarray analysis of pooled RNA samples. Gene set enrichment analysis revealed several clusters of processes being enriched as a result of HP feeding independent of the background diet and the time point . A firstThis study confirms that mice fed a high fat-normal protein diet gained significantly more weight, adipose tissue mass and liver fat compared to all other groups despite a similar energy intake The present results are in agreement with the idea that in order to handle the overflow of incoming AA under HP feeding, AA uptake capacity in the liver increases. Also hepatic genes involved in AA deamination and metabolism are induced whereas the use of AA for liver protein synthesis mainly remains unaffected. The results of GSEA revealed that HP feeding both in context of a high fat or a high carbohydrate diet strongly affects hepatic gene expression in mice. This effect includes induction of gene sets belonging to AA processing pathways such as AA transport and catabolism, transamination processes and nitrogen metabolism. In previous observations HP diet enhanced liver transporter activity and AA uptake in rats de novo lipogenesis De novo lipogenesis was repeatedly demonstrated to be reduced during high fat feeding In contrast to earlier findings, in the present study GSEA did not reveal any effects of the HP diet on It was also previously reported in rats that in response to HP feeding hepatic gluconeogenesis remains induced in the fed state Interestingly, the HP diet increased plasma β-hydroxybutyrate (BHB) levels, suggesting the induction of a fasting-like situation and a channelling of the excess of acetyl-CoA to BHB synthesis. It is likely that during HP feeding the increased catabolism of AA acids induces an overflow of acetyl-CoA exceeding the cellular energy requirements. In the liver it is not converted into fat but into BHB that is excreted and may act as energy source for extrahepatic organs. The HP diet increased postprandial plasma TG which suggests a larger excretion of hepatic lipids via VLDL which at the same time decreases lipid storage. Indeed VLDL production rate decreases with a HF-NP diet but this effect is restored under HF-HP conditions. In humans increased postprandial TG levels in response to a HP adaptation were likewise observed de novo lipogenesis seems absent, these carbon skeletons are metabolized and transferred to acetyl-CoA. Thirdly, as result of an overload of acetyl-CoA and limited capacities to further metabolise it, energy is excreted from the liver in form of BHB and can act as a substrate to extrahepatic organs. In addition, more energy has to be used for detoxification of electrophiles produced during protein catabolism. Thus, handling of large amounts of amino acids leads to an increased energy utilisation in the liver. At the same time hepatic lipid storage is reduced. Additional effects contributing to the leaner phenotype of HP fed mice might be a slight, non-significant reduction in food intake, modifications in intestinal microbiota resulting in reduced nutrient availability as well as increased thermogenesis and therefore easier maintenance of body temperature at 20°C. This, however, has to be subject to further investigation. At last, HP in comparison with NP feeding promoted down regulation of both adaptive and innate immunity. Fatty liver is often associated with inflammation Taken together, the present data suggest that at a similar energy intake, mice ingesting HP as compared to NP diets possess a lower body weight, adiposity and hepatic fat due to a combination of modulated processes . FirstlyIn conclusion, the liver plays a buffering role in preventing the extrahepatic tissues from an overload of AA. Moreover, a shift in energy from the liver to peripheral tissues and its use for example in muscle are suggested and should be further investigated also on a longer term.Figure S1Enrichment map for HP versus NP feeding to identify biological functions. The map displays the enriched gene-sets and their labels in HP fed vs. NP fed mice, independent of background diet and time. Nodes represent gene-sets while edges represent overlapping genes. Gene-sets that did not pass the enrichment significance threshold  ≤0.1) are not shown. Red node colour represents enrichment in HP fed animals (or induction by HP diet), whereas green represents enrichment in NP fed animals (or suppression by HP diet). Clusters of functionally related gene-sets were manually circled and assigned a label.(PDF)Click here for additional data file.Table S1Heat maps of genes contributing to the enrichment of the gene sets within selected clusters of processes. For each of those genes, fold changes (FC) of signal intensity of the treatment group in comparison with the reference group at week 0 were calculated and expressed in a heat map. Red colour represents up regulation and green colour represents down regulation of a gene in comparison with the reference group. Additionally, genes contained in gene sets describing fatty acid oxidation are expressed.(XLSX)Click here for additional data file.


Hyperglycemia is common after stroke, and it is well known to worsen its outcome. However, it is important to consider that blood glucose (BG) levels can undergo dynamic changes during the acute stage of ischemic stroke. We sought to investigate the clinical significance of various glucose parameters within first 24 hours in acute ischemic stroke (AIS). The study focused on hyperacute stage patients who underwent IVT and investigated which parameters of glucose demonstrated to be helpful for predicting outcome.This was a retrospective study of consecutive patients with AIS at a single stroke center. Patients were consecutively enrolled if they were treated with IV-tPA within 3 hours of symptom onset. BG was measured immediately upon arrival in ER, after IVT and every 6–8 hours during the first 24 hours after IVT. The various parameters of BG were the following: BG before IVT, BG after IVT, mean BG (mBG), maximal BG (max BG), standard deviation of BG (sdBG), and standard deviation of mean BG (sdmBG).207 patients (127 men and 80 women) were included in this study. Seventy seven of 207 patients had favorable outcomes at 3 months. High BG after IVT, mBG and max BG were independently associated with mRS>2 at 3 months . Several parameters of BG were also independently associated with early mortality within 3 months . BG after IVT and mBG over 180 mg/dL were independently associated with early mortality within 3 months.Serial measurements of BG might be a better predictor of clinical outcome in patients with AIS treated with IVT than single BG measurements before IVT. Therefore, these results suggest that variable parameters of BG could be important for the prediction of clinical outcome in AIS treated with IVT. Hyperglycemia is common after stroke, and it is well known to worsen its outcome A recent study found that blood pressure variability rather than mean blood pressure was more associated with outcome in ischemic stroke We sought to investigate the clinical significance of various glucose parameters in the acute stage of ischemic stroke. In order to eliminate the effect of stroke severity on glucose in delayed examinations, the study focused on hyperacute stage patients who underwent IVT, investigating which parameters of glucose demonstrated to be helpful for predicting outcome.This was a retrospective study of prospectively registered patients with acute ischemic stroke at our tertiary stroke center between January 2011 and July 2012. Patients were consecutively enrolled if they (1) had an acute ischemic stroke within 3 hours of symptom onset, (2) had a National Institute of Health Stroke Scale (NIHSS) score of ≥4, (3) had acute ischemic lesions on diffusion-weighted imaging (DWI), and (4) were treated with IV-tPA. We excluded patients (1) who had other etiologies, such as Moyamoya disease, (2) with loss of follow-up evaluations, (3) with a previous modified Rankin Scale of >1 and (4) without at least 2 results of blood glucose between the first 24 hours after admission. This study was approved by the Institutional Review Board of Chonnam National University Hospital and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Written informed consent was not obtained due to the retrospective design of this study; therefore, the IRB of the hospital waived the need for written informed consent from participants.We previously described our emergency imaging protocol for patients with acute ischemic stroke Blood glucose was measured by the Accu Check® test. It was measured immediately upon arrival in ER and after IV-tPA administration. Although this was a retrospective study, in our acute stroke protocol, blood glucose was measured every 6–8 hours (at least 4–6 times) during the first 24 hours after admission. Hyperglycemia was defined as a glucose level >180 mg/dL and managed by short-acting insulin in cases that exceeded 200 mg/dL according to our stroke protocol.The various parameters of blood glucose were the following: blood glucose before IV-tPA administration (BG before IVT), blood glucose after IV-tPA administration (BG after IVT), mean blood glucose (mBG), maximal blood glucose (max BG), standard deviation of blood glucose (sdBG), and standard deviation of mean blood glucose (sdmBG).Baseline data collected from all patients included age, gender, NIHSS scores, and onset-to-treatment time. Stroke pathomechanisms were stratified according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria after a complete diagnostic workup t-test or the Mann–Whitney U test when appropriate. To evaluate which parameters of blood glucose were associated with early outcomes after IV-tPA administration, each parameter of blood glucose was adjusted by covariates of favorable outcomes at 3 months and death within 3 months . In addition, multiple logistic regression analysis was used to evaluate independent factors associated with PH; model 1 was adjusted by age, NIHSS score, hypertension, and atrial fibrillation; model 2 was adjusted by variables of model 1 and history of coronary artery disease. The associations between early outcomes and each parameter of the various blood glucoses levels were also analyzed by multiple logistic regression analysis. Adjustment was made for the variables of age, SBP, atrial fibrillation, NIHSS scores and white matter hyperintensities (WMH) for death at 3 months and age, NIHSS, atrial fibrillation and WMH for mRS 0–2 at 3 months. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. A p value of <0.05 was considered to be statistically significant. All of the statistical analyses were performed using SPSS for Windows, version 17 .The percentage, mean , or median  are reported depending on variable characteristics. Categorical variables were analyzed using the χ2-test and Fisher's exact test when appropriate. Continuous variables were analyzed using the independent samples A total of 232 patients treated with IV-tPA were screened during the study period. Of these patients, 25 were excluded: 11 were excluded due to loss of follow-up, 8 were excluded due to incomplete work-up , 4 were excluded due to a previous mRS score of >1, and 2 were excluded due to Moyamoya disease. Ultimately, 207 patients (127 men and 80 women) were included in this study, and the mean age of the patients was 70.6±11.11 years. The general characteristics of the patients are summarized in Blood glucose was measured at 2 intervals for 24 patients (11.4%), 3 intervals for 37 patients (17.9%), 4 intervals for 86 patients (41.5%), and 5 intervals or more for 60 patients (29.0%). The association between blood glucose parameters and clinical outcome (mRS>2 at 3 months and death within 3 months) is summarized in In addition, we analyzed the relationship between various parameters of blood glucose and clinical outcomes according to the levels of blood glucose; <100 mg/dL, 100–180 mg/dL, and >180 mg/dL . All parOur data also analyzed the association between parameters of blood glucose and PH  and 5. EThe results of our study showed that high levels of BG after IVT, mBG and max BG were independently associated with mRS>2 at 3 months and death within 3 months. Among the parameters exceeding 180 mg/dL of blood glucose, BG after IVT was independently associated with mRS>2 at 3 months and mBG and max BG with death within 3 months while sdBG showed a non-significant yet positive trend with death within 3months. These results suggest that a single measurement of BG levels after IVT could be better for predicting prognosis than admission blood glucose and that mBG, max BG, or sdBG may better predict death within 3 months than single BG before and after IVT. In other words, it is suggested that serial blood glucose exams rather than single BG exams before IVT can possibly be more useful in predicting outcome in acute ischemic stroke.Our study presents different implications compared to previous studies. Most previous studies pointed to admission hyperglycemia as a predictor of poor outcome In our study, mBG was more associated with clinical outcome than single shot blood glucose before IVT. According to other studies, mean glucose during ICU admission is also related to mortality in critical illnesses like sepsis and trauma Ischemic penumbra is likely to be found in the early hours after stroke onset but may also be seen up to 24 hours Although the investigators sought to evaluate the effect of glucose lowering through intensive treatment as attempted by several clinical studies, they were unable to verify the effect of intensive insulin therapy (IIT) Importantly, the results of our study reveal the clinical significance associated with early max BG. Max BG was indeed associated with death at 3 months and mRS>2. Fuentes et al showed that hyperglycemia ≥155 mg/dL at any time within the first 48 hours from stroke onset, is associated with poor outcome In our study, there was furthermore a higher likelihood of future occurrence of PH in patients with higher BG after IVT. On the other hand, BG before IVT was not associated with PH, where mBG>180 mg/dL was significantly associated with PH. These results are different from previous studies that reported that BG before IVT could predict PH. Therefore, our study suggests the need for blood glucose lowering even if it is after IVT.There are several limitations in this study. First, it has the inherent limitations of a retrospective observational study in a single center with a modest sample size. Although this was a retrospective study, we prospectively collected data in consecutive patients. Second, recanalization, which could affect prognosis, was not analyzed in this study. Because we performed NCCT-based IVT, we could not confirm recanalization in the patients. Previously, in models with reperfusion, hyperglycemia increased infarct size, while in animals without reperfusion, hyperglycemia seemed to have no adverse effect and might even have been beneficial In conclusion, serial measurements of blood glucose might be a better predictor of clinical outcome in patients with acute ischemic stroke treated with IVT than single blood glucose measurements. Among the variable parameters, BG before IVT was not associated with clinical outcome in this study. Therefore, these results suggest that variable parameters of blood glucose could be important for the prediction of clinical outcome in acute ischemic stroke treated with IVT.