Time Course Of Inhibition Research Articles

Target Site of Prepulse Inhibition of the Trigeminal Blink Reflex in Humans.

Despite the clinical significance of prepulse inhibition (PPI), the mechanisms are not well understood. Herein, we present our investigation of PPI in the R1 component of electrically induced blink reflexes. The effect of a prepulse was explored with varying prepulse test intervals (PTIs) of 20-600 ms in 4 females and 12 males. Prepulse-test combinations included the following: stimulation of the supraorbital nerve (SON)-SON [Experiment (Exp) 1], sound-sound (Exp 2), the axon of the facial nerve-SON (Exp 3), sound-SON (Exp 4), and SON-SON with a long trial-trial interval (Exp 5). Results showed that (1) leading weak SON stimulation reduced SON-induced ipsilateral R1 with a maximum effect at a PTI of 140 ms, (2) the sound-sound paradigm resulted in a U-shaped inhibition time course of the auditory startle reflex (ASR) peaking at 140 ms PTI, (3) facial nerve stimulation showed only a weak effect on R1, (4) a weak sound prepulse facilitated R1 but strongly inhibited SON-induced late blink reflexes (LateRs) with a similar U-shaped curve, and (5) LateR in Exp 5 was almost completely absent at PTIs >80 ms. These results indicate that the principal sensory nucleus is responsible for R1 PPI. Inhibition of ASR or LateR occurs at a point in the startle reflex circuit where auditory and somatosensory signals converge. Although the two inhibitions are different in location, their similar time courses suggest similar neural mechanisms. As R1 has a simple circuit and is stable, R1 PPI helps to clarify PPI mechanisms.SIGNIFICANCE STATEMENT Prepulse inhibition (PPI) is a phenomenon in which the startle response induced by a startle stimulus is suppressed by a preceding nonstartle stimulus. This study demonstrated that the R1 component of the trigeminal blink reflex shows clear PPI despite R1 generation within a circuit consisting of the trigeminal and facial nuclei, without startle reflex circuit involvement. Thus, PPI is not specific to the startle reflex. In addition, PPI of R1, the auditory startle reflex, and the trigeminal late blink reflex showed similar time courses in response to the prepulse test interval, suggesting similar mechanisms regardless of inhibition site. R1 PPI, in conjunction with other paradigms with different prepulse-test combinations, would increase understanding of the underlying mechanisms.

Transient Inhibition of the JAK1/3-STAT5 Pathway By Ruxolitinib Is Insufficient to Produce Clinical Benefit in Patients with Indolent Adult T-Cell Leukemia

▪ INTRODUCTIONAdult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of regulatory T cells caused by human T-cell leukemia virus type 1 (HTLV-1) infection. Viral protein Tax is a key factor in malignant transformation. Peripheral blood mononuclear cells (PBMCs) collected from patients with smoldering and chronic-subtype ATL exhibited ex vivo spontaneous proliferation associated with Tax-dependent transactivation of two autocrine loops (IL-2/IL-2Rα, IL-15/1L-15Rα), and one paracrine loop (IL-9/IL-9Rα). Accordingly, antibodies directed against IL-2Rα, IL-9, and IL-15 inhibited proliferation of ATL cells. As the three cytokine/cytokine receptor loops act through downstream activation of the JAK1/3 and STAT5 signaling pathways, addition of the pan-JAK inhibitor tofacitinib and JAK1/2 inhibitor ruxolitinib also inhibited proliferation. Furthermore, ruxolitinib, when administered by continuous intravenous infusion, inhibited ATL cell proliferation in NOG mice. METHODSThese observations led to a phase 2 open-label trial evaluating the safety and efficacy of ruxolitinib in patients with smoldering, chronic, or clinically indolent acute subtype ATL. Ruxolitinib was given at the FDA-approved dose of 20 mg orally twice daily for 28 days. Patients who responded to initial treatment could restart at the time of progression and be treated until progression. Primary objective was the clinical response rate per Revised Cheson Criteria and the International Consensus Meeting Criteria for ATL. Secondary objectives were 1) safety profile, 2) time to progression, 3) overall survival, and 4) duration of response off treatment. RESULTS5 patients with smoldering, 3 with chronic, and 2 with previously treated acute ATL were enrolled between October 2012 and July 2016. There were no grade 3/4 toxicities attributable to the study drug. Cytopenias were the most commonly reported adverse events, including two grade 3 neutropenias (table 1). One patient had a partial response lasting 2.4 months. Median time to progression was 1.6 months and median overall survival was 22.3 months. Three patients with partial or near-partial response were re-treated after a median of 5.7 months. To determine the time course of inhibition of the JAK-STAT pathway the concentration of phosphorylated STAT3 or STAT5 in ex vivo PBMCs was determined after an oral dose of 20 mg of ruxolitinib. Levels of phosphorylated STAT5 returned to baseline after 3-6 hours following the oral dose (figure 1), indicating that the pathway remained active for up to 18 hours per day when the drug was given twice daily. DISCUSSIONBoth of ruxolitinib's FDA approvals-for polycythemia vera and myelofibrosis-were based on reduction in spleen size. Its inhibitory effect on JAK2-mediated thrombopoietin signaling lead to thrombocytopenia as the main dose-limiting toxicity and precluded aggressive dose titration. In our study, single-agent ruxolitinib at the FDA-approved dose was safe but not effective in patients with indolent subtypes of ATL, likely due to lack of persistent JAK1/STAT5 inhibition. Further studies should therefore focus on 1) combinations of ruxolitinib with agents showing synergy on high-throughput screens, such as the Bcl-XL inhibitor navitoclax, and 2) more potent inhibitors of JAK1/3, like baricitinib, which was not associated with significant anemia or thrombocytopenia in a phase 3 trial. Ultimately, the future of indolent ATL treatment may lie in a multidrug combination that includes a specific JAK1 or JAK3 inhibitor. Considering the pervasive activation of the gamma cytokine JAK/STAT pathway in all T-cell malignancies, with up to 86% of patients manifesting pSTAT in the nucleus, the future of all T-cell malignancies may involve a specific JAK1 or JAK3 inhibitor. [Display omitted] DisclosuresMiljkovic:Pfizer Oncology: Research Funding.

Human serine racemase is inhibited by glyceraldehyde 3-phosphate, but not by glyceraldehyde 3-phosphate dehydrogenase

Murine serine racemase (SR), the enzyme responsible for the biosynthesis of the neuromodulator d-serine, was reported to form a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in SR inhibition. In this work, we investigated the interaction between the two human orthologues. We were not able to observe neither the inhibition nor the formation of the SR-GAPDH complex. Rather, hSR is inhibited by the hGAPDH substrate glyceraldehyde 3-phosphate (G3P) in a time- and concentration-dependent fashion, likely through a covalent reaction of the aldehyde functional group. The inhibition was similar for the two G3P enantiomers but it was not observed for structurally similar aldehydes. We ruled out a mechanism of inhibition based on the competition with either pyridoxal phosphate (PLP) – described for other PLP-dependent enzymes when incubated with small aldehydes – or ATP. Nevertheless, the inhibition time course was affected by the presence of hSR allosteric and orthosteric ligands, suggesting a conformation-dependence of the reaction.

A Novel Study Design Using Continuous Intravenous and Intraduodenal Infusions of Midazolam and Voriconazole for Mechanistic Quantitative Assessment of Hepatic and Intestinal CYP3A Inhibition.

The extent of a drug-drug interaction (DDI) mediated by cytochrome P450 (CYP) 3A inhibitors is highly variable during a dosing interval, as it depends on the temporal course of victim and perpetrator drug concentrations at intestinal and hepatic CYP3A expression sites. Capturing the time course of inhibition is therefore difficult using standard DDI studies assessing changes in area under the curve; thus, a novel design was developed. In a 4-period changeover pilot study, 6 healthy men received intraduodenal or intravenous infusions of the CYP3A substrate midazolam (MDZ) at a rate of 0.26 mg/h for 24 hours. This was combined with intraduodenal or intravenous infusion of the CYP3A inhibitor voriconazole (VRZ), administered at rates of 7.5 mg/h from 8 to 16 hours and of 15 mg/h from 16 to 24 hours, after starting midazolam administration. Plasma and urine concentrations of VRZ, MDZ, and its major metabolites were quantified by liquid chromatography-tandem mass spectrometry and analyzed by semiphysiological population pharmacokinetic nonlinear mixed-effects modeling. A model including mechanism-based inactivation of the metabolizing enzymes (maximum inactivation rate constant kinact , 2.83 h-1 ; dissociation rate constant , 9.33 μM) described the pharmacokinetics of VRZ well. By introducing competitive inhibition by VRZ on primary and secondary MDZ metabolism, concentration-time profiles, MDZ and its metabolites were captured appropriately. The model provides estimates of local concentrations of substrate and inhibitor at the major CYP3A expression sites and thus of the respective dynamic extent of inhibition. A combination of intravenous and intraduodenal infusions of inhibitors and substrates has the potential to provide a more accurate assessment of DDIs occurring in both gut wall and liver.

Effect of sound pressure level on contralateral inhibition underlying duration-tuned neurons in the mammalian inferior colliculus.

Duration tuning in the mammalian inferior colliculus (IC) is created by the interaction of excitatory and inhibitory synaptic inputs. We used extracellular recordings and paired tone stimulation to measure the strength and time course of the contralateral inhibition underlying duration-tuned neurons (DTNs) in the IC of the awake bat. The onset time of a short, best duration (BD), excitatory probe tone set to +10 dB (re threshold) was varied relative to the onset of a longer-duration, nonexcitatory (NE) suppressor tone whose sound pressure level (SPL) was varied. Spikes evoked by the roving BD tone were suppressed when the stationary NE tone amplitude was at or above the BD tone threshold. When the NE tone was increased from 0 to +10 dB, the inhibitory latency became shorter than the excitatory first-spike latency and the duration of inhibition increased, but no further changes occurred at +20 dB (re BD tone threshold). We used the effective duration of inhibition as a function of the NE tone amplitude to obtain suppression-level functions that were used to estimate the inhibitory half-maximum SPL (ISPL50). We also measured rate-level functions of DTNs with single BD tones varied in SPL and modeled the excitatory half-maximum SPL (ESPL50). There was a correlation between the ESPL50 and ISPL50, and the dynamic range of excitation and inhibition were similar. We conclude that the strength of inhibition changes in proportion to excitation as a function of SPL, and this feature likely contributes to the amplitude tolerance of the responses of DTNs. NEW & NOTEWORTHY Duration-tuned neurons arise from excitatory and inhibitory synaptic inputs offset in time. We measured the strength and time course of inhibition to changes in sound level. The onset of inhibition shortened while its duration lengthened as the stimulus level increased from 0 to +10 dB re threshold; however, no further changes were observed at +20 dB. Excitatory rate-level and inhibitory suppression-level response functions were strongly correlated, suggesting a mechanism for level tolerance in duration tuning.

Human serine racemase is nitrosylated at multiple sites

Serine racemase is a pyridoxal 5′‑phosphate dependent enzyme responsible for the synthesis of d‑serine, a neuromodulator of the NMDA receptors. Its activity is modulated by several ligands, including ATP, divalent cations and protein interactors. The murine orthologue is inhibited by S-nitrosylation at Cys113, a residue adjacent to the ATP binding site. We found that the time course of inhibition of human serine racemase by S-nitrosylation is markedly biphasic, with a fast phase associated with the reaction of Cys113. Unlike the murine enzyme, two additional cysteine residues, Cys269, unique to the human orthologue, and Cys128 were also recognized as S-nitrosylation sites through mass spectrometry and site-directed mutagenesis. The effect of S-nitrosylation on the fluorescence of tryptophan residues and on that of the pyridoxal phosphate cofactor indicated that S-nitrosylation produces a partial interruption of the cross-talk between the ATP binding site and the active site. Overall, it appears that the inhibition results from a conformational change rather than the direct displacement of ATP.

Time course of inhibition of return in a spatial cueing paradigm with distractors

Studies of endogenous and exogenous attentional orienting in spatial cueing paradigms have been used to investigate inhibition of return, a behavioral phenomenon characterized by delayed reaction time in response to recently attended locations. When eye movements are suppressed, attention is covertly oriented to central or peripheral stimuli. Overt orienting, on the other hand, requires explicit eye movements to the stimuli. The present study examined the time course of slowed reaction times to previously attended locations when distractors are introduced into overt and covert orienting tasks. In a series of experiments, manual responses were required to targets following central and peripheral cues at three different cue-target intervals, with and without activated oculomotor systems. The results demonstrate that, when eye movements are suppressed, behavioral inhibition is reduced or delayed in magnitude by the presence of a distractor relative to conditions without distractors. However, the time course of behavioral inhibition when eye movements are required remains similar with or without distractors.

The Time Course of Inhibition of Return: Evidence from Steady-State Visual Evoked Potentials.

Inhibition of return (IOR) refers to slower responses to targets at a previously cued location than that at an uncued location. The time course of IOR has long been a topic of interest in the field. Investigations into the time course of IOR are typically performed by examining the magnitude of IOR under various cue-target onset asynchrony (CTOA) conditions. Therefore, the results are vulnerable to influence of factors that could affect the target processes (e.g., the frequency of the target type). In the present study, steady-state visual evoked potentials (SSVEPs) were implemented to directly take a continuous measurement of the degree to which cued location is processed, eliminating the influence mentioned above. The results indicate that, relative to the baseline interval (−400 to 0 ms), the presence of peripheral cues generated a typical two-stage effect on the SSVEP amplitude evoked by a 20 Hz flicker. Specifically, after the onset of the peripheral cues, the SSVEP amplitude first showed a significant increase, which subsequently turned into a significant inhibition effect after 200 ms. These results provide a continuous time course diagram of the cueing effect and suggest an effective way for future investigations of controlling the masking effects of target stimuli processing on IOR.

Abstract 4351: Effects of epigenetic agents on methylation of DNA in vitro and in vivo, as measured with stable isotopically labeled methionine

Abstract Background: Modulation of cytidine methylation in DNA continues to be actively investigated as a target for chemotherapy. The primary source of methyl groups is methionine, via S-adenosyl-methionine (SAM) as the direct methyl donor for DNA methyl transferases, e.g., DNMT1. Methods were developed using stable isotopes and mass spec detection to examine the flux of the DNMT reaction and modification to this flux by cytidine analogs such as decitabine, an established hypomethylation agent, and 5-aza-4’-thio-2’-deoxy-cytidine (AzaTdC), currently under development at NCI. Methods: For in vitro studies; A549, H23, HCT116, H522, and OVCAR3 cell lines grown in RPMI1640 media supplemented with 10% FBS were exposed to D3-methionine(20uM) and either decitabine or AzaTdC (NSC77586) at 0 to 5uM in the media for 0 to 48hr. For in vivo studies; mice were maintained under standard ACUC protocols and practices. Tumors were grown SC in the flank area. Stable labeled methionine, decitabine and AzaTdC were administered as an oral bolus. At set times, mice were sacrificed and tumors, tissues and plasma samples collected. DNA was extracted and purified from cells and tissues using PureLink Genomic DNA kits (Invitrogen) according to manufacturer’s instruction. The purified DNA was digested to nucleosides for analysis. The isotopic enrichment in 5-methyl-deoxy-cytidine (mdC) was determined by analysis using HPLC coupled to a QE mass spectrometer (Thermo Scientific). Results: D3-methionine was found to be an efficient precursor via SAM for the methylation of cytidine in DNA. Fortification of basal media (100uM methionine) with 20uM D3-methionine yielded D3-mdC enrichment of 5-15% in 24 hr, depending on cell line. All cell lines were found to have some enrichment. Treatment of cells with either decitabine or AzaTdC caused a concentration dependent reduction of the enrichment for mdC in DNA. In mice, after an oral dose of D3-methionine, enrichment of methionine and SAM in plasma peaked within 1hr and fell to 5% of peak levels within 5hr. Methionine and SAM enrichment in tissues followed similar time curves. At 50mg/kg of D3-methionine, no perturbation was observed for endogenous levels of either methionine or SAM. Enrichment of mdC in DNA was detectable in 1hr, maximal in 5hr, and remained constant for up to 48hr. When mice were treated with 1-2 mg/kg of either decitabine or AzaTdC along with D3-methionine, the level of enrichment of mdC in DNA was reduced by 20-70% in tumors, bone marrow and intestine. Conclusions: Stable labeled methionine can be efficiently used to monitor the flux of methyl groups into mdC in DNA, both in vitro and in vivo. Changes in the flux of the methylation reaction can be used to assess the extent and time course for inhibition caused by cytidine analogs. These techniques may have relevance for improvement of doses and schedules to produce epigenetic modulation. Funded by NCI Contract No. HHSN261200800001E Citation Format: Lawrence Anderson, Eva Majerova, Kimberly D. Hill, John Carter, Jesse Stottlemyer, Howard Stotler, Melinda G. Hollingshead, Jerry M. Collins. Effects of epigenetic agents on methylation of DNA in vitro and in vivo, as measured with stable isotopically labeled methionine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4351. doi:10.1158/1538-7445.AM2017-4351

Prefrontal functional connectivity measured with near-infrared spectroscopy during smoking cue exposure.

Cue reactivity (CR) is an important concept for relapse in substance use disorders (SUD). Although cue exposure (CE) therapy is discussed as relapse prevention, current approaches still need improvement considering its efficacy. From a neurobiological perspective, CR is related to an over-activation in sensitized subcortical structures, their projections to motivationally relevant cortical structures (e.g. orbitofrontal cortex, OFC) and deficient prefrontal inhibitory control. Therefore, we analyzed prefrontal cortical activation and its relation to craving during smoking CE. We focused on the OFC-as a projection area of sensitized subcortical structures-due its importance in the processing of reinforcement value and the dorsolateral prefrontal cortex (dlPFC) based on its importance for behavioral inhibition. Functional near-infrared spectroscopy (fNIRS) was used to assess hemodynamics in prefrontal regions during smoking CE in 24 subjects (n = 12 occasional smokers, n = 12 controls). Subjective craving intensity (minimum craving as marker of baseline inhibition, range as marker of inhibition time course) was additionally assessed. Craving ratings indicated that CR was elicited solely in smokers, not controls. Those subjective ratings correlated with hemodynamic activity in OFC (craving range) and dlPFC (minimum craving). OFC activation was found earlier throughout the CE in smokers compared to controls. Connectivity (seed-based correlation) between OFC and dlPFC was increased in smokers. fNIRS can capture prefrontal hemodynamic activity involved in CR elicited during CE and is therefore a promising method to investigate CR and its implications for relapse prevention in SUD.

Peripheral vision, perceptual asymmetries and visuospatial attention in young, young-old and oldest-old adults

ObjectivesThe present study investigated possible changes occurring in peripheral vision, perceptual asymmetries and visuospatial attention in oldest-old adults and compared their performance with that of young and young-old adults. MethodWe examined peripheral vision (PV) and perceptual asymmetries in the three age groups for stimuli varying in eccentricity (Experiment 1). In Experiment 2, designed to investigate possible changes in spatial attention, the same participants performed an exogenous orienting attention task. ResultsExperiment 1 showed that the three age groups performed the task similarly but differed in processing speed. Importantly, the oldest-old group showed a different perceptual pattern than the other groups suggesting a lack of specificity in visual asymmetries. Experiment 2 indicated that the validity effects emerged later in the young-old and even later in the oldest-old participants, showing a delayed time course of inhibition of return (IOR). Orienting effects, however, were preserved with age. DiscussionTaken together, these results indicate that the three age groups displayed similar perceptual and orienting attention patterns, but with differences in processing speed. Importantly, age (only in the oldest-old adults) altered perceptual visual asymmetries. These results suggest that some neural plasticity is still present even in oldest-old adults, but a lack of specificity occurs in advanced age.

Different types of retinal inhibition have distinct neurotransmitter release properties.

Neurotransmitter release varies between neurons due to differences in presynaptic mechanisms such as Ca(2+) sensitivity and timing. Retinal rod bipolar cells respond to brief dim illumination with prolonged glutamate release that is tuned by the differential release of GABA and glycine from amacrine cells in the inner retina. To test if differences among types of GABA and glycine release are due to inherent amacrine cell release properties, we directly activated amacrine cell neurotransmitter release by electrical stimulation. We found that the timing of electrically evoked inhibitory currents was inherently slow and that the timecourse of inhibition from slowest to fastest was GABAC receptors > glycine receptors > GABAA receptors. Deconvolution analysis showed that the distinct timing was due to differences in prolonged GABA and glycine release from amacrine cells. The timecourses of slow glycine release and GABA release onto GABAC receptors were reduced by Ca(2+) buffering with EGTA-AM and BAPTA-AM, but faster GABA release on GABAA receptors was not, suggesting that release onto GABAA receptors is tightly coupled to Ca(2+). The differential timing of GABA release was detected from spiking amacrine cells and not nonspiking A17 amacrine cells that form a reciprocal synapse with rod bipolar cells. Our results indicate that release from amacrine cells is inherently asynchronous and that the source of nonreciprocal rod bipolar cell inhibition differs between GABA receptors. The slow, differential timecourse of inhibition may be a mechanism to match the prolonged rod bipolar cell glutamate release and provide a way to temporally tune information across retinal pathways.

Mechanism of β-lactam action in Streptococcus pneumoniae: the piperacillin paradox.

The human pathogen Streptococcus pneumoniae has been treated for decades with β-lactam antibiotics. Its resistance is now widespread, mediated by the expression of mosaic variants of the target enzymes, the penicillin-binding proteins (PBPs). Understanding the mode of action of β-lactams, not only in molecular detail but also in their physiological consequences, will be crucial to improving these drugs and any counterresistances. In this work, we investigate the piperacillin paradox, by which this β-lactam selects primarily variants of PBP2b, whereas its most reactive target is PBP2x. These PBPs are both essential monofunctional transpeptidases involved in peptidoglycan assembly. PBP2x participates in septal synthesis, while PBP2b functions in peripheral elongation. The formation of the "lemon"-shaped cells induced by piperacillin treatment is consistent with the inhibition of PBP2x. Following the examination of treated and untreated cells by electron microscopy, the localization of the PBPs by epifluorescence microscopy, and the determination of the inhibition time course of the different PBPs, we propose a model of peptidoglycan assembly that accounts for the piperacillin paradox.

Abstract 1835: Transcriptome and metabolome reprogramming in EGFR-mutant NSCLC early adaptive drug-escape against erlotinib

Abstract Background. Advanced NSCLC with sensitizing EGFR mutation is being treated with EGFR inhibitor first-line. Despite initial tumor response, targeted therapy invariably fails due to acquired resistance. Classical studies of acquired drug resistance focusing on late clinical events in rebiopsy studies have identified some resistance mechanisms including T790M-EGFR, MET amplification and AXL upregulation. Methods. HCC827 and PC-9 NSCLC cells (sensitizing EGFR exon 19-del) were used in vitro and in vivo drug-sensitive models. MTS viability assay, TLVM cell mobility assay, immunoblot and immunofluorescence and in vivo xenograft IHC analysis were performed. Transcriptome analysis of HCC827 cells under erlotinib inhibition time-course was performed using Affymetrix microarray gene expression profiling (Human GeneChip 1.0 ST arrays) in triplicate at 0 hr, 8 hr, 9 days of erlotinib-treatment, and 9 days pretreatment with erlotinib followed by 7 days drug-washout. Data analysis was performed using PCA, heatmap, BAMarray and PathwayStudio 6.0 analysis. We also performed mass-spectrometry-based global profiling analysis of cellular metabolome in 5 replicate. Results. We analyzed drug-sensitive HCC827 and PC-9 cells under erlotinib inhibition to characterize the adaptive response that engenders drug resistance. We identified an early adaptive drug-escape that emerged after 9 days of erlotinib, characterized with MET-independent mitochondrial BCL-2/BCL-xL prosurvival priming to result in 100-fold IC50 increase towards resistance. These cells displayed a quiescence state associated with retarded cell proliferation/cytoskeletal functions. Transcriptome profiling analysis of the early adaptive resistant HCC827 cells revealed a remarkable genome-wide adaptive reprogramming of gene expression signature, involving pathways of cell adhesion, cell cycle regulation, cell division/mitosis, glycolysis and gluconeogenesis, response to DNA damage stimulus, and DNA repair. Metabolomic profiling revealed a global adaptive metabolic reprogramming also, with resultant suppression of glucose and TCA cycle metabolism, and lipid bioenergetics. Our studies also identified autocrine TGFβ2 signaling pathway in mediating the early adaptive resistance in the mutated-EGFR lung cancer cells that escaped erlotinib inhibition through a combination of quiescence-state and EMT, cellular metabolic reprogramming and STAT3/BCL-2/BCL-xL mitochondrial prosurvival priming. Conclusion. Early adaptive drug-escape emerges within a subpopulation of EGFR-mutant NSCLC under erlotinib inhibition. This constitutes an early-stage minimal residual disease leading to eventual resistant tumor relapse. These early drug-escaping tumor cells undergo global cellular reprogramming with upregulated mitochondrial prosurvival priming, representing attractive therapeutic targets to eradicate drug resistance. Citation Format: Patrick C. Ma, Praveena S. Thiagarajan, Patrick Leahy, Rakesh Bagai, Ivy Shi, Wei Zhang, Yan Feng, Martina L. Veigl, Daniel Lindner, David Danielpour, Lihong Yin. Transcriptome and metabolome reprogramming in EGFR-mutant NSCLC early adaptive drug-escape against erlotinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1835. doi:10.1158/1538-7445.AM2014-1835

Tracing the time course of n - 2 repetition costs in task switching.

In order to flexibly adapt to a permanently changing environment, it is necessary to inhibit previously activated but now irrelevant processing pathways. Empirically, this inhibition manifests itself only indirectly in terms of a cost of reengaging a previously inhibited pathway (n - 2 repetition costs). While imaging studies suggest an involvement of the prefrontal cortex in this type of inhibition, it has recently been argued that the underlying processes are implicated not in triggering inhibition, but in overcoming it. To disentangle these processes on a behavioral level, we investigated the time course of inhibition using a cued task switching paradigm. The response-cue interval (between the response of trial n - 1 and the cue of trial n) was varied in five steps to capture its influence on inhibition in a fine-grained manner. The results suggest that the impact of inhibitory processes increases during the first 200-300 ms after the response of the previous trial, reaches its full extent with about 300 ms, and starts to diminish after that. Therefore, future research on the neural correlates of n - 2 repetition costs should employ techniques with a high temporal resolution that are able to capture this presumed time course of inhibitory processes.

Opposite effects of weak transcranial direct current stimulation on different phases of short interval intracortical inhibition (SICI)

Short interval intracortical inhibition (SICI) is a common paired-pulse TMS technique that is used to measure GABAa-ergic inhibition in the cerebral motor cortex. However, inhibition evaluated with an interstimulus interval (ISI) between the TMS pulses of 2.5ms has quite different properties from that seen at 1ms. It is thought that the latter may represent either (or both) a different type of synaptic inhibition or refractoriness of neural membranes. The present experiments provide further evidence about the early and late components of SICI using transcranial direct current stimulation (tDCS), a technique thought to change neural excitability by polarising the nerve membranes. We assessed SICI using a threshold tracking method at a range of ISIs during concurrent application of tDCS in 11 healthy volunteers (8 males, 27-43years old). Each subject underwent both anodal and cathodal tDCS with two different intensities of stimulation (1 and 2mA). Because there was no significant difference between the results at the two intensities, the data were combined. Principal component analysis was used to separate the contributions of early and late SICI to the time course of inhibition from 1 to 5ms tDCS had opposite effects on early and late SICI. Anodal tDCS reduced late SICI but enhanced early SICI, whereas cathodal tDCS had the opposite effect. This is further evidence that the two phases of SICI are produced by different mechanisms, perhaps involving different sets of neurones or different locations on the same neurone that respond oppositely to tDCS.

Liver-stage malaria parasites vulnerable to diverse chemical scaffolds

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. All high-throughput malaria drug discovery efforts have focused on the cyclic blood stage, which has limited potential for the prophylaxis, transmission blocking, and eradication efforts that will be needed in the future. To address these unmet needs, a high-throughput phenotypic liver-stage Plasmodium parasite screen was developed to systematically identify molecules with liver-stage efficacy. The screen recapitulates liver-stage infection by isolating luciferase-expressing Plasmodium berghei parasites directly from the salivary glands of infected mosquitoes, adding them to confluent human liver cells in 384-well plates, and measuring luciferase activity after a suitable incubation period. Screening 5,375 known bioactive compounds identified 37 liver-stage malaria inhibitors with diverse modes of action, as shown by inhibition time course experiments. Further analysis of the hits in the Food and Drug Administration-approved drug subset revealed compounds that seem to act specifically on the liver stage of infection, suggesting that this phase of the parasite's life cycle presents a promising area for new drug discovery. Notably, many active compounds in this screen have molecular structures and putative targets distinctly different from those of known antimalarial agents.

Setting the Time Course of Inhibitory Synaptic Currents by Mixing Multiple GABAAReceptor α Subunit Isoforms

The kinetics of IPSCs influence many neuronal processes, such as the frequencies of oscillations and the duration of shunting inhibition. The subunit composition of recombinant GABA(A) receptors (GABA(A)Rs) strongly affects the deactivation kinetics of GABA-evoked currents. However, for GABAergic synapses, the relationship between subunit composition and IPSC decay is less clear. Here we addressed this by combining whole-cell recordings of miniature IPSCs (mIPSCs) and quantitative immunolocalization of synaptic GABA(A)R subunits. In cerebellar stellate, thalamic relay, and main olfactory bulb (MOB) deep short-axon cells of Wistar rats, the only synaptic α subunit was α1, and zolpidem-sensitive mIPSCs had weighted decay time constants (τ(w)) of 4-6 ms. Nucleus reticularis thalami neurons expressed only α3 as the synaptic α subunit and exhibited slow (τ(w) = 28 ms), zolpidem-insensitive mIPSCs. By contrast, MOB external tufted cells contained two α subunit types (α1 and α3) at their synapses. Quantitative analysis of multiple immunolabeled images revealed small within-cell, but large between-cell, variability in synaptic α1/α3 ratios. This corresponded to large cell-to-cell variability in the decay (τ(w) = 3-30 ms) and zolpidem sensitivity of mIPSCs. Currents evoked by rapid application of GABA to patches excised from HEK cells expressing different mixtures of α1 and α3 subunits displayed highly variable deactivation times that correlated with the α1/α3 cDNA ratio. Our results demonstrate that diversity in the decay of IPSCs can be generated by varying the expression of different GABA(A)R subunits that alone confer different decay kinetics, allowing the time course of inhibition to be tuned to individual cellular requirements.

Characterization of Plasmodium Liver Stage Inhibition by Halofuginone

Malaria is a devastating parasitic disease that afflicts one-third of the world's population. Antimalarial drugs in common use address few targets, and their efficacy is being undermined by parasite resistance. Most therapeutics target blood-stage malaria, whereas only few compounds are active against malaria's liver stage, the first stage of the Plasmodium parasite's life cycle within the human host. The identification of inhibitors active against liver-stage malaria would benefit the development of chemical probes to elucidate the poorly understood biology of this phase of the parasite life cycle and could provide agents to prevent and eliminate the disease. Herein we report the development of a live-cell parasite traversal assay in 384-well format amenable to high-throughput screening that exploits the wounding of liver cells by the parasite. This method identifies small molecules that may inhibit the parasite's actin-myosin motor system. The traversal assay, in addition to established methods, was used to evaluate the activity of halofuginone, a synthetic halogenated derivative of the natural alkaloid febrifugine, against liver-stage Plasmodium berghei parasites. Halofuginone was found to inhibit P. berghei sporozoite load in HepG2 cells with an IC(50) value of 17 nM. While the compound does not affect parasite traversal through human liver cells, an inhibition time course assay indicates that it affects essential processes in both early- and late-stage parasite development.

Casiopeina II-gly and bromo-pyruvate inhibition of tumor hexokinase, glycolysis, and oxidative phosphorylation

The copper-based drug Casiopeina II-gly (CasII-gly) shows potent antineoplastic effect and diminishes mitochondrial metabolism on several human and rodent malignant tumors. To elucidate whether CasII-gly also affects glycolysis, (a) the flux through the complete pathway and the initial segment and (b) the activities of several glycolytic enzymes of AS-30D hepatocarcinoma cells were determined. CasII-gly (IC₅₀ = 0.74-6.7 μM) was more effective to inhibit 24-72 h growth of several human carcinomas than 3-bromopyruvate (3BrPyr) (IC₅₀ = 45-100 μM) with no apparent effect on normal human-proliferating lymphocytes and HUVECs. In short-term 60-min experiments, CasII-gly increased tumor cell lactate production and glycogen breakdown. CasII-gly was 1.3-21 times more potent than 3BrPyr and cisplatin to inhibit tumor HK. As CasII-gly inhibited the soluble and mitochondrial HK activities and the flux through the HK-TPI glycolytic segment, whereas PFK-1, GAPDH, PGK, PYK activities and HPI-TPI segment flux were not affected, the data suggested glycogenolysis activation induced by HK inhibition. Accordingly, glycogen-depleted as well as oligomycin-treated cancer cells became more sensitive to CasII-gly. The inhibition time-course of HK by CasII-gly was slower than that of OxPhos in AS-30D cells, indicating that glycolytic toxicity was secondary to mitochondria, the primary CasII-gly target. In long-term 24-h experiments with HeLa cells, 5 μM CasII-gly inhibited OxPhos (80%), glycolysis (40%), and HK (42%). The present data indicated that CasII-gly is an effective multisite anticancer drug simultaneously targeting mitochondria and glycolysis.