Time Course Of Induction Research Articles

The subtilisin-like protease SBT3 contributes to insect resistance in tomato.

Subtilisin-like proteases (SBTs) constitute a large family of extracellular plant proteases, the function of which is still largely unknown. In tomato plants, the expression of SBT3 was found to be induced in response to wounding and insect attack in injured leaves but not in healthy systemic tissues. The time course of SBT3 induction resembled that of proteinase inhibitor II and other late wound response genes suggesting a role for SBT3 in herbivore defense. Consistent with such a role, larvae of the specialist herbivore Manduca sexta performed better on transgenic plants silenced for SBT3 expression (SBT3-SI). Supporting a contribution of SBT3 to systemic wound signaling, systemic induction of late wound response genes was attenuated in SBT3-SI plants. The partial loss of insect resistance may thus be explained by a reduction in systemic defense gene expression. Alternatively, SBT3 may play a post-ingestive role in plant defense. Similar to other anti-nutritive proteins, SBT3 was found to be stable and active in the insect's digestive system, where it may act on unidentified proteins of insect or plant origin. Finally, a reduction in the level of pectin methylesterification that was observed in transgenic plants with altered levels of SBT3 expression suggested an involvement of SBT3 in the regulation of pectin methylesterases (PMEs). While such a role has been described in other systems, PME activity and the degree of pectin methylesterification did not correlate with the level of insect resistance in SBT3-SI and SBT3 overexpressing plants and are thus unrelated to the observed resistance phenotype.

Time lapse imaging analysis of the effect of ER stress modulators on apoptotic cell assessed by caspase3/7 activation in NG108-15 cells.

This paper reports the data from the long term time lapse imaging of neuronal cell line NG108-15 that were treated with apoptosis inducer or various ER stress inducers. Use of the fluorescent reporter for activated caspase3/7 in combination with the conventional light microscope allowed us to investigate the time course of apoptosis induction at the single cell level. Quantitative as well as qualitative data are presented here to show the effect of two different ER stress modulating chemical compounds on caspase3/7-dependent apoptosis in neuronal cell line NG108-15 cells. Additional results and interpretation of our data concerning ER stress and apoptosis in NG108-15 cells can be found in Suga et al. (2015) [1] and in Suga et al. (2015) [2].

IGF-I and IGFBP-2 Stimulate AMPK Activation and Autophagy, Which Are Required for Osteoblast Differentiation.

IGF-I/insulin-like growth factor binding protein 2 (IGFBP-2) coordinately stimulate osteoblast differentiation but the mechanisms by which they function have not been determined. AMP-activated protein kinase (AMPK) is induced during differentiation and AMPK knockout mice have reduced bone mass. IGF-I modulates AMPK in other cell types; therefore, these studies determined whether IGF-I/IGFBP-2 stimulate AMPK activation and the mechanism by which AMPK modulates differentiation. Calvarial osteoblasts and MC-3T3 cells expressed activated AMPK early in differentiation and AMPK inhibitors attenuated differentiation. However, expression of constitutively activated AMPK inhibited differentiation. To resolve this discrepancy we analyzed the time course of AMPK induction. AMPK activation was required early in differentiation (day 3-6) but down-regulation of AMPK after day 9 was also necessary. IGF-I/IGFBP-2 induced AMPK through their respective receptors and blocking-receptor activation blocked AMPK induction. To determine the mechanism by which AMPK functioned we analyzed components of the autophagosome. Activated AMPK stimulated ULK-1 S555 phosphorylation as well as beclin-1 and microtubule-associated protein 1A/1B light-chain phosphatidylethanolamine conjugate (LC3II) induction. Inhibition of AMPK attenuated these changes and direct inhibition of autophagy inhibited differentiation. Conversely, expression of activated AMPK was associated with persistence of these changes beyond day 9 and inhibited differentiation. Blocking AMPK activation after day 9 down-regulated these autophagosome components and rescued differentiation. This allowed induction of mechanistic target of rapamycin and AKT, which suppressed autophagy. The results show that early induction of AMPK in response to IGF-I/IGFBP-2 followed by suppression is required for osteoblast differentiation. AMPK functions through stimulation of autophagy. The findings suggest that these early catabolic changes are important for determining the energy source for osteoblast respiration and down-regulation of these components may be required for induction of glycolysis, which is required during the final anabolic stages of differentiation.

On the time course of rapid and repeatable response induction

As reinforcers are delivered for a particular response, that response increases. Other similar responses also increase, with the extent to which this increase is seen being positively related to the similarity between the responses. This process, called induction, is pronounced during initial response acquisition, and dissipates as the target response is established. The present experiment examined this process in a paradigm wherein responses to one of five nose poke receptacles results in reinforcement for six sessions in each condition and the target location changes between conditions. Consistent patterns of induction were seen across conditions, with patterns of induction dissipating within each condition.

40. Transgene Product-Specific Treg Are Initially Induced in the Celiac Lymph Node During Tolerance Induction By Hepatic AAV Gene Transfer

Previous work by us and several other laboratories has shown that liver-directed gene transfer can induce transgene product-specific immune tolerance. Induction of CD4+CD25+FoxP3+ regulatory T cells (Treg) is a critical component of the tolerance mechanism. Gaps in knowledge exist about the identity of the antigen-presenting cells (APCs) and the origin of peripherally induced Treg. To characterize the interaction of adeno-associated virus (AAV) antigen expression in the liver with immune cells, we used AAV8-EF1a vectors, which have a high tropism for murine liver, expressing either a secreted ovalbumin (AAV8-ova) or cytoplasmic ovalbumin (AAV8-Cytoova). In Treg-deficient mice transgenic for an ova-specific CD4+ T cell receptor (DO11.10-tg Rag-2-/- BALB/c), Treg were induced with either vector. For secreted ova, multiple organs (liver, spleen, lymph nodes [LN]) showed a similar level and time course of Treg induction, with greater than 1% of the CD4+ T cells being Treg starting at 4 weeks. Substantial T cell activation using the very early activation marker CD69 was also observed in these tissues starting at 3 weeks after AAV8-ova. However, Treg induction was observed in the celiac LN (one of the liver-draining LN) as early as 3 weeks after AAV8-Cyto-ova gene transfer, followed by induction of Treg in other tissues by 4 weeks. Interestingly, T cell activation was notably higher in liver and celiac LN after AAV8-Cyto-ova with over 70% activated CD4+ T cells after 4 weeks. In addition, T cell activation was observed earlier in all tissues after AAV8-Cyto-ova when compared to AAV8-ova. Literature data document a role for tolerogenic antigen presentation in the liver itself via sinusoidal endothelial cells and possibly hepatocytes. Our data suggest an important role of conventional mechanisms of adaptive immunity involving draining LNs that survey tissue antigens leading to early T cell activation and Treg induction of hepatic expressed non-systemic antigens. To start defining the critical APCs, BALBc mice were given AAV8-ova vector two weeks prior to adoptive transfer of CellTrace Violet-labeled CD4+ T cells from DO11.10 donor mice and their proliferation was measured using flow cytometry after 5 days. Half of these mice had their Kupffer cells and M1 macrophages inactivated with Gadolinium chloride for two days prior to DO11.10 adoptive transfer. DO11.10 cells proliferated in an antigen-specific manner, and proliferation was blocked by Gadolinium chloride, supporting a role for professional APCs in presentation of hepatic transgene product. Our data suggest and reinforce the importance of Kupffer cells as key APCs for hepatic antigens and further characterize the impact of the route of antigen expression on T cell activation and Treg induction.

Levetiracetam accelerates the onset of supply rate depression in synaptic vesicle trafficking.

To determine if levetiracetam (LEV) enhances the impact in excitatory presynaptic terminals of a rate-limiting mechanism in vesicle trafficking termed supply rate depression that emerges to limit synaptic transmission during heavy, epileptiform use. The effect of LEV was measured with electrophysiologic assays of monosynaptic connections in ex vivo hippocampal slices from wild-type and synapsin knockout mice, and in primary cell culture neurons from wild-type and synaptic vesicle glycoprotein 2a (SV2a) knockout mice. LEV enhanced the impact of supply rate depression at Schaffer collateral synapses by shortening the time course for induction. The LEV effect was selective for supply rate depression because other presynaptic vesicle trafficking mechanisms were not affected. The half maximal effective concentration (EC50 ) was ~50 μm. The maximal effect was ~15% and occurred at 100 μm, which is a clinically relevant concentration. An experimental protocol is established for distinguishing atypical antiepileptic drugs (AEDs) that affect supply rate depression, such as LEV, from typical AEDs, such as carbamazepine, that affect upstream mechanisms. The LEV effect was abolished at synapses from knockout mice lacking SV2a and from synapses lacking synapsin 1 and 2. The findings are consistent with the new hypothesis that LEV acts to treat epilepsy by accelerating the induction of supply rate depression at excitatory synapses during incipient epileptic activity. The absence of the effect in the knockouts confirms that presynaptic function is the target. More specifically, the absence in SV2a knockouts is consistent with previous binding studies suggesting that SV2a is the target for LEV. The absence in synapsin knockouts indicates that the phenotypic target intersects with the biochemical pathway that is altered in synapsin knockouts. The results from synapsin knockouts additionally suggest that development of functional analogs with increased potency might be possible because induction of supply rate depression is faster in synapsin knockouts compared to wild-type synapses treated with LEV.

Therapeutic window for cyclooxygenase-2 related anti-inflammatory therapy after status epilepticus

As a prominent inflammatory effector of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) mediates brain inflammation and injury in many chronic central nervous system (CNS) conditions including seizures and epilepsy, largely through its receptor subtype EP2. However, EP2 receptor activation might also be neuroprotective in models of excitotoxicity and ischemia. These seemingly incongruent observations expose the delicacy of immune and inflammatory signaling in the brain; thus the therapeutic window for quelling neuroinflammation might vary with injury type and target molecule. Here, we identify a therapeutic window for EP2 antagonism to reduce delayed mortality and functional morbidity after status epilepticus (SE) in mice. Importantly, treatment must be delayed relative to SE onset to be effective, a finding that could be explained by the time-course of COX-2 induction after SE and compound pharmacokinetics. A large number of inflammatory mediators were upregulated in hippocampus after SE with COX-2 and IL-1β temporally leading many others. Thus, EP2 antagonism represents a novel anti-inflammatory strategy to treat SE with a tightly-regulated therapeutic window.

Exercise-induced irisin secretion is independent of age or fitness level and increased irisin may directly modulate muscle metabolism through AMPK activation.

Irisin has been proposed to be a myokine mediating the effect of exercise on adipocyte browning. The physiology of irisin in humans is not completely understood. To study the physiology of irisin in healthy individuals with different age and fitness levels and to explore the direct effects of irisin on muscle metabolism. Treadmill exercise studies were conducted to measure circulating irisin at baseline and in response to exercise among old and young, physically active and sedentary individuals. Also, high- and moderate-intensity swimming was performed in adolescent men and women to study the effect of exercise intensity and the time course of irisin induction by acute bouts of exercise. Human myotubes were treated with recombinant irisin, and the effect on gene expression, cell signaling, and metabolism was examined. Baseline circulating irisin was lower in old (vs young) and physically active (vs sedentary) subjects. Despite differences in basal levels, the percentage increase of irisin by acute bouts of exercise was not related to age or fitness level. The time course study revealed that circulating irisin increased immediately after high-intensity interval exercise and declined 1 hour thereafter. In vitro experiments showed that irisin facilitates glucose and lipid metabolism in human muscle through AMP kinase phosphorylation. Despite the differences in basal irisin levels, exercise-induced irisin secretion is independent of age or fitness level. Increased irisin can directly modulate muscle metabolism through AMP kinase activation.

Carbonic anhydrase induction in euryhaline crustaceans is rate-limited at the post-transcriptional level

The transfer of euryhaline crustaceans from full-strength seawater to low salinity results in both a rapid up-regulation of carbonic anhydrase (CA; EC 4.2.1.1) mRNA and a slow induction of CA activity. There is a delay of several days between the two processes, which is attributed to the time required to synthesize new enzyme. These delays may also be due to limitations in the cellular uptake of Zn, which is a required post-translational active site modification to CA. To investigate these processes, the euryhaline crabs, Callinectes sapidus and Carcinus maenas, were acclimated to salinities below their isosmotic points (22.5 and 25ppt, respectively) for 7days to activate the physiological and molecular mechanisms of osmoregulation. CA mRNA increased 90-fold in C. sapidus and 2-fold in C. maenas within 6h; whereas it took 48h for the initial increases in CA activity (120% and 31%), and 4 to 7days for new acclimated levels (300% and 100%, respectively). Crabs were then transferred to lower salinities (10 and 15ppt) to induce further CA activity and to determine if previous increases in CA mRNA reduced the time required for subsequent CA induction. Additionally, the expression of the Zn transporter ZIP1 was examined in C. sapidus at 35 and 22.5ppt. In both species, prior CA mRNA elevation failed to accelerate the rate of CA induction. Levels of CA mRNA did not change in either crab following transfer from intermediate to low salinity. Taken together, these results show that the timecourse of CA induction at low salinity is not limited by the expression of CA mRNA, but by the synthesis of new enzyme from an existing pool of mRNA. No increases in ZIP1 expression occurred at low salinity, therefore these delays may be due to the limits of cellular Zn uptake.

Macromolecular Crowding Amplifies Adipogenesis of Human Bone Marrow-Derived Mesenchymal Stem Cells by Enhancing the Pro-Adipogenic Microenvironment

The microenvironment plays a vital role in both the maintenance of stem cells in their undifferentiated state (niche) and their differentiation after homing into new locations outside this niche. Contrary to conventional in-vitro culture practices, the in-vivo stem cell microenvironment is physiologically crowded. We demonstrate here that re-introducing macromolecular crowding (MMC) at biologically relevant fractional volume occupancy during chemically induced adipogenesis substantially enhances the adipogenic differentiation response of human bone marrow-derived mesenchymal stem cells (MSCs). Both early and late adipogenic markers were significantly up-regulated and cells accumulated 25-40% more lipid content under MMC relative to standard induction cocktails. MMC significantly enhanced deposition of extracellular matrix (ECM), notably collagen IV and perlecan, a heparan sulfate proteoglycan. As a novel observation, MMC also increased the presence of matrix metalloproteinase -2 in the deposited ECM, which was concomitant with geometrical ECM remodeling typical of adipogenesis. This suggested a microenvironment that was richer in both matrix components and associated ligands and was conducive to adipocyte maturation. This assumption was confirmed by seeding undifferentiated MSCs on decellularized ECM deposited by adipogenically differentiated MSCs, Adipo-ECM. On Adipo-ECM generated under crowding, MSCs differentiated much faster under a classical differentiation protocol. This was evidenced throughout the induction time course, by a significant up-regulation of both early and late adipogenic markers and a 60% higher lipid content on MMC-generated Adipo-ECM in comparison to standard induction on tissue culture plastic. This suggests that MMC helps build and endow the nascent microenvironment with adipogenic cues. Therefore, MMC initiates a positive feedback loop between cells and their microenvironment as soon as progenitor cells are empowered to build and shape it, and, in turn, are informed by it to respond by attaining a stable differentiated phenotype if so induced. This work sheds new light on the utility of MMC to tune the microenvironment to augment the generation of adipose tissue from differentiating human MSCs.

Nigerian bonny‐light crude oil induces alteration in testicular stress response proteins and caspase‐3 dependent apoptosis in albino wistar rats

In the past few decades, there has been much concern about the adverse health effects of environmental contaminants in general and Crude Oil in particular around the Niger Delta region of Nigeria where all the crude Oil exploration is taking place. Studies have shown the repro-toxic effects of Bonny-light crude oil (BLCO). However, the insight into the mechanisms of gonadal toxicity induced by BLCO is not well known. In this study, we sought to elucidate the mechanism(s) underpinning the gonadal effects within hours of exposure to BLCO. Experimental rats were divided into five groups of four each. Animals were orally administered with a single dose of BLCO (800 mg/kg body weight) and killed at 0, 6, 12, 24, and 72 h post-treatment. The levels and time-course of induction of stress response proteins and apoptosis-related proteins like cytochorome C, caspase 3 and procaspase 9, Fas-FasL, NF-kB and TNF-α were determined to assess sequential induction of apoptosis in the rat testis. DNA damage was assessed by TUNEL assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptotis- related proteins as early as 6 h following exposure. Time-dependent elevations in the levels of the proteins were observed. The DNA damage was measured and showed time-dependent increase in the TUNEL positive cells of testicular cells. The study demonstrates induction of testicular apoptosis in adult rats following exposure to a single dose of BLCO.

Process for Production of Recombinant Baboon Uricase in Escherichia Coli Rosetta (DE3)

ABSTRACTHyperuricaemia is a serious purine metabolism disorder which may lead to gout, nephrolithiasis and other serious health problems. As a key enzyme responsible for the hydrolysis of uric acid to allantoin in the purine degradation pathway, uricase, especially recombinant uricase, shows great potential in the therapy of hyperuricaemia. In this paper, baboon uricase gene attached with Trx and hexahistidine tags was cloned and expressed in Escherichia coli Rosetta (DE3). The target protein formed a soluble structure in the cytoplasm at 25°C, with a gradual enrichment in the induction time course, reaching its highest level at 6 h. After purification and maturation, the final yield of mature baboon uricase was 136.0 mg/L with enzyme activity of 17.93 U/mg. The Km of recombinant uricase was 6.15 7mu;mol/L. And the optimum temperature and pH of the prepared uricase were 37 ⪚C and 8.2, respectively. MALDI-TOF-MS/MS characterization confirmed the prepared protein was identical with the native baboon uricase. These results showed that the baboon uricase gene had been expressed effectively in E. coli and the obtained protein possessed promising enzymatic properties, which gives a new candidate for the therapy of hyperuricaemia.

Dynamic changes in genomic histone association and modification during activation of the ASNS and ATF3 genes by amino acid limitation

Amino acid deprivation of mammalian cells triggers several signalling pathways, the AAR (amino acid response), that results in transcriptional activation. For the ASNS (asparagine synthetase) and ATF3 (activating transcription factor 3) genes, increased transcription occurs in conjunction with recruitment of ATF4 to the gene. In HepG2 cells, analysis of the ASNS and ATF3 genes during AAR activation revealed increases in histone H3K4me3 (histone 3 trimethylated Lys4) and H4Ac (acetylated histone 4) levels, marks associated with active transcription, but a concurrent loss of total H3 protein near the promoter. The dynamic nature of AAR-regulated transcription was illustrated by a decline in ASNS transcription activity within minutes after removal of the AAR stress and a return to basal levels by 2h. Reversal of ASNS transcription occurred in parallel with decreased promoter-associated H4Ac and ATF4 binding. However, the reduction in histone H3 and increase in H3K4me3 were not reversed. In yeast, persistence of H3K4me3 has been proposed to be a 'memory' mark of gene activity that alters the responsiveness of the gene, but the time course and magnitude of ASNS induction was unaffected when cells were challenged with a second round of AAR activation. The results of the present study document changes in gene-associated nucleosome abundance and histone modifications in response to amino-acid-dependent transcription.

Subunit-specific Incorporation Efficiency and Kinetics in Mitochondrial Complex I Homeostasis

Studies employing native PAGE suggest that most nDNA-encoded CI subunits form subassemblies before assembling into holo-CI. In addition, in vitro evidence suggests that some subunits can directly exchange in holo-CI. Presently, data on the kinetics of these two incorporation modes for individual CI subunits during CI maintenance are sparse. Here, we used inducible HEK293 cell lines stably expressing AcGFP1-tagged CI subunits and quantified the amount of tagged subunit in mitoplasts and holo-CI by non-native and native PAGE, respectively, to determine their CI incorporation efficiency. Analysis of time courses of induction revealed three subunit-specific patterns. A first pattern, represented by NDUFS1, showed overlapping time courses, indicating that imported subunits predominantly incorporate into holo-CI. A second pattern, represented by NDUFV1, consisted of parallel time courses, which were, however, not quantitatively overlapping, suggesting that imported subunits incorporate at similar rates into holo-CI and CI assembly intermediates. The third pattern, represented by NDUFS3 and NDUFA2, revealed a delayed incorporation into holo-CI, suggesting their prior appearance in CI assembly intermediates and/or as free monomers. Our analysis showed the same maximum incorporation into holo-CI for NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS4, NDUFA2, and NDUFA12 with nearly complete loss of endogenous subunit at 24 h of induction, indicative of an equimolar stoichiometry and unexpectedly rapid turnover. In conclusion, the results presented demonstrate that newly formed nDNA-encoded CI subunits rapidly incorporate into holo-CI in a subunit-specific manner.

Time Course, Distribution and Cell Types of Induction of Transforming Growth Factor Betas following Middle Cerebral Artery Occlusion in the Rat Brain

Transforming growth factor-βs (TGF-β1–3) are cytokines that regulate the proliferation, differentiation, and survival of various cell types. The present study describes the induction of TGF-β1–3 in the rat after focal ischemia at 3 h, 24 h, 72 h and 1 month after transient (1 h) or permanent (24 h) middle cerebral artery occlusion (MCAO) using in situ hybridization histochemistry and quantitative analysis. Double labeling with different markers was used to identify the localization of TGF-β mRNA relative to the penumbra and glial scar, and the types of cells expressing TGF-βs. TGF-β1 expression increased 3 h after MCAO in the penumbra and was further elevated 24 h after MCAO. TGF-β1 was present mostly in microglial cells but also in some astrocytes. By 72 h and 1 month after the occlusion, TGF-β1 mRNA-expressing cells also appeared in microglia within the ischemic core and in the glial scar. In contrast, TGF-β2 mRNA level was increased in neurons but not in astrocytes or microglial cells in layers II, III, and V of the ipsilateral cerebral cortex 24 h after MCAO. TGF-β3 was not induced in cells around the penumbra. Its expression increased in only a few cells in layer II of the cerebral cortex 24 h after MCAO. The levels of TGF-β2 and -β3 decreased at subsequent time points. Permanent MCAO further elevated the levels of all 3 subtypes of TGF-βs suggesting that reperfusion is not a major factor in their induction. TGF-β1 did not co-localize with either Fos or ATF-3, while the co-localization of TGF-β2 with Fos but not with ATF-3 suggests that cortical spreading depolarization, but not damage to neural processes, might be the mechanism of induction for TGF-β2. The results imply that endogenous TGF-βs are induced by different mechanisms following an ischemic attack in the brain suggesting that they are involved in distinct spatially and temporally regulated inflammatory and neuroprotective processes.

When herbivores come back: effects of repeated damage on induced resistance

Summary Plants are known to respond to damage with subsequent changes in resistance. The consequences of these changes for plant fitness and herbivore populations will depend on both the response of a plant to a particular attack at a given moment and on how plants respond through time to varying levels of damage and varying numbers of attacks. While a small number of studies document how induced resistance changes with time after attack (time course of induction) and different levels of damage, few studies have examined the cumulative level of resistance after multiple attacks, and little is known about how the plant's response to damage changes with subsequent attacks. Two experiments were conducted to address the consequences of repeated damage for resistance of tomato (Solanum lycopersicum, var Castlemart) to a common pest, the beet armyworm (Spodoptera exigua). The first experiment documented the time course of resistance following a single damage event using both growth and choice bioassays. The second experiment examined whether the plant responded differently to one versus two damage events. Results show that plants were significantly induced by day 1 and remained induced until 15 or 20 days later, suggesting that repeated damage during the response to initial damage is possible. Plants receiving a second bout of damage were able to further increase their resistance level over the level reached in response to a first bout of damage, but the magnitude of response to the second damage event was initially smaller and slower than the response to a single damage event. There was no evidence in this study for immune‐like memory in induced resistance. Results of this study suggest that plants can respond to repeated damage, but that there is some limit on responses to repeated damage. Such limits on total plant resistance will affect the influence of induced resistance on herbivore populations and are consistent with assumptions of existing models of induced resistance and herbivore population dynamics, although models have not yet considered the consequences of slower rather than smaller responses to repeated damage.

Using GFP Video to Track 3D Movement and Conditional Gene Expression in Free-Moving Flies

BackgroundIn vivo imaging and quantification of fluorescent reporter molecules is increasingly useful in biomedical research. For example, tracking animal movement in 3D with simultaneous quantification of fluorescent transgenic reporters allows for correlations between behavior, aging and gene expression. However implementation has been hindered in the past by the complexity of operating the systems.ResultsWe report significant technical improvements and user-friendly software (called FluoreScore) that enables tracking of 3D movement and the dynamics of gene expression in adult Drosophila, using two cameras and recorded GFP videos. Expression of a transgenic construct encoding eGFP was induced in free-moving adult flies using the Gene-Switch system and RU486 drug feeding. The time course of induction of eGFP expression was readily quantified from internal tissues including central nervous tissue.ConclusionsFluoreScore should facilitate a variety of future studies involving quantification of movement behaviors and fluorescent molecules in free-moving animals.

Different brain potentials evoked at distinct phases of rule learning

The neural mechanisms of rule learning are of interest to cognitive neuroscientists, but the time course of rule induction and the related brain potential remain unclear. In this study, event-related brain potentials (ERPs) were measured during the distinct phases of rule induction. Participants in two experiments were presented with a series of Arabic numbers and were asked to detect the hidden rules. The ERP results revealed that (a) the rule-discovery trials elicited a larger P3 component than the nondiscovery trials, reflecting the initial identification of the regularity of number series, and (b) when a new instance was incongruent with the previously acquired rule, a larger N2 and enhanced late positive component were elicited, reflecting the process of mismatch detection and the updating of working memory context.

Abstract 3980: Computational analysis of paclitaxel-induced tumor priming that leads to enhanced deposition and efficacy nanoparticulate doxorubicin

Abstract Purpose: Paclitaxel (PAC) has been shown to cause tumor priming (TP), which increases delivery of subsequently-administered drugs. A wave of PAC-mediated apoptosis results in a transient reduction in tumor cell density, expansion of the interstitial space, and vascular permeability compromise that enhances tumor deposition of nanoparticulate formulations such as liposomal doxorubicin (L-dox). Our aim was to develop an integrated quantitative pharmacokinetic/pharmacodynamic (PK/PD) analysis approach to optimize combination chemotherapy of PAC with L-dox. Methods: Data were extracted from the study of Lu et al. (J Pharmacol Exp Ther 322: 80, 2007) in which mice bearing human tumor xenografts were treated with PAC (40 mg/kg), L-dox (20 mg/kg), TP dosing (PAC followed by L-dox at 48h, the peak of the priming effect), or the reverse sequence (L-dox then PAC). Data included plasma and tumor drug concentrations, kinetics of PAC and L-dox induced apoptosis, and % change in tumor volume from baseline. Drug profiles were described with a PK model for carrier-mediated PAC, and a two-compartment model for L-dox, with first-order release of free drug from the liposome. Intratumor concentrations were described using a hybrid well-stirred model with estimated partition coefficients (kp) for each drug. Tumor PK profiles were used to drive apoptotic responses, which were modeled using nonlinear time dependent signal transduction functions. The tumor growth PD model consisted of two transit compartments, with a first order net growth rate constant and the apoptotic signals from each agent driving cytotoxic effects. PAC induced TP was modeled using a feedback loop, with the apoptosis signal of PAC enhancing the deposition of L-dox (i.e. increasing kp for dox). Model fittings were performed using MATLAB, and simulations were conducted to explore priming sequences. Results: The final model captured well the PK of PAC and L-dox in plasma and tumors, and the time course of apoptosis induction, and tumor growth for each treatment sequence. With single agent dosing, kp values for PAC and L-dox were estimated at 0.044 and 0.085. L-dox kp increased 2-fold after the TP treatment. Apoptosis signals exhibited a delayed onset that was well captured, and the intratumor concentrations producing maximum effects (Emax) and 50% Emax were 18 and 7.2 μg/mL (PAC) and 17.6 and 14.3 μg/mL (L-dox). The duration of drug induced apoptosis was 27.4 h for PAC and 15.8 h for L-dox. Simulation with the PK/PD model suggested that earlier administration of L-dox would increase efficacy markedly. Conclusions: A model was successfully developed that captured the priming effect on PK and efficacy. Simulation suggested that administration of L-dox 24 h prior to the priming peak would enhance efficacy further. This model could be adapted for evaluating other combination chemotherapies using PAC as a TP agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3980. doi:1538-7445.AM2012-3980

Kinetic profiling of the c-Myc transcriptome and bioinformatic analysis of repressed gene promoters

Mammalian c-Myc is a member of a small family of three related proto-oncogenic transcription factors. c-Myc has an unusually broad array of regulatory functions, which include roles in cell cycle and apoptosis, a variety of metabolic functions, cell differentiation, senescence, and stem cell maintenance. c-Myc modulates the expression of a very large number of genes, but the magnitude of the majority of the regulatory effects is only 2-fold or less. c-Myc can both activate and repress the promoters of its target genes. Identification of genes directly regulated by c-Myc has been an enduring question in the field. We report here microarray expression profiling of a high resolution time course of c-Myc induction, using fibroblast cells in which c-Myc activity can be modulated from null to physiological. The c-Myc transcriptome dataset presented is the largest reported to date with 4,186 differentially regulated genes (1,826 upregulated, 2,360 downregulated, 1% FDR). The gene expression patterns fit well with the known biological functions of c-Myc. We describe several novel findings and present tools for further data mining. Although the mechanisms of transcriptional activation by c-Myc are well understood, how c-Myc represses an even greater number of genes remains incompletely described. One mechanism involves the binding of c-Myc to other, positively acting transcription factors, and interfering with their activities. We identified rapid-response genes likely to be direct c-Myc targets, and analyzed the promoters of the repressed genes to identify transcription factors that could be targets of c-Myc repression.