To investigate whether this leads to functional differences in DNA repair abilities measurable in patients, we adapted two plasmid-based host-cell reactivation assays for use in primary lymphocytes. Prior to applying these assays to patients who underwent aHCT, we wanted first to verify whether sample preparation affected repair measurements, as patient samples were simply depleted of erythrocytes (with hetastarch) prior to freezing, which is not the classical way to prepare lymphocytes prior to DNA repair experiments (with a density gradient). We show here that lymphocytes from healthy donors freshly prepared with hetastarch show systematically a higher level of double-strand break repair as compared to when prepared with a density gradient, but that most of this difference disappears after samples were frozen. Several observations points to granulocytes as the source for this effect of sample preparation on repair: 1) removal of granulocytes makes the effect disappear, 2) DSB repair measurements for the same individual correlate to the percentage of granulocytes in the sample and 3) nucleofection in presence of granulocytes increases the level of reactive oxygen species (ROS) in neighboring lymphocytes in a dose-dependent manner (R2 of 0.95). These results indicate that co-purified granulocytes, possibly through the release of ROS at time of transfection, can lead to an enhanced repair in lymphocytes that obfuscates any evaluation of inter individual differences in repair as measured by host-cell reactivation. As a result, hetastarch-prepared samples are likely unsuitable for the assessment of DSB repair in primary cells with that type of assay. Granulocyte contamination that exists after a density gradient preparation, although much more limited, could have similar effects, but might be circumvented by freezing cells prior to analysis.Patients who develop therapy-related myelodysplasia/acute myeloid leukemia after autologous-hematopoietic stem cell (aHCT) transplant show lower expression levels of DNA repair genes in their pre-aHCT CD34 Our ultimate goal is to verify if these differences result in functional changes in DNA repair capacities that could be more easily evaluated in a clinical setting.Therapy-related myelodysplasia/acute myeloid leukemia (t-MDS/AML) is a major complication of autologous-hematopoietic stem cell transplant (aHCT). Samples from patients who received aHCT for a relapsed or refractory Hodgkin's or non-Hodgkin's lymphoma have been collected for a prospective longitudinal study with the objective to identify new markers that help predict patients at risk of t-MDS/AML in vitro prior to the introduction in the cells where the repair will be measured by the reactivation of a transgene, avoiding as much as possible concerns about cytotoxicity associated with the damage. Host-cell reactivation assays can be performed on any cell type that can be transfected, including cryopreserved primary lymphocytes Many assays exist that can be used to evaluate inter-individual differences in repair abilities. Among those, host-cell reactivation assays have the advantage to directly measure repair and can be adapted to study specific repair pathways. Moreover, the damage is generated Use of human blood samples was approved by the City of Hope Internal Review Board: IRB protocol #98117 entitled “The Molecular Pathogenesis of Therapy-Related Myelodysplasia/Acute Myelogenous Leukemia”. Volunteers provided their written consent.in vitro by a double-digestion with ApaI and XhoI (pSF-tdTomato-END (NHEJ assay) and pSF-tdTomato-HOM (SSA assay) are modified versions of pCMS-end and pCMS-hom plasmids kindly donated by R. Schiestl and XhoI . Completand XhoI . Deletedand XhoI  were als2+ or Mg2+ (Cellgro) and the culture medium is Iscove's modified Dulbecco's medium  with 20% heat-inactivated fetal bovine serum (FBS) (Omega Scientific).In all the experiments, Dulbecco's phosphate buffered saline (DPBS) is without Ca6 cells in 1 ml of 90% FBS with 10% DMSO . Prior to all DNA repair experiments, cells from an aliquot were thawed and washed once in culture medium, put back in culture and allowed to recover for >7 h before nucleofection.GM01953 lymphoblastoid cell line (LCL) was obtained from the Coriell Cell Repository . Cells growing exponentially in culture medium were frozen in aliquots containing 3×10Human blood samples (30–35 ml in heparin tubes) were obtained from 6 healthy volunteers (identifiers LYM10031 to 10036 shortened for convenience as LYM1 to LYM6) and processed ∼3 h after being drawn, except for some of the blood from LYM3 and 4 that were processed the next day when ∼29 h old. The blood was first diluted 1∶1 in DPBS containing 2% FBS. For density gradient separation, the diluted blood was transferred onto a SepMate-50 tube (Stem Cell Technologies) containing 15 ml of HISTOPAQUE-1077 (Sigma) and further processed as recommended by the tube manufacturer. For hetastarch preparation, 1 volume of HESPAN hetastarch  was added to the diluted blood and mixed thoroughly. RBCs were then let to sediment for 45 min. The upper phase, containing WBCs and 5–10% of RBCs, is transferred to a new tube and washed once with DPBS and once with culture medium. For every type of preparation tested, aliquots of cells were made containing cells recovered from 2.5–3 ml of blood, either to be analyzed immediately as fresh cells or frozen aliquots for later analysis. Cell counts based on cell morphology were determined using the Hemavet 950FS (Drew Scientific).+ cells were recovered by rapidly flushing the column with 5 ml of cold buffer before cells were centrifuged and resuspended in culture medium.Cells recovered from ∼12 ml of blood processed with hetastarch (LYM5 and 6) were subjected to an RBC lysis (see below) and then resuspended in 1.2 ml of cold buffer . To those cells, 280 μl of CD15 MicroBeads were added and incubated at 4°C for 15 min before wash with the buffer and passage through a 25 LS MACS separation column placed on a MACS Quadro as recommended by the manufacturer (Miltenyi Biotec). Cells that flowed through the column, mostly lymphocytes, were resuspended in culture medium. When indicated (LYM6 sample), CD15For each aliquot obtained after hetastarch or density gradient (cells recovered from 2.5–3 ml of blood), cells were resuspended in 1 ml of culture medium and 1 ml of cold freezing medium was added . Cell temperature was progressively decreased to −80°C in a Mr Frosty container  and then transferred the next day to the vapor phase of a liquid nitrogen storage tank. For thawing, 30 ml of medium containing 4,000 U/ml heparin and 62.5 μg/ml DNase were added dropwise to a rapidly thawed aliquot and cells were allowed to recover 4 h in a 37°C incubator before a 15 min spin at 200 g and resuspension in culture medium.RBC lysis on hetastarch-prepared samples was performed by adding 9 volumes of cold ammonium chloride solution (Stem Cell technologies) to 1 volume of cells in culture medium. After 6–8 inversions, cells were centrifuged for 15 min at 200 g at 4°C before 2 washes in culture medium. Depending on the experiment, RBC lysis was performed on fresh cells or after recovery from thawing and prior to nucleofection.Both GM01953 cells and primary lymphocytes were transfected with the P3 primary cells nucleofection kit (Lonza) using the 96-E0-115 program recommended for “high functionality” transfection of uninduced T-cells on the Amaxa Nucleofector 96-well Shuttle system. Typical transfections used 150,000–190,000 GM01953 or 125,000–200,000 primary lymphocytes (plus a variable amount of other WBCs depending on the sample preparation method). One individual aliquot of frozen GM01953 or primary lymphocytes was used for each experimental day to generate 3 measurements of each type of repair (NHEJ and SSA) plus all controls for the FACS analysis .LIN, ENDDSB, HOMLIN or HOMDSB) were allowed to repair DSB and express transfection (tdTomato) and repair (EYFP) reporters for 12 h before being harvested and resuspended in HBSS (compensation controls) or HBSS containing 0.3 μg/ml DAPI. Cells were then kept on ice until analysis on a BD LSRFortessa cell analyzer using FACSDiva software . Fluorescent signals were analyzed as follow: DAPI with a 355 nm UV laser (filter 450/50 nm), tdTomato with 561 nm Yellow/Green laser (filter 610/20 nm) and EYFP signal with a 488 nm blue laser (filter 525/50 nm). The PMT voltages for FSC, SSC and DAPI were kept constant for all experiments at 466 V, 237 V and 302 V respectively for lymphocytes, FSC being reduced to 350 V for GM01953 cell acquisition. The voltages for tdTomato and EYFP signals were adjusted according to the baseline established using the Cytometer Setup & Tracking beads the day of the acquisition (410 – 434 V for tdTomato and 429 – 435 V for EYFP), except for GM01953 were EYFP voltage was reduced and maintained at 370 V to compensate for a slight autofluorescence of the cells. After the acquisition, the population of lymphocytes was first selected based on their FSC and SSC values , the absolute recombination efficiency (ARE)  =  tdTom+EYFP+/(tdTom+EYFP+ + tdTom+EYFP−) was determined (DSB with ARE of the ENDLIN plasmid (represents 100% repair) (AREDSB/ARELIN) and for SSA by subtracting the ARE for HOMLIN plasmid (AREDSB - ARELIN). ARELIN represents the background of cells that show reactivation of EYFP by other means than SSA. This kind of background is almost nonexistent when analyzing normal cells such as lymphocytes or LCLs, but that is not the case for all cells types.Cells transfected by nucleofection with 0.4 μg of digested plasmid DNA  in lymphocytes 1 h after or not a mock nucleofection (cells resuspended in solution and electroporated in absence of DNA) in presence of granulocytes was performed using the CellROX Deep Red Flow Cytometry Assay kit (Molecular probes). Selection by FACS of the lymphocytes as the population of interest was performed as described above, except that dead cells were excluded using the SYTOX Blue Dead Cell Stain . The presence of ROS was then detected through the oxidation of the CellROX Deep Red Reagent that leads to a red fluorescent compound . A ROS-free negative control was generated by adding N-acetylcysteine antioxidant to lymphocytes 1 h prior to the mock transfection in order to determine the level of background Cy5 fluorescence in stained cells  for measurements of RRE (NHEJ and SSA) after density gradient or hetastarch. RTo evaluate DSB repair in primary lymphocytes, two constructs were used that allow the separate investigation of repair by NHEJ or SSA, in order to potentially identify effects specific to one pathway or the other. The original host reactivation assay system used (pCMS-end and pCMS-hom plasmids), kindly provided by Robert Schiestl Undigested supercoiled plasmids are generally used as controls for host cell reactivation assays for DSB repair to indicate 100% repair (NHEJ assay) or 0% repair (SSA assay) Based on the design described above, nucleofection of all digested constructs was performed on GM01953 cells (lymphoblastoid cell line), and on primary lymphocytes prepared from blood using a density gradient (LYM1). The same transfection conditions could be used for both cell types and there was no significant toxicity due to the transfection method  in relation to this same ratio for the control linearized plasmid , which represents the proportion of cells that express EYFP6 per ml of blood), but result in major differences in the cell composition after preparation: both RBCs and granulocytes are expected to sediment at the bottom of the tube during a density gradient isolation, whereas RBCs and granulocytes constitute the vast majority of the cells in hetastarch prepared samples. RBCs constitute ∼95% of all blood cells, so even though the hetastarch can eliminate up to 95% of them, they can still outnumber WBCs by a factor 10. RBCs hemolysate and hemoglobin have been previously reported to affect DNA repair in peripheral blood mononuclear cells The purification of peripheral blood mononuclear cells with a density gradient is the classical way to prepare lymphocytes to measure DNA repair A potentially greater problem is the presence of other types of WBCs in the environment, even though flow cytometry can focus our analysis specifically on the cell type of interest (lymphocytes) by selecting specific forward and side scatter values . Notably6/ml blood) of lymphocytes (LYM) but with different levels of granulocyte (GRA) contamination in their environment  were prepared using several procedures  that leaironment . Hetastaironment .+ cells were depleted  after hetastarch treatment and prior to the DNA repair measurement (LYM 5 and 6), indicating that the effect on repair is not related to the incubation with hetastarch, but rather linked to the presence of other cell types in the environment of the lymphocytes. Monocytes, being similar in proportion for all types of preparation, are unlikely to be the cause of the effect. There was also no correlation between DNA repair measurements and corresponding transfection efficiencies (data not shown). However, for all individuals analyzed (LYM3-6), there was a correlation between DSB repair measurements (% RRE) and the percentage of granulocytes in the corresponding sample, especially so for NHEJ  recovered from the column during the CD15+ depletion step for LYM6 . In samples that were mock transfected . Either way, there was clearly a de novo formation of ROS 1 h after mock nucleofection of cells in a granulocyte amount-dependent manner  is expected. Such a burst of ROS into the medium could affect neighboring lymphocytes even if the nucleofection is not toxic to them. To test whether nucleofection of granulocytes affect ROS content in lymphocytes, LYM6 hetastarch-prepared cells were depleted of CD15phocytes . F reprephocytes . An extrnsfected  right, tt manner . This ROt manner , which it manner . Therefoi.e. starting from different frozen aliquots), but different preparation protocols starting from the same blood sample can lead to different repair measurements for the same individual. Notably, fresh hetastarch-prepared cells  show systematically higher levels of repair in lymphocytes compared to their density gradient-prepared counterparts, although the analysis is focused in both cases on the same cell type. The single most likely factor responsible for this effect is the presence of granulocytes at the time of transfection in the vicinity of the lymphocytes in which the repair is measured. Granulocytes carry granules in their cytoplasm that contain toxic materials that help eliminate unwanted microorganisms after phagocytosis. They are short-lived in the bloodstream and activated granulocytes are thought to be responsible for the suppression of T-cell function that is observed in advanced cancer patients The host-cell reactivation assays modified for primary lymphocytes that we describe here successfully measure DSB repair by NHEJ and SSA starting from a limited number of cells in both lymphoblasts and density gradient-prepared primary lymphocytes. Reproducible measurements can consistently be achieved when repeating measurements from the same preparation  was within the range measured for other individual's primary lymphocytes (RRE between 16.2% and 42.4%). However, NHEJ in lymphoblasts (RRE = 58.4%) seemed radically more efficient compared to the repair in lymphocytes (RRE between 21.3% and 36.7%). Although one cannot exclude that the individual whose cells were used to generate the GM01953 cells line had higher repair capacities, the high repair could reflect a better ability for NHEJ in cycling and/or transformed cells where the risk of DSB is expected to be higher than in GThe protocol used to prepare blood cells for phenotypic DSB repair assays in peripheral primary lymphocytes can alter the results to the point where inter individual differences can be completely obfuscated. This effect is likely caused by the variable presence of granulocytes in the sample and precludes the use of alternatives to density gradient to recover lymphocytes. As a consequence, great care should be taken to minimize the level of granulocyte contamination if DNA repair by host cell reactivation assay is the purpose of any cell preparation.Figure S1Verification of the complete XhoI + ApaI double digestion prior to transfection. (A) The complete double digestion can be verified by studying the ability to recircularize the plasmids or not after linearization. The theoretical products are shown here for the pSF-tdTomato-END plasmid but would be the same when verifying the pSF-tdTomato-HOM double digestion, except that a SalI site is used instead of EcoRI for the secondary digestion. Plasmids digested with a single enzyme (ApaI) can be recircularized as a single plasmid (*) or a dimer (*Δ), whereas double-digested plasmid (ApaI + XhoI) can only generate linear dimers (Δ) or trimers after ligation. The removal of the excised XhoI - ApaI fragment is further verified by a secondary digestion of the ligation products cutting inside of this fragment. Recircularized plasmids (*) can be linearized again with this secondary digestion, whereas the ApaI + XhoI double-digested DNA have lost the target restriction site and are not affected. Linear dimers are obtained with the secondary EcoRI digestion of the recircularized dimers (*Δ) when the plasmids were religated in head-to-head orientation. (B) The results of the religation experiment can be analyzed on an agarose gel where the different ligation products have different migration patterns. The secondary digestion is EcoRI for END or SalI for HOM.(TIF)Click here for additional data file.Figure S2Map of plasmids used in the host cell reactivation assays. (A) pSF-tdTomato-END used to measure NHEJ. (B) pSF-tdTomato-HOM used to measure SSA. (C) and (D) Deleted plasmids expressing a single fluorescent protein used as compensation controls for the FACS analysis.(TIF)Click here for additional data file.Figure S3Typical FACS data. (A) Lymphocytes (P1) in red are the population of interest for the DNA repair assays (in this example: frozen hetastarch-prepared LYM5). DAPI staining is used to eliminate dead cells (in blue) from the analysis and to delineate the quadrants separating negative and positive populations. Control single color plasmids are used to verify that compensation is appropriate. For each digested construct , the absolute recombination efficiency (ARE  =  Q2/(Q1+Q2)) is determined. The relative recombination efficiency (RRE) is then calculated for NHEJ by normalizing data for ENDDSB with ARE of the ENDLIN plasmid (represents 100% repair) (AREDSB/ARELIN) and for SSA by subtracting the ARE for HOMLIN plasmid (represents no repair) (AREDSB – ARELIN). (B) Effect of a mock nucleofection on fresh granulocytes. After elution from the CD15+ depletion column, LYM6 granulocytes were put back into culture and mock nucleofected (electroporated without DNA) or not in conditions identical to those used for the DNA repair assays. In a FACS analysis, CD15+ cells (mostly granulocytes) present as two populations that differ mainly by their forward scatter: P1 (in red) is mostly live cells (>95% are DAPI negative) and P5 (in blue) is mostly dead cells (>90% are DAPI positive). Untransfected cells are mostly in the P1 population, whereas mock transfected cells are overwhelmingly in the P5 population, indicating massive level of granulocyte cell death upon mock nucleofection.(TIF)Click here for additional data file.Figure S4ROS measured in LYM6. Samples were depleted of CD15+ cells in freshly prepared cells (A) or after thawing (B). For both types of preparation (from the same donor LYM6), cells in culture show a subpopulation of cells that have a Cy5 signal above background measured as the % Cy5+ cells (P5 gate). This specific population tends to disappear in presence of an antioxidant (NAC) and/or after mock nucleofection. However, nucleofection in presence of increasing number of CD15+ cells added back in the cell mix leads to a dose-dependent general shift of the lymphocyte population towards higher level of ROS as measured by a change in the median Cy5 value in the whole population. The estimated cell composition of the tested samples is shown (bottom right).(TIF)Click here for additional data file.Figure S5Effect of linearization on transfection efficiency. For all DNA amount tested, the transfection efficiency in primary lymphocytes LYM1 of XmnI-linearized pSF-tdTomato is decreased compared to the same amount of supercoiled undigested plasmid.(TIF)Click here for additional data file.Figure S6Time-dependent toxicity associated with DNA after nucleofection. (A) GM01953 LCLs and (B) LYM1 primary lymphocytes were transfected with the same amount of XmnI-linearized END control (ENDLIN) that expresses both tdTomato and EYFP constitutively. Live (DAPI negative) cells in the populations of interest are shown in red. For both cell types, the population of transfected cells (Q1+Q2+Q4) decreased with time after transfection , whereas mock or untransfected cells (Q3) were not affected, indicating toxicity specifically associated with the expression of the transgenes and not the transfection protocol per se.(TIF)Click here for additional data file.Figure S7Cell composition of analyzed samples. The proportion of lymphocytes (in blue) and granulocytes (in red), as determined with a Hemavet 950FS, are indicated for each sample analyzed for DNA repair.(TIF)Click here for additional data file.


The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0–4], pre-stable stage [day 5–7] and the stable stage [day 8–10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine. Extracorporeal bioartificial liver devices (BAL) are perhaps among the most promising technologies for the treatment of liver failure, but significant technical challenges remain in order to develop in vitro systems with sufficient functional capacity in vitro systems are also imperative for drug metabolism and toxicity evaluation studies Hepatocytes constitute about 70% of the cellular population of the liver and play an indispensable role in over 500 metabolic, regulatory and immune functions in vitro include effect of extracellular matrix topology, cellular environment and medium composition on function in vitro is the collagen I sandwich configuration Various methodologies to maintain hepatocytes In vivo, the hepatic ECM also known as the Space of Disse is a 1–1.5 mm complex macromolecular deposition of collageneous proteins, fibronectin, entactin and heparin sulfate proteoglycans in vitro systems for hepatic functional upregulation in Bioartificial Liver Devices and drug toxicity studies is necessary. From a mechanistic standpoint, these systems are useful to unravel the effect of ECMs on hepatic function and metabolism.Thus, influence of ECM on hepatocyte culture systems is important from two perspectives. From an application standpoint, development of scalable, simple, “in vivo” like and cost-effective in vivo microenvironment Variation in ECM compositions including addition of glycosaminoglycans and hepatic proteoglycans that promotes formation of gap junctions in vitro system will provide a platform to assess the effect of hepatic-like ECM on cell metabolism and function. The inter-relationship between hepatic intracellular pathways and the ECM is currently less studied and the elucidation of the effect of the ECMs on the metabolism is critical to identify the mechanism of in vivo hepatocellular functional and morphological integrity. This necessitates a suitable in vitro model system that can closely mimic the in vivo hepatic microenvironment.Moreover, the developed in vitro model for investigating the interactions between hepatic ECM and metabolism.We have previously developed a system to synthesize mammalian preadipocyte cell secreted extracellular matrix proteins termed Adipogel in copius amounts. Adipogel has been shown to exhibit at least fibronectin [the most abundant ECM in the Space of Disse] and collagen IV that is present in the Space of Disse. We have also shown that Adipogel laden on top of collagen single gel hepatocyte cultures increased albumin differentiated function as compared to collagen double gel cultures Numerous researchers have investigated the intermediary metabolism of hepatocytes, the primary functional cells of the liver Dulbecco's Modified Eagle Medium [DMEM] containing 4.5 g/L glucose, fetal bovine serum [FBS], penicillin, streptomycin, and trypsin–EDTA were obtained from Invitrogen Life technologies . Dexamethasone, isobutyl-methylxanthine, epidermal growth factor, insulin, glucagon, rosiglitazone and hydrocortisone were purchased from Sigma-Aldrich . 100 kDa cutoff protein centrifugal filters were purchased from Millipore Technologies .2 flasks in Dulbecco's Modified Eagle's media supplemented with 10% FBS, 2% Penicillin and Streptomycin till the cells attained confluency. 48 hours post confluency, the cells were differentiated in culture media supplemented with 1 µM dexamethasone, 0.1 µM isobutyl-methylxanthine and 1 µM rosiglitazone for 2 days with media changes every two days. On the second day post-differentiation, cells were exposed to culture medium supplemented with 1 µM rosiglitazone for an additional 2 days. Media supernatant was collected on days 2 and 4 of differentiation and stored at 4°C prior to further processing.Adipogel was generated from differentiating murine preadipocytes as described previously. Briefly, 3T3-L1 murine preadipocytes were cultured in T-175 cmg for 1.5 hr using an Amicon 100 kDa centrifugal filter. Since the concentrate was derived from adipocyte-related cell type and had a gel-like configuration, it was termed as ‘Adipogel’. To elucidate the effect of Adipogel on hepatocellular metabolism, four different conditions were tested as described in In order to purify the extracellular matrix rich material, the differentiated preadipocyte conditioned media was centrifuged at 4000Female Lewis rats  weighing 180 to 200 g [2 to 3 months old] were used as a hepatocyte source and were maintained in accordance with National Research Council guidelines. Experimental protocols were approved by the Subcommittee on Animal Care, Committee on Research, Massachusetts General Hospital (Protocol # 2005N000130). Using a modification of the two-step collagenase perfusion method 5 primary hepatocytes in suspension in 0.5 ml standard hepatocyte culture medium, which consisted of DMEM supplemented with 14 ng/ml glucagon, 7.5 µg/ml hydrocortisone, 0.5 U/ml insulin, 20 ng/ml EGF, 200 U/ml penicillin, 200 µg/ml streptomycin, and 10% FBS. Cultures were incubated in 90% air/10% CO2 at 37°C. Cells were rinsed with 1× PBS to remove non-adherent cells 4–6 hours after seeding. For the double collagen gel culture configuration, a second layer of 250 µl collagen was laden on top of the cells 48 hours post-seeding . Albumin protein and the antibody were purchased from MP Biomedicals. Urea concentration was determined via its specific reaction with diacetyl monoxime with a commercially available assay kit . The absorbance was measured with a Thermomax microplate reader The biochemical assays were performed at the recovery, pre-stable and stable stages of culture with media samples. Amino acids were fluorescently labeled using the AccQ-Tag system , separated by high performance liquid chromatography , and quantitated by a fluorescence detector . Glucose and lactate levels were measured with commercially available kits [Sigma], the former based on the reaction of glucose catalyzed by glucose oxidase and the latter based on the conversion of lactate to pyruvate catalyzed by lactate oxidase. Acetoacetate and β-hydroxybutyrate were measured using a commercially available kit [Bioassay Systems].MFA is a useful methodology to characterize the differential activation of metabolic pathways in hepatocyte cultures. Thus, based on a stoichiometric model for the metabolic reaction network prevalent in hepatocytes, intracellular reaction fluxes are estimated by mass balances around each intracellular metabolite and extracellular flux measurements. This gives the possibility to calculate intracellular metabolite fluxes, which are difficult to measure from relatively few measurements and to corroborate the metabolic network. The model used in this work was originally developed for perfused liver The metabolic network is based on known stoichiometry of hepatic intermediary metabolism with consideration of carbon and nitrogen balances.Albumin is a major protein product of hepatocytes and hence only this protein is considered.The cellular uptake/secretion rates of metabolites are distinct from the intracellular fluxes of the corresponding metabolites. Thus, the intracellular and extracellular pools of substrates have been distinguished. Also, the mechanisms of active and passive transport have not been incorporated.The metabolite pools are at pseudo steady-state with a single pool in the cell. The influx and efflux of metabolites into/from hepatocytes are calculated from the amount of metabolites remaining in the extracellular media after 24 h.ij of S is the coefficient of metabolite i in reaction j, and each vj of vector v is the net flux or conversion rate of reaction j. Equation 1 is separated into measured and unknown fluxes, vm and vu, respectively, as follows:Following the above assumptions, the mathematical model consists of mass balances around 45 intracellular metabolites  considerEach data point represents the mean of two or three experiments [each with three biological replicates], and the error bars represent the standard error of the mean. Data was normalized to number of cells initially seeded and expressed as µmol/million cells/day.For extracellular metabolite measurements, the data-sets for each metabolite flux for each experimental condition were averaged from the sum of all replicates per experimental condition. The standard error of the mean was calculated from replicate data-set for each experimental condition. The mean and standard error of the mean of the extracellular metabolite measurements is quantitatively represented in the The MFA framework was applied to each biological experimental data-set of extracellular metabolite measurements in µmol/million cells/day input units. The output of the MFA for each biological experimental data-set was averaged using the sum over all computationally derived replicates for the unknown [non-measured] fluxes. The standard error of the mean was calculated from the computationally derived replicates for the unknown [non-measured] fluxes.The summary statistics were calculated using t-test by comparing data from all experiments from one experimental condition for (e.g.: CSG+solASG) vs. data from all experiments from another experimental condition (for e.g.: CDG). This statistical method was used for extracellular metabolite measurements and computed intracellular fluxes while comparing a specific metabolite flux between different experimental conditions. Statistical significance was determined using the Student's t-test for unpaired data. Differences were considered significant when the probability was less than or equal to 0.05.We have previously performed a preliminary characterization of the extracellular matrix components derived as a basement membrane extract from preadipocytes during the differentiation process 2 flasks in Dulbecco's Modified Eagle's media supplemented with 10% FBS, 2% Penicillin and Streptomycin till the cells attain confluency. 48 hours post confluency, the cells are differentiated in culture media supplemented with 1 µM dexamethasone, 0.1 µM isobutyl-methylxanthine and 1 µM rosiglitazone for 2 days with media changes every two days. On day 2, the differentiation medium is supplemented with rosiglitazone only.The preadipocytes are cultured in T-175 cmg for 1.5 hr using an Amicon 100 kDa or 10 kDa centrifugal filters. The concentrate, primarily composed of media constituents with molecular cut-off of 100 kDa or 10 kDa comprises the cell culture supernatant derived protein rich extracellular matrix.During the differentiation process, cell exposed media is collected and processed further for generation of cell derived extracellular matrix. We have previously identified a highly viscoelastic material on days 2 and 4 of adipocyte differentiation resembling extracellular matrix components secreted by preadipocytes to maintain adipose tissue cell-cell contact, morphological induction of adipocytes and functional and gene expression indicative of mature adipocyte lineage. In order to purify the extracellular matrix-rich material, the cell exposed media is centrifuged at 4000Routine culture of primary hepatocytes is difficult and cumbersome due to their ability to develop compromised function. We have developed a primary hepatocyte culture system that supersedes the traditional methodology of maintaining hepatocyte function and polarity in collagen double gel sandwich systems. Hepatocytes when cultured on single collagen gel with a soluble matrix of Adipogel in the culture media showed comparable urea secretion rates  but signWe have performed a metabolic analysis of the different culture configurations viz. the collagen single gel [CSG], collagen double gel [CDG], collagen-Adipogel sandwich [CSG+ASG] and collagen single gel+soluble Adipogel [CSG+solASG]. To elucidate the effect of Adipogel on hepatocyte metabolism, we have compared the CDG to the CSG+solASG conditions as described below. We have chosen day 4 [recovery stage], day 7 [pre-stable stage] and day 10 [stable stage] of analysis to investigate the delayed effect  as well as the immediate effect [day10] of Adipogel supplementation on hepatic metabolism. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation 1] at the recovery stage and stable stage of culture respectively with glucose production at the pre-stable stage [14] is significantly higher for the CSG+solASG condition as compared to the CDG condition only at the stable stage.As shown in le stage . On the 55] is significantly higher for CSG+solASG vs. CDG condition. At the recovery stage, glutamine [v67] and arginine synthesis rate [v68] is also significantly higher for CSG+solASG vs. CDG condition. At the stable stage, serine [v69], glycine [v59], isoleucine [v61], lysine [v62], leucine [v63], methionine [v64], arginine [v68], valine [v71] and tyrosine [v72] uptake rates are significantly higher for CSG+solASG condition as compared to CDG condition. Thus, we can correlate increased amino acid uptake rates and changes in glucose metabolism to hepatocellular function at the stable stage of culture.At the recovery stage and pre-stable stage, cysteine synthesis [v17–v20] for the recovery stage comparison [2–v9] and reduction in gluconeogenic pathway [v11–v13], glutamine metabolism [v36–v38] and acetyl-CoA synthesis  and clearance  with increased citrate synthesis rate [v15] for the CSG+solASG condition as compared to the CDG condition. At the stable stage, increased amino acid uptake rates, decreased acetyl-CoA synthesis  with increased TCA cycle fluxes [v17–v20] were observed between the two conditions. Overall, from a functional standpoint the urea synthesis fluxes increased  and there were numerous differences in various pathway fluxes between the two conditions including an increase in albumin secretion for the CSG+solASG vs. CDG condition on all days of analysis.Using a previously developed hepatic metabolic network comprising of 72 reactions and 27 extracellular metabolite measurements mparison . At the mparison , there wWe also performed a comparison of the CSG+solASG condition at different time points of the differentiation process. A comparison of the recovery stage and pre-stable stage cultures revealed significant increase in PPP and decrease in TCA cycle fluxes for the CSG+solASG condition with no significant difference in albumin synthesis rate . A signiin vitro using various techniques viz. collagen double gel sandwich Primary hepatocytes are cultured Particularly, there is some literature evidence for the effects of ECM proteins on enhanced albumin synthesis of hepatocytes While hepatic function is enhanced by utilizing the above mentioned culture systems, the albumin synthetic functional capacity of the cells is not maximal. In this study, Adipogel, a natural cell secreted extracellular matrix, can be added in soluble media format to hepatocyte cultures  and has 69], glycine [v59], isoleucine [v61], lysine [v62], leucine [v63], methionine [v64], arginine [v68], valine [v71] and tyrosine [v72] uptake rates as compared to the CDG condition at the stable stage of culture.In this context, we have performed a metabolic analysis of the different culture configurations viz. the collagen single gel [CSG], collagen double gel [CDG], collagen-Adipogel sandwich [CSG+ASG] and the collagen single gel+soluble Adipogel [CSG+solASG]. The analysis shows that the key amino acids upregulated for the CSG+solASG conditions are serine  model, such that the distribution of fluxes can be predicted with a limited number of values [active flux bounds] We have shown that Adipogel can be utilized for augmenting hepatocyte differentiated function Table S1List of reactions in metabolic network utilizing Gluconeogenic fluxes. The common pathways include pentose phosphate pathway; lipid, glycerol and fatty acid metabolism; lactate metabolism and tricarboxylic acid (TCA) cycle; urea cycle; amino acid metabolism; oxygen uptake and electron transport and albumin protein metabolism.(DOC)Click here for additional data file.Table S2List of reactions in metabolic network utilizing Glycolytic fluxes. The common pathways include pentose phosphate pathway; lipid, glycerol and fatty acid metabolism; lactate metabolism and tricarboxylic acid (TCA) cycle; urea cycle; amino acid metabolism; oxygen uptake and electron transport and albumin protein metabolism.(DOC)Click here for additional data file.


Aim: Describe the use of 320 row detector CT scanner for 4 Dimensional CT acquisition on a specialized platform designed for wrist kinematic evaluation and to demonstrate the utility of 4 Dimensional CT in the assessment of wrist biomechanics before and after surgical repair. Materials and Methods: Six wrists (1 volunteer and 4 patients) were uniformly imaged with conventional X-rays and 4 Dimensional CT on a 320 row detector CT scanner . A dedicated custom designed wrist platform was used for kinematic imaging. Three subjects (3 wrists) had prior fixation of the complex wrist injury. Clinical correlations were obtained. Results: All subjects were successfully scanned in various wrist motions. 4 Dimensional CT image quality was adequate and carpal kinematic behavior was easily assessed in various wrist motions both before and after surgical repair. The normal and altered carpal kinematic behaviors correlated well with the clinical findings. In the operated wrists, while X-rays demonstrated slight gapping after scapholunate ligament repair, kinematic imaging demonstrated no abnormal widening on dynamic motion and showed normal dorsally hinged “scissoring” type scapholunate motion with active wrist motion. However, normal mid-carpal initiated wrist motion on flexion/extension was replaced by radiocarpal initiated motion, likely because of midcarpal stiffness/scarring. Conclusion: 4 Dimensional CT provides adequate and novel assessment of wrist biomechanics both before and after surgical repair. Twenty-eight percent of all injuries to the musculoskeletal are hand/wrist injuries3“4 Dimensional” CT kinematics were obtained on 6 wrists, 1 volunteer and 4 patients (both wrists in 1 patient) . All subThe patient demographics, clinical findings, X-ray, and 4D CT imaging findings as well as final patient disposition are depicted in The human carpus is a complex system of 7 load sharing bones plus a sesamoid (pisiform) linked between the distal radius and ulna and the metacarpal bases with various static (ligament) and dynamic (musculotendinous units) stabilizers. Conventional static X-rays are used to follow patients longitudinally after injury, along with symptom development and physical examination, to determine whether or not a patient may have a ligamentous injury requiring intervention. The early insensitivity of static radiography for scapholunate ligament instability is well known.-21-19,20,28Patients with wrist instability variably present with symptoms of pain, wrist clunk, and decreased grip strength similar to 18Our understanding of the biomechanics involved in allowing stable motion with and without loading continues to develop. Its evolution has progressed substantially from a tightly linked ball-and-socket type joint to the central column theory described by Navarro,18,19Spiral and multislice imaging built great milestones in the history of CT. 4D kinematic CT offers the next leap forward in CT technology that allows acquisition of isotropic volumes of an entire wrist with a single rotation of the gantry. The ability to acquire the entire wrist from distal forearm to metacarpal heads with one volume scan opens the door to new diagnostic possibilities and can revolutionize patient care. It allows real time visualization of the wrist dynamics during various unconstrained motions, such as supination-pronation, flexion-extension, fist clenching, and radialulnar deviation/dart throwing motions.32Patients with obvious findings necessitating surgical intervention such as the radiographic findings already mentioned, or more complex injuries such as a perilunate or lunate dislocation, may have questionable radiographic findings even after surgical repair/reconstruction. A persistent 3- to 4-mm scapholunate gap suggesting persistent ligamentous instability despite repair on multiple unchanging postoperative X-rays following fixation removal can be common finding that delays initiation of wrist motion rehabilitation and return to normal function, as it was observed in ,The ability to rapidly deliver high-resolution images has made CT a core imaging modality across healthcare today. However, there have been increasing concerns over patient safety due to radiation exposure and ALARA (As Low As Reasonably Achievable) principle should be followed. The extended coverage provided by the 16-cm wide area detector enables high-quality scanning of the entire wrist within one rotation, eliminating the need for helical scanning, which in turns lowers the dose dramatically. Although the extremities are more exposure resistant, there are a number of approaches used in these scans to reduce exposure dose to the patient. These include active collimator, AIDR 3D (Adaptive Iterative Dose Reduction 3D), SUREExposure 3D (automated exposure control) and quantum denoising software. The Active Collimator prevents extra radiation dose by eliminating rays that are not used for image reconstruction. It operates automatically at the start and end of scan range limiting the extent of the X-Ray beam. AIRD 3D is an advanced iterative reconstruction algorithm used in our scanner that reduces noise in both the 3D reconstruction data and raw data domains. SUREExposure 3D is another function that continuously modulates the exposure dose  in all 3 directions based on the patient's wrist shape. With the integration of AIDR 3D into SUREExposure controls, radiation exposure is automatically reduced before the scan, ensuring that the lowest possible dose is employed for the specific diagnostic objective irrespective of the size or shape of the patient. For routine clinical use, with the use of these functions, one is able to remove up to 50% of image noise that corresponds to a dose reduction of up to 75%, ultimately leading to improved spatial resolution and excellent artifact (streak/beam hardening) reduction that are otherwise routinely present at low dose acquisition. There is also minimal penalty in reconstruction times. Quantum Denoising Software is a powerful tool to remove noise in reconstructed images. It works in all dimensions and selectively smoothens out areas of uniform density while preserving the edge information within the image. It also reduces noise and increases the signal-to-noise ratio, while preserving contrast resolution. Finally, Boost3D is a 3D technology that automatically detects streak artifacts, and it eliminates them by reducing the photon starvation effects in the raw data domain. As a result, the image quality is improved in all viewing planes. A technologist training program ensures that operators become proficient in taking advantage of all the dose-reduction technologies available for every scan, providing clinicians with maximum image quality at the lowest possible dose to the patient. We achieved a dose of CT dose index of 10.5mGy and a dose length product of 84.3mGy-cm, close to 2× to 3× that of a static high-resolution scan. The effective dose was only 0.07mSv per scan maneuver . With us28This study had certain limitations. In this pilot study, we did not do reader reliability evaluation or direct comparison of scan quality between 2D, 3D, and 4D reconstruction images, with static high-resolution scan. We did not perform any computational analysis for detection of subtle abnormalities and we only relied on gross visual evaluation of abnormality by the experienced reader. Further longitudinal study is warranted with this technique and should include preoperative and postoperative evaluation of ligamentous injuries in the same subjects for the evaluation of incremental value of this technique over conventional CT examination.To conclude, kinematic 4 dimensional computed tomography (4DCT) provides adequate and novel assessment of wrist biomechanics both before and after surgical repair.


To the Editor: During May and June 2011, a large outbreak of hemolytic uremic syndrome (HUS) and diarrhea caused by Shiga toxin–producing Escherichia coli (STEC) occurred, centered on northern Germany  of 320 of the responders stated they used the cafeterias; 60 employees fulfilled our case definition. Case-patients’ median age was 33 years (range 22–60 years); 36 (60%) of 60 were female. Thirty case-patients were hospitalized; HUS developed in 18 (30%) . DiseaseWe used billing data from the cafeterias’ obligatory cashless payment system to ascertain risk factors for disease. A nested case–control study design was chosen, limited to a fraction of the cohort to obtain rapid risk estimates. Exposures included were purchases of any fruit, salad bar item, dessert, or asparagus dish in either cafeteria from May 2 through May 13. On the basis of customer identification numbers, the caterer provided billing information for persons with early cases (n = 23). Controls were randomly chosen persons from the caterer’s database whose disease status was checked against the survey information (n = 30) and who did not report symptoms of diarrhea (nonbloody), vomiting, or nausea during the same period. Univariable logistic regression was performed.In univariable analysis, salad bar purchases were highly associated with illness , and desserts, fruit, and asparagus dishes were not . Three (These results and the identification of the same rare serotype of O104:H4 renders this a satellite outbreak to the larger outbreak in northern Germany, which is the largest outbreak in terms of HUS ever described worldwide. Sprouts are believed to be the food vehicle (Salmonella enterica serovar Montevideo (Cafeteria billing information allowed for a rapid investigation while avoiding exposure misclassification attributable to ill-remembered food purchases (


Camellia sinensis L.). Dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR) are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of CsDFR and CsANR produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in transgenic lines. The free radical scavenging activity of CsDFR and CsANR transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura. Results suggested that the overexpression of CsDFR and CsANR cDNA in tobacco has improved flavonoids content and antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and biotic stress tolerance.Flavan-3-ols contribute significantly to flavonoid content of tea ( Flavonoids comprise one of the largest groups of plant secondary metabolite. These are widespread throughout the plant kingdom and are found to be accumulated in different organs and tissues of plants. They are mainly involved in providing protection to plants against predation, pathogen (bacteria and fungi) attack, and act as effective repellant and prevent feeding by herbivores Medicago truncatula (MtDFR) has been produced and studied with respect to flavonoids content MtDFR has been reported for altered metabolites profile MtANR has been generated and analyzed for its effect on PAs content ANR1 and ANR2 in Glycine maxANR1 and ANR2 genes from leaf tissue of a blister blight-resistant tea cultivar TRI2043 have been documented for different level of epimerase activity and exhibited similar kinetic properties The genetics and biochemistry of flavonoid biosynthetic pathway has been extensively studied in number of plant species. The broad outline of flavonoid biosynthesis pathway in plants is shown in Camellia sinensis) plant contains an extraordinarily high ∼25–30% of flavan-3-ols on leaf dry weight basis. Recently, progress has been made towards elucidation of flavonoid biosynthesis in tea. A cDNA encoding for DFR  has been cloned from tea cultivar UPASI 10. CsDFR is 1,413 bp full-length cDNA with ORF of 1,044 bp (115–1158) and encoding a protein of 347 amino acid residues + oxidoreductase: EC 1.3.1.77) has been cloned from tea cultivar UPASI 10. CsANR is 1,233 bp full-length cDNA with ORF of 1,014 bp (79–1092) and encoding a protein of 337 amino acid residues. The CsANR cDNA from tea cultivar UPASI 10 showed 97% and 80% homology at nucleotide level with previously reported CsANR2 (Accession no. GU992400) and CsANR1 (Accession no. GU992402) from blister blight-resistant tea cultivar TRI2043, respectively. Hence, this has suggested that CsANR used in this study is CsANR2. Interestingly, CsANR activity has been found to be directly correlated with epicatechin content in tea Flavan-3-ols are the building block of PAs. They are also reported for their antioxidant potential and free radical scavenging activity CsF3H, CsANR and CsDFR encoding enzymes are crucial for flavonoid biosynthesis in tea CsDFR and CsANR encoding for DFR and ANR. To see the influence of CsDFR and CsANR cDNA overexpression, transgenic tobaccos were analyzed for flavonoids content and antioxidant potential. They were further assessed for the influence of these transgenes on their development and protective abilities against biotic stress.Previously, a number of transgenic plants have been developed and characterized with respect to engineer flavonoid biosynthetic pathway CsDFR and CsANR is available at NCBI with Accession number AY64027 and AY641729, respectively. The isolated cDNAs of CsDFR and CsANR from C. sinensis cultivar UPASI-10 were cloned into pCAMBIA 1302 plasmid between Nco I and Bgl II restriction sites. This was resulted in the formation of recombinant construct pCAMBIA-CsDFR and pCAMBIA-CsANR. These constructs were transferred into Agrobacterium tumefaciens strain LBA4404 by triparental mating. A. tumefaciens harboring pCAMBIA-CsDFR and pCAMBIA-CsANR constructs were used for leaf disc transformation of tobacco (Nicotiana tabacum cv Xanthi nc) following the standard transformation protocol CsDFR and CsANR transgene by carrying out PCR with genomic DNA and gene specific primers . The reaction conditions were as follow: 94°C for 1 min, and then 35 cycles of 30 s at 94°C, 30 s at 54°C (for CsDFR) and 58°C (for CsANR), and 2 min at 72°C. PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide. T1 transformants were self-pollinated and the seeds obtained from T1 were analyzed for segregation by germinating on half strength Murashige and Skoog medium supplemented with hygromycin (50 mg/ml). One month old plants were transferred in green house in pots containing the manure, sand and soil in 1∶1∶2 ratios. Tobacco plant transformed with pCAMBIA vector having no transgene CsDFR or CsANR was used as control (mock). The third leaf of three plants of each independent transgenic line as well as control tobacco plant was harvested and used for various sample preparation.The cDNA sequence of 12–18, 200 U of superscript III RT, and 10 mM dNTPs in a 20 µl reaction volume. Equal quantity of cDNA was used as template in PCR with gene-specific primer sets for probing the expression of NtPAL, NtC4H, Nt4CL, NtCHS, NtCHI, NtF3H, NtFLS, NtDFR, NtANR1, NtANR2 and NtANS genes. All primer sequences used in this study are shown , 54°C , 58°C  for 30 s, and 72°C for 30 s for 27 cycles. The amplified products were separated on 1% agarose gel and visualized with ethidium bromide staining. A gel documentation system  was used to scan the gel and changes in the gene expression were analyzed by calculating integrated density values (IDV) using AD-1000 software. The 26S rRNA-based gene primers were used as internal control for relative gene expression studies The 100 mg of leaf tissue of transgenic lines as well as control tobacco plants was ground in liquid nitrogen and total RNA was isolated using RNeasy Plant Mini Kit . The total RNA from each sample was treated with DNase I to remove DNA contamination. cDNA was prepared according to manufacturer's protocol  using 2 µg of total RNA, 250 ng oligo dTre shown . The priCsDFR and CsANR overexpressing tobaccos as well as control tobacco plants. The capsules were counted at their maturation time and seed yield and weight of thousand seeds was measured after harvesting the capsules of CsDFR and CsANR overexpressing transgenic lines as well as control tobacco plants.The parameters namely days to flowering, capsules number, seed yield and thousand seed weight were analyzed. These parameters were analyzed in eight plants of each selected lines of The 250 mg of freeze-dried leaf powder of transgenic lines as well as control tobacco plants was taken for extract preparation. Total flavonoids content in the extract was determined using the previously described method Antioxidant activity of the methanolic extract of leaves of three plants from each transgenic as well as control tobacco plants was performed. The ability of plant extract to oxidize stable DPPH (diphenylpicryl-hydrazyl) free radicals was estimated as described earlier 2O2 for 2 h as described previously Fresh leaves from three plants of each transgenic line as well as control tobacco plants were treated with 5 mM HSpodoptera litura), leaf discs of 6 cm diameter were prepared from transgenic lines as well as control tobacco plants and experiment was performed with some modification as described earlier S. litura and allowed to feed for 7 consecutive days. Experiment was performed in three replicates for each transgenic line. The petri dishes were kept at 27±2°C, 60±5% RH and a photoperiod of 16 h: 8 h (Light: Dark). The growth behavior of cutworm caterpillar was represented as percentage growth inhibition and calculated asTo check the possible anti-feeding behavior of tobacco cutworms (3) and auxin in the form of free indole-3-acetic acid (IAA) content of control as well as transgenic tobacco lines were determined by HPLC method as described earlier 3 and 265 nm for IAA. The GA3 and IAA from Sigma were used as standard for estimation of respective constituent.Endogenous gibberellin  and CsANR (A) flowered after an average of 86 days (D-01), 95 days (D-15), 86 days (D-17), 90 days (A-05), 88 days (A-08), and 85 (A-27) days of seed germination compared to 105 days to flower after seed germination in control tobacco plants -Cat, (−)- EC and (−)-EGC flavan-3-ols were detected in tobacco. The Cat, EC and EGC were found to be increased in both CsDFR and CsANR overexpressing transgenic lines. The Cat level was estimated in range of 0.38–0.5 mg g−1 DW in CsDFR and CsANR overexpressing transgenic lines as compared to 0.07 mg g−1 DW in control tobacco plants  was determined in the transgenic lines vis-à-vis control tobacco plants. Total flavonoids content of control tobacco was 39.8 mg quercetin equivalent (QE) gic lines . To checo plants . Similaro plants . EGC cono plants . Spectrao plants .CsDFR and CsANR overexpressing transgenic lines and control tobacco to scavenge a stable free radical DPPH was measured. Methanolic extract of the leaves of CsDFR and CsANR overexpressing transgenic lines showed higher free radical scavenging activity for DPPH by 70–185% as compared to control tobacco plants  that catalyzes the condensation of 4-coumaroyl-CoA and malonyl-CoAs. CHS catalyzes the first regulatory step and regulates the diversion of carbon flux towards flavonoid biosynthesis pathway. Silencing and overexpression of CHS encoding gene has resulted in a successful alternation of flavonoids in petunia and tomato, respectively ANR genes (ANR1 and ANR2) have also redirected the metabolic flux either towards anthocyanins or PAs biosynthesis PAR (proanthocyanidin regulator). Hence, the overexpression of CsDFR and CsANR might have affected the expression of selected endogenous genes through influencing the regulators or only the genes encoding endogenous regulatory enzymes of flavonoid pathway showed influence at expression level. Further, CsDFR and CsANR were continuously expressed by a constitutive promoter 35S and need continuous substrate in transgenic tobacco. This might be avoiding the feedback inhibition of their expression which is otherwise natural possible situation. The upregulation of transcript level of NtCHS and NtANR2 in transgenic tobacco overexpressing CsDFR and CsANR might further aid to the accumulation of total flavonoids including PAs and monomeric flavan-3-ols.Altered flavonoids content has been reported through metabolic engineering of flavonoid pathway genes in several transgenic plants CsDFR and CsANR overexpressing transgenic lines was histochemically confirmed by using DMACA staining. The elevation in EC and GC  content has also been reported earlier in tobacco co-expressing PAP1 and MtANRMtANR and PAP1 tobacco plants was not affected FLS silencing in tobacco plants. The FLS silencing has inhibited the production of flavonols and directed the carbon flux towards biosynthesis of Cat, EC and EGC The accumulation of flavan-3-ols in CsDFR and CsANR overexpressing transgenic lines showed higher levels of flavonoids, they were evaluated for their free radical scavenging activity. Both types of transgenics have shown higher total free radical scavenging activities than control tobacco plants. Furthermore, the improved total free radical scavenging activity has encouraged us to check the tolerance of plant against oxidative stress. The overexpression of CsDFR and CsANR in transgenic lines has lowered H2O2 mediated cell death, confirming their tolerance against oxidative stress. Similarly, the overexpression of CsDFR in transgenic rice has also been reported to provide cell death tolerance Flavan-3-ols act as antioxidant by scavenging free radicals as well as by breaking the free radical chain reactions S. lituraVaccinium myrtillus infection Helicoverpa zea2 groups S. litura upon feeding on leaves of CsDFR and CsANR overexpressing transgenic tobacco. Higher levels of monomeric and polymeric flavan-3-ols could be responsible for such an anti-herbivores activity. Their higher content in transgenic tobacco might be acting as anti-nutrient and therefore, reduced the digestibility of protein by S. litura either by precipitation of protein or by inhibiting the enzyme activity CsDFR and CsANR overexpressing transgenic tobacco could be the possible reason for providing protection against feeding by herbivores S. litura.Level of phenolic compounds, especially flavonoids in leaf extracts of various plant species have been documented for plant anti-herbivore defense CsDFR and CsANR encoding DFR and ANR enzymatic proteins in transgenic tobacco has induced early flowering and improved seed yield. The CsDFR/CsANR overexpression has increased the accumulation of flavonoids, thereby improved antioxidant potential and redox state of transgenic tobacco plants. Ultimately, improved antioxidant potential upon CsDFR and CsANR overexpression in transgenic tobacco has provided the biotic stress tolerance against S. litura.In conclusion, overexpression of tea cDNA Figure S1Transcript expression analysis of genes encoding various enzymes of flavonoid biosynthetic pathway in CsDFR and CsANR overexpressing transgenic lines as well as control tobacco plants.(TIF)Click here for additional data file.Figure S2HPLC spectra of standard catechin (Cat), epicatechin (EC) and epigallocatechin (EGC) are on the left hand side. On the right hand side, peaks are identified for Cat, EC and EGC from the extracts of transgenic as well as control tobacco plants by comparing with their respective standard.(TIF)Click here for additional data file.Figure S3DMACA stained leaf of CsDFR and CsANR overexpressing transgenic tobacco plant vis-à-vis control tobacco plants.(TIF)Click here for additional data file.Figure S43) and indole-3-acetic acid (IAA) are on the left hand side.HPLC spectra of standard gibberellin Click here for additional data file.Table S1List of primers used for expression analysis of various genes encoding enzymes of flavonoid biosynthetic pathway.(TIF)Click here for additional data file.


P≤2.8E-10 versus 0.018). When compared to other prognosis factors  in a multivariate analysis, the 22-gene predictor remained significantly associated with overall survival. Early prediction of high risk patients (3% of WHO grade II), and low risk patients (29% of WHO grade III) in clinical routine will allow the development of more appropriate follow-up and treatments.Diffuse gliomas are incurable brain tumors divided in 3 WHO grades  based on histological criteria. Grade II/III gliomas are clinically very heterogeneous and their prognosis somewhat unpredictable, preventing definition of appropriate treatment. On a cohort of 65 grade II/III glioma patients, a QPCR-based approach allowed selection of a biologically relevant gene list from which a gene signature significantly correlated to overall survival was extracted. This signature clustered the training cohort into two classes of low and high risk of progression and death, and similarly clustered two external independent test cohorts of 104 and 73 grade II/III patients. A 22-gene class predictor of the training clusters optimally distinguished poor from good prognosis patients  in the validation cohorts. This classification was stronger at predicting outcome than the WHO grade II/III classification ( IDH1 gene are associated with a longer survival than astrocytomas Gliomas are the most frequent primary tumors of the CNS  Over the past ten years, transcriptome profiling has largely been used in cancer to explore patient heterogeneity, define tumor subclasses and predict prognosis. Gene expression profiling of gliomas has been recognized to produce a more robust classification than the conventional histological diagnosis P≤2.8E-10, P = 0.018), it was less efficient at distinguishing the short-surviving population revealed by our signature  and poor prognostic values .To validate our predictor, the NL and NIH datasets were used after normalizing and scaling to fit with QPCR expression level. Grade II gliomas represented a quarter of the NL cohort and half of the NIH cohort according to the WHO classification. This grouping almost superimposed the survival curves of patients with grade II/III for the NL cohort  and sepaP≤5.4E-14, P≤7.7E-05, Applying the predictor built on the training cohort to the independent NL and NIH cohorts separated two groups of patients in a proportion very similar to that obtained in clustering the training cohort (two thirds of good versus one third of poor prognosis). Seventeen out of the 22 predictive genes were individually significant for survival in the NL validation cohort. This model led to a staging with highly significant differential survival  was analyzed using the NL validation cohort for which these molecular data were available. As expected, the absence of 1p19q codeletion or the amplification of EGFR presented a significant higher risk of poor survival in univariate analysis. In this cohort, the absence of IDH1 mutation was surprisingly not associated with a poor outcome. In multivariate analysis of each factor and the PAM prediction, only EGFR amplification remained an independent prognostic factor . Using the latter, grade III gliomas represented more than half of the patients in our training cohort, while our classification showed that only one third was at risk of shorter survival. Thus the grade II/III WHO classification appears to overestimate the number of bad prognosis gliomas. This conclusion is substantiated in the larger validation cohorts, in which half to three-quarter of patients are classified by WHO as grade III while predicted classification only delineates one third of high risk patients. With the longer follow-up in the test populations, all uncensored patients have died by twenty years, but contrary to the WHO classification, PAM prediction was able to delineate a poor and a better prognosis group of gliomas  as well as an early detection of good prognosis patients among grade III gliomas , with a significantly better predictive value than the WHO histological classification as evidenced by cross-tabulation . Such a The “MPL” training cohort included 65 adult patients diagnosed with WHO grade II/III glioma undergoing surgery at Montpellier, Nice and Lille Hospitals during 2004–2007 without prior chemical or radiation therapy. All samples were processed in accordance with European bioethics laws regarding patient information: written consent was obtained from participants, tumor collection was accepted by the Center for Biological Collections of the Montpellier University Hospital (#AC-2009–889) and the ethics committee CPP Sud-Méditerranée IV approved this study (#CPP030601). Affymetrix U133 Plus 2.0 microarray data, histological staging and outcome for two validation cohorts of WHO grade II/III glioma patients “NL” , n = 73 At the time of resection, one sample for each tumor was immediately frozen and stored at −80°C and another sample was fixed in 4% formalin, embedded in paraffin, sectioned (3 µm) and then stained with hematoxylin-eosin. The histopathological subtypes and grades of glioma were determined by two independent pathologists following the revised WHO 2007 classification TBP gene (termed Ntarget), were determined with the following formula: Ntarget = 2ΔCtsample, where the ΔCt value of the sample was determined by subtracting the average Ct value of the target gene from the average Ct value of the TBP gene. Missing values were replaced by the minimal value of the gene expression across the cohort, and expression signals were scaled positive as log2(Ntarget ×1000).Quantitative real-time RT-PCR (QPCR) was performed as described previously P-value, their QPCR reproducibility and their relevance to cancer and glioma. In addition, we included 5 genes  which displayed a strong variation of expression from one glioma sample to another within the same grade, suggesting that these genes could identify glioma subgroups. Expression of this 38-gene list was then analyzed by QPCR on the whole MPL cohort including the five grades II/III samples of the preliminary screening. Elimination of genes not significantly relevant to overall survival was achieved using a univariate Cox-model analysis of the expression of each of these genes on overall survival with multiple testing correction We first quantified the expression of a list of 365 preselected genes in 5 samples of grade II glioma (3 oligodendrogliomas and two mixed tumors), 5 grade III gliomas , 5 control brain tissues derived from epileptic resections and 5 GBM samples using QPCR as described above. Analysis of this quantification revealed 45 genes with a 2 fold increase between grade II and III gliomas while 47 genes displayed a 2 fold decrease. In addition, we performed Mann-Whitney tests between grade II and III for these 365 genes to identify those showing a minimal fold change but with a strong discrimination score between the two glioma grades. This allowed us to select 11 additional genes. From this first list of 103 (45+47+11) genes, we selected 33 of them based on their level of expression, their fold change and individual http://www.R-project.org) and Bioconductor Computations were performed using R  Individual gene expression in the training cohort. (B and C) The optimal number of genes in the predictor corresponds to the minimum number of misclassification errors. (D) The class score of each selected centroid is plotted according to its class incidence. In all plots, the red color represents poor prognosis genes.(PDF)Click here for additional data file.Figure S2Incidence of histopathological subtypes of gliomas on overall survival. Kaplan-Meier curves were designed and log-rank tests performed on both WHO and our PAM classifications for all cohorts either unseparated  or separated into their histological components, astrocytomas , mixed  or oligodendrogliomas .(PDF)Click here for additional data file.Table S1List of 365 initially selected genes with annotations.(XLS)Click here for additional data file.Table S2Primers and expression of 38 genes in the training cohort. Values represent n-fold differences in target gene expression relative to the TBP reference gene.(XLS)Click here for additional data file.Table S3Clinical characteristics of patient cohorts.(PDF)Click here for additional data file.Table S4Twenty-seven genes significant in univariate Cox model analysis of overall survival in training cohort after multiple testing correction.(PDF)Click here for additional data file.


A-dependent excitation in isolated post-synaptic CA1 hippocampal neurons because it may constitute a trigger for pathological synchronous epileptiform discharge. In particular, the interplay intracellular chloride accumulation via the GABAA receptor and extracellular potassium accumulation via the K/Cl co-transporter KCC2 in promoting GABAA-mediated excitation is complex. Experimentally it is difficult to determine the ionic mechanisms of depolarizing current since potassium transients are challenging to isolate pharmacologically and much GABA signaling occurs in small, difficult to measure, dendritic compartments. To address this problem and determine plausible ionic mechanisms of GABAA-mediated excitation, we built a detailed biophysically realistic model of the CA1 pyramidal neuron that includes processes critical for ion homeostasis. Our results suggest that in dendritic compartments, but not in the somatic compartments, chloride buildup is sufficient to cause dramatic depolarization of the GABAA reversal potential and dominating bicarbonate currents that provide a substantial current source to drive whole-cell depolarization. The model simulations predict that extracellular K+ transients can augment GABAA-mediated excitation, but not cause it. Our model also suggests the potential for GABAA-mediated excitation to promote network synchrony depending on interneuron synapse location - excitatory positive-feedback can occur when interneurons synapse onto distal dendritic compartments, while interneurons projecting to the perisomatic region will cause inhibition.GABA is the key inhibitory neurotransmitter in the adult central nervous system, but in some circumstances can lead to a paradoxical excitation that has been causally implicated in diverse pathologies from endocrine stress responses to diseases of excitability including neuropathic pain and temporal lobe epilepsy. We undertook a computational modeling approach to determine plausible ionic mechanisms of GABA GABAA-mediated excitation has been suggested as a potential mechanism in diseases of neuronal hyperexcitability, such as epilepsy and neuropathic pain, and in neuroendocrine responses to stress A-mediated excitation and its role in triggering pathological excitation may allow identification of new therapeutic targets to treat diseases of excitability.Despite widespread acceptance of GABA as the transmitter of inhibition in the central nervous system, there is plentiful evidence that GABA can also cause pathological A receptors at frequencies that mimic rapid physiological firing leads to a reproducible cellular level phenomenon in isolated post-synaptic CA1 pyramidal neurons - paradoxical excitatory depolarization following the expected hyperpolarization In experimental slice preparations with glutamatergic blockade, synaptic activation of GABAA-mediated excitation in CA1 pyramidal neurons, the precise contributions of intracellular chloride accumulation and extracellular potassium accumulation to depolarization remain unclear. Some experiments show substantial accumulation of intracellular chloride through the GABAA receptor which causes depolarization of the GABAA reversal potential (EGABA(A)) and may lead to GABA-mediated excitation A-mediated excitation, since these levels are coupled through the primary chloride efflux mechanism, the potassium-chloride co-transporter, KCC2 A-mediated excitation is necessary.Although numerous experimental studies have attempted to reveal the underlying ionic mechanisms of GABAA-mediated excitation we have developed a biophysically detailed computational model of a CA1 pyramidal cell that accounts for the myriad processes contributing to ionic homeostasis. To our knowledge, no computationally based morphological neuron model yet exists that incorporates both chloride and potassium homeostasis mechanisms to allow for simultaneous study of these essential coupled (via KCC2) ionic subsystems To determine ionic mechanisms of GABAA-mediated excitation and represents a foundation for future studies to investigate other disease mechanisms stemming from disruptions in ion homeostasis. The model may also be spatially extended to networks that can be used to investigate how GABAA-mediated depolarization in pyramidal cells triggers and supports sustained epileptiform activity in hippocampal circuits.The model that we present suggests plausible ionic mechanisms of GABAhttp://www.compneuro.org/CDROM/nmorph/index/n123_t.html. The model was modified to include an axon, as described in Poirazi et al. +, K+, Cl−, Ca2+, and HCO3− were based on the intracellular and extracellular concentrations described in Smirnov et al. +, K+, Cl−, and Ca2+ were allowed to fluctuate in both the intracellular and extracellular compartments, except where specified.The computationally based model neuron was morphologically reconstructed from hippocampal pyramidal neuron n123 taken from the published Duke Southhampton neuronal morphology The cable equation A channel and fast sodium channel (described below). All other currents were updated using the explicit Euler method at every time step.The implicit Euler method was also used to update the state of the two currents described by Markov models, the GABAion is the transmembrane current carried by each ion and F is the Faraday constant. Extracellular potassium concentration was buffered to represent glial buffering as in previous models At each time step, intracellular and extracellular ion concentrations were calculated based on the sum of ionic current (from channels and transporters).2/ms: DNa = 1.33, DK = 1.96, DCl = 2.08, DCa = 0.60 The model allowed for longitudinal diffusion of all ions between compartments with the following diffusion constants, in mmnsurface is the connecting surface across which compartment i has a concentration gradient +-Cl- transporter KCC2 was based on a 2-state version of potassium-chloride cotransport previously developed to describe ion homeostasis in the renal distal tubule + and Cl- are from neuronal KCC2 in a heterologous expression system The model for KKCC2 and the current density carried by chloride ions is –IKCC2.The current density carried by potassium ions transported by KCC2 is IThe NKCC1 model is from 1)p(E represents the extracellular probability with no ions bound and 1NaClKCl)pp(E and 2NaClKCl)p/250 if distance <250 and fraction = 1 if distance >250. Therefore, sodium channel conductance is calculated:6state) or five state (O5state) model.Total fast sodium conductance densities were as follows: in the axon, 2 is K,ATPaseI = −2*pumpI and Na,ATPaseI = 3*pump. imaxI was determined by the model algorithm to be 0.0024 mA/cm2 in the soma.The Na/K pump was based on Kager et al. The resting membrane potential of the model neuron is assumed to be constant at steady-state, and so requires balanced ionic homeostasis. The minimum NaKATPase current density at rest must balance sodium and potassium flux via the voltage gated currents and ion transporters described above. We set the NaKATPase current to be twenty percent above this minimum in order to have a sufficient potassium leak to allow the cell to respond to extracellular potassium transients by depolarization, as observed experimentally. Sodium and potassium leak current amplitudes were the final parameters determined in the model, set at rest (−66 mV) to balance ion flux from voltage-gated channels and ion transporters. This led to both a membrane resistance and responses to extracellular potassium transients that fell within experimental values: the membrane resistance is 53 MΩ see , and theA current was modeled using a 6-state Markov model as was done previously by Bai et al. A current was carried by chloride and bicarbonate ions, based on measurements of a bicarbonate:chloride permeability ratio of 1∶4 of GABAA channels GABARate constants were taken from the α1β2γ3 receptor rates in Bai et al. Synaptic density is in in vitro slice preparation experiments, we modeled brief square pulses of GABA transmitter with a maximum concentration of 1 mM for 1 ms, and an exponential decay time constant of 100 microseconds based on experimental evidence stratum radiatum of CA1.To simulate high-frequency electrode stimulation of interneurons in ceclancy@ucdavis.edu.Simulation of “voltage clamp” experiments assumed that only somatic compartments were clamped; voltage in the dendrites were not held constant, based on empirical evidence that voltage clamp in CA1 pyramidal neurons is imperfect and that there is significant attenuation of voltage in the dendritic tree A-mediated depolarization in post-synaptic CA1 pyramidal cells, we developed a computational model that incorporates components critical for ion transport to account for electrical activity in the cell, and importantly, for ion homeostasis. The morphology of the CA1 pyramidal neuron is derived from geometry of a real neuron extracted from cell n123 in the hippocampal neuron database as described in the Methods and shown in In order to better understand the underlying ionic mechanisms of GABA+ homeostasis is maintained by influx via the voltage-gated Na+ channel, a leak current, and the sodium-calcium exchanger NaCaX; efflux occurs via the sodium-potassium pump NaKATPase and the sodium-dependent potassium-chloride transporter NKCC1. K+ homeostasis is preserved by efflux via voltage-gated channels, a K+ leak current, and the potassium chloride transporter KCC2, and influx via the Na-K ATPase and NKCC1. Cl- homeostasis is maintained by influx via GABAA channels and NKCC1 and efflux via KCC2. Ca2+ homeostasis is maintained by influx via voltage-gated channels and efflux via NaCaX and the membrane calcium pump. The intracellular and extracellular bicarbonate concentrations are assumed constant based on experimental data Detailed descriptions of all ionic processes are in the Methods and depicted schematically in h) . Ih density was adjusted to match experimental input resistance and voltage attenuation observed in the dendrites (bottom compared to top panel in A). The model has a somatic input resistance of 53 MΩ, compared to experimental values of 54+/−2 MΩ in vitro CA1 pyramidal neuron We next used experimental data that describe functional properties stemming from ion flux, ion channel kinetics and ion channel densities in CA1 pyramidal cells to guide parameter estimation of key components in our model. + and Cl−, we incorporated a model of the dominant K+-coupled Cl− extrusion mechanism in CA1 cells as suggested by experiments, the neuronal K+-Cl– cotransporter (KCC2) In order to properly account for homeostasis of KA notable feature of CA1 pyramidal cells is activity-dependent attenuation of back-propagating action potentials + channel inactivation in the dendrites Experiments suggest that attenuation of back-propagating action potentials results from an increasing fraction of slow cumulative Nawithout any further tuning of parameters - was sufficient to reproduce experimentally observed post-synaptic GABAA-mediated depolarization in CA1 cells following high frequency stimulation of synaptic GABAA receptors.A-mediated excitation observed experimentally, we applied high frequency GABA pulses in the model to mimic the stimulation protocol in a semi-intact network as in experiments (glutamatergic and GABAB synapses blocked): 40 pulses at 100 Hz were applied to the soma and apical dendrites in the model neuron (applied to stratum radiatum in experiments) A synaptic conductance in the soma and apical dendrites in the model neuron (to simulate a barrage of interneuron activity) during each pulse led to currents with similar time-course and kinetics to those observed experimentally under voltage clamp conditions at −75 mV and −55 mV . Note that the precise magnitude and distribution of the stimulation of GABAA receptors is unknown and will vary from cell to cell. Hyperpolarizing and depolarizing currents of the magnitude observed experimentally were achieved in the model by stimulation of GABAA conductance that was within the experimental estimates of GABAA conductance in CA1 pyramidal cells To determine whether our model had the elements necessary to reproduce the GABAA receptor conductance in the apical dendrites during high frequency stimulation led to membrane potential measurements comparable to those observed experimentally , but was much smaller than in the experiment , suggesting that quinine may have resulted in partial inhibition of GABAA receptors. We estimated, given measured IC50 values, that the experimental quinine concentration of 0.5 mM would have caused a 60% maximum inhibition of the GABAA current. Simulation of this 60% inhibition of the GABAA receptor by quinine resulted in a voltage timecourse very similar to the experimental recording , we observe similar attenuation of GABAA-mediated depolarization as under conditions of fixed extracellular potassium. The model predicts that attenuation of GABAA-mediated depolarization in the absence of extracellular K+ transients results from an increase in KCC2 activity due to a steeper K+ gradient and a consequent increase in Cl- extrusion. Increased Cl- extrusion prevents depolarization of the GABAA reversal potential and diminishes GABAA-mediated depolarization.In the absence of any extracellular potassium transient , reveals that in the dendrites the GABAA reversal potential is depolarized relative to the membrane potential, although the membrane potential is still positive to the reversal potential for chloride (the chloride current component remains inward). The driving force for chloride is nonetheless sufficiently reduced so that chloride current through the GABAA receptor is smaller in magnitude than bicarbonate current, which causes further depolarization  applied to the distal dendrites (greater than 200 µm from the soma) following HFS caused an excitatory response manifested as somatic depolarization and action potential firing . In contnfluence .A synapse stimulation (A conductance (A conductance (100%), the membrane in the distal dendrites is “short-circuited” , spiking probability dropped throughout the course of 400 ms long HFS of GABA receptors , spiking probability was n of HFS .increased KCC2 activity might promote excitability. Indeed, KCC2 transporter activity has two competing effects on the membrane potential. On the one hand, maintenance of EGABA following GABAA stimulation depends on KCC2 activity to extrude chloride from the intracellular space. In this manner, KCC2 activity promotes hyperpolarization by maintaining a hyperpolarized GABA reversal potential. On the other hand, KCC2 chloride transport is coupled to potassium transport out of the cell, leading to increased extracellular potassium, which directly depolarizes the membrane.It has been widely proposed that pathological reductions in KCC2, and the consequent reduction of chloride extrusion, is a likely mechanism of hyperexcitability in the setting of diverse ailments including anxiety, neuropathic pain, and epilepsy A-mediated depolarization. Inhibition of KCC2 activity in the model neuron increases and prolongs GABA-mediated depolarization  in distal dendrites. The model simulations suggest strong depolarization mediated by GABAA current in the distal dendrites is the source of depolarizing current flowing to the soma , extracellular potassium may nevertheless be required for persistent network bursting Our results, do not however, rule out the possible contribution of extracellular potassium transients in sustaining network synchrony following GABAFollowing high-frequency stimulation with intact glutamatergic and GABAergic synaptic transmission, Isomura et al. in vitro? 2) Can such heterogeneity underlie the progression from gamma to beta frequency network firing observed in experimental seizure models, as suggested by Vreugdenhill et al. The implications of the model prediction for network behavior are as follows: Simulations suggest a transient positive-feedback network will be established between the pyramidal cell and bistratified or O-LM cells. A more stable negative-feedback network will occur between the basket cells and the pyramidal cells. Key questions for future studies include: 1) Can the interaction between heterogeneous networks cause seizure-like dynamics seen Despite the drastic reduction in membrane resistance in postsynaptic CA1 pyramidal cells following a brief application of HFS to GABAergic interneurons , CA1 celSelf-sustaining network activity following HFS of GABAergic interneurons is known to be dependent on glutamatergic transmission, yet it is unknown whether it is glutamatergic input to the interneurons or recurrent glutamatergic input to pyramidal cells that is required. Our model simulations predict that during the application of HFS to GABAergic interneurons, only excitatory input to non-tetanized CA1 dendrites will cause phasic excitation, while excitatory input to tetanized CA1 dendrites will be shunted. Immediately following a bout of HFS to GABAergic interneurons, excitatory input anywhere along the CA1 dendritic tree will be enhanced . What roA-mediated chloride accumulation, simulations predict that loss of KCC2 actually increases firing and prolongs GABAA-mediated depolarization in single cells  Drugs targeting extracellular potassium and KCC2 directly affect GABAAThe question of which molecular mechanisms underlie GABAA-mediated depolarization. Our model predicts that functional removal of dendrites beyond the perisomatic region will eliminate the depolarization. This could be tested experimentally by dissociating apical and basal dendrites at various points along the dendritic tree A-mediated depolarization. Alternately, localized application of bicuculline would help to elucidate the role of synapses in different locations. This approach would also allow for testing the model prediction that inhibition of GABAA in the perisomatic region enhances depolarization.The model simulations presented in this study give rise to a set of potentially experimentally testable predictions. One major prediction of our model is the importance of the dendrites in generating the GABAThe model predicts that a secondary stimulus from a basket cell, projecting onto the perisomatic region will be inhibitory while a secondary stimulus from a bistratified cell projecting to apical and basal dendrites may promote further excitation. This could potentially be tested experimentally by recordings in a post-synaptic CA1 cell in response to secondary stimulation of specific subtypes of interneurons.Location dependence of responses may also be relevant to understanding underlying mechanisms of epilepsy. For example, models of epilepsy have shown differential changes in somatic and dendritic GABA synapses A-mediated depolarization in isolated post-synaptic neurons. In other words, KCC2 blockers would be expected to increase seizure trigger events. As more specific KCC2 blockers are developed, this will be testable experimentally. At present, it would be interesting to determine whether tissue from human temporal lobe epilepsy patients is more susceptible to the seizure-like network activity observed in vitro following tetanic stimulation as compared to normal human tissue, since chloride homeostasis mechanisms have been found to be disrupted in such tissue A-mediated excitation because of the diminished capacity for chloride extrusion. Of course, it would also be critical to consider how elevated extracellular potassium affects other network structures and populations of neurons. The less critical role of increased extracellular potassium in mediating the response in our post-synaptic cell model does not preclude the possibility that reductions in KCC2 may be therapeutic by reducing extracellular potassium sufficiently to prevent persistent network bursting A-mediated depolarization in seizure generation.Simulations support the claim that decreases in KCC2 transport will lead to increased GABA