Cortical computations are critically dependent on interactions between pyramidal neurons (PNs) and a menagerie of inhibitory interneuron types. A key feature distinguishing interneuron types is the spatial distribution of their synaptic contacts onto PNs, but the location-dependent effects of inhibition are mostly unknown, especially under conditions involving active dendritic responses. We studied the effect of somatic vs. dendritic inhibition on local spike generation in basal dendrites of layer 5 PNs both in neocortical slices and in simple and detailed compartmental models, with equivalent results: somatic inhibition divisively suppressed the amplitude of dendritic spikes recorded at the soma while minimally affecting dendritic spike thresholds. In contrast, distal dendritic inhibition raised dendritic spike thresholds while minimally affecting their amplitudes. On-the-path dendritic inhibition modulated both the gain and threshold of dendritic spikes depending on its distance from the spike initiation zone. Our findings suggest that cortical circuits could assign different mixtures of gain vs. threshold inhibition to different neural pathways, and thus tailor their local computations, by managing their relative activation of soma- vs. dendrite-targeting interneurons. Establishing how inhibitory neurons shape the computing functions of neural circuits is crucial to understanding both normal function and dysfunction in the human brain. It has been known for over a century that different classes of inhibitory interneurons project to different sub-regions of the neurons they contact – some primarily target cell bodies, others the dendrites, still others the axon. It remains poorly understood, however, how these different projection patterns influence synaptic integration in the target neuron populations. By providing new data from intracellular recordings in brain slices, and a simple but powerful model of the location-dependent effects of inhibition on dendritic spike generation, our study (1) demonstrates the importance of the absolute and relative locations of excitatory and inhibitory inputs to pyramidal neurons, the principal cells of the cerebral cortex, and (2) helps to establish a more solid theoretical understanding of these complex integrative phenomena. As high resolution mapping of the cortical “connectome” becomes available in the coming years, our work will be helpful in interpreting the computing functions of cortical tissue both at the single neuron and circuit levels. The sensory, motor, and cognitive functions of neocortical circuits depend critically on synaptic interactions between pyramidal neurons (PN), the principal excitatory neurons of the neocortex, and a multitude of inhibitory interneuron types One of the most salient features distinguishing cortical interneurons, however, is the spatial distribution of the synaptic contacts they form onto their PN targets. For example, basket cells target the soma and peri-somatic region Several studies have explored the location-dependence of excitatory-inhibitory (E-I) interactions under passive conditions in a variety of cell types, mainly focusing on the effectiveness of inhibition at different locations relative to an excitatory input. In the first systematic study of this issue, Koch et al. Much less is known about E-I synaptic location effects under “active” response conditions, that is, when PN dendrites are driven to generate local spikes We tested the effects of the location of inhibition in experiments in neocortical somatosensory slices. Whole cell somatic recordings were made from layer 5 pyramidal neurons. Excitation was delivered to a dendritic site ranging from 85 to 200 µm from the soma either by electrical stimulation or glutamate uncaging . InhibitTo examine the effects of inhibition co-localized with the excitatory stimulus, we applied increasing levels of dendritic excitation until a local spike was evoked, both under control conditions without inhibition , dendritWhen higher levels of excitation were applied, local dendritic spikes were generated . Based oLike dendritic inhibition, somatic inhibition also suppressed subthreshold EPSP peaks recorded at the soma . In contrast, blocking NMDA channels eliminated local spikes altogether in the model, as in previous experimental studies The location-dependent effects of inhibition in the compartmental model were very similar to the experimental results described above. Dendritic inhibition substantially increased the threshold level of excitation needed to initiate an NMDA spike, but had little effect on spike height as measured at the soma . In contA key difference between dendritic and somatic inhibition conditions was the observation of full-height spikes at the soma under increasing levels of dendritic inhibition, in contrast to a gradual reduction in the peak response at the soma under increasing levels of somatic inhibition . The graThe similarity of our experimental and modeling data, despite the much slower time course of synaptic action in our slice experiments compared to the compartmental simulations, suggested that inhibitory location effects might depend mainly on the voltage-dependence of the NMDA conductance rather than its time course. To test this hypothesis and to probe the biophysical mechanisms underlying the location-dependent effects we had observed, we analyzed the input-output behavior of a time-invariant 2-compartment model as in Vu and Krasne synN), covering the range from subthreshold to suprathreshold responses. We asked whether such a simplified model, which captures the spatial separation of the NMDA spike initiation zone in the dendrite and the soma, but suppresses many details including all temporal dynamics, could replicate the location-dependent effects of inhibition on NMDA spike generation described above. We found the 2-compartment model closely matched both the slice data and the results of our detailed compartmental simulations, including the reduction in slope in the subthreshold response range  an analysis of its steady state solutions allowed the effects of dendritic vs. somatic inhibition seen in the slice data to be explained based on 3 simple principles:Inhibition anywhere increases the leak conductance at the site of spike initiation, which proportionally raises the NMDA spike threshold. But once triggered, the NMDA spike is invariant in height at the site of initiation regardless of the effective total leak conductance that had to be overcome.1. Details. In a single electrical compartment  with corresponding distal inhibition at +60 µm (open green squares) and on-path inhibition at −20 µm (open orange squares).At equal distances from the excitatory stimulus, inhibition distal to the site of excitation is more effective than on-the-path inhibition at increasing the spike threshold (compare open green squares on the +60 µm arc to open orange squares on the −40 µm arc at the soma (compare same strength inhibitory cases at various locations). This reiterates the finding of Koch et al. (1983) for inhibition of passive synaptic responses, and can be explained by slight elaborations of the electric circuit principles discussed above . Once triggered by a sufficiently large excitatory stimulus, however, dendritic spikes are of full (pre-inhibition) height at the site of spike generation. In turn, the spiking dendrite counterpart of absolute inhibition is absolute magnitude inhibition, which includes not only somatic but on-the-path inhibition: any inhibition proximal to the site of spike generation increases the voltage attenuation that the dendritic spike experiences as it propagates to the soma, putting an absolute limit on the peak response that can be measured at the soma.In active dendrites, the closest counterpart of relative inhibition is relative is not synonymous with weak, and absolute is not synonymous with strong. Inhibition placed at, or more distal, than the site of excitation, though relative, can under some circumstances have a stronger gain-suppressing effect measured at the soma than the same inhibitory conductance placed directly at the soma. For example, a dendritic inhibitory conductance that cuts the input resistance by a factor of two at the site of excitation, and thus cuts the subthreshold response at the soma by half, may have a negligible effect on the response at the soma when it is placed directly at the soma. Similarly, a dendritic inhibitory input is much better situated to veto dendritic spikes than an inhibitory input of the same size delivered to the soma It is important to note that in vitro and in vivoRhodes' Though it is a straightforward outcome of our time-invariant 2-compartment model, the fact that synaptically evoked NMDA spikes are essentially unchanged in height even when powerful inhibitory conductances are activated directly at the site of spike generation seems surprising in the context of classical synaptic integration effects. In particular, inhibition is generally expected to reduce the magnitude of an excitatory synaptic response in a graded fashion, especially when the excitatory and inhibitory synapses are co-localized. The all-or-none character of NMDA spikes in the presence of inhibition seen both in our models and our electrophysiological data is less surprising, however, when it is recalled that conventional fast action potentials are also stereotyped in height despite orders-of-magnitude differences in input resistance both within (axon vs. soma) and between  cells. Our observations here support the conclusion that NMDA currents, like other types of spiking mechanisms, produce relatively stereotyped responses once they are driven into the regenerative range, despite the substantial differences in input resistance found in different cellular locales at different moments in time.increases in spike height in the presence of strong dendritic inhibition  to describe the effects of inhibition targeted to other parts of the cell , (2) to determine whether excitatory and inhibitory location effects combine in predictable ways when they occur together, and (3) to assess the degree to which location effects generalize across neuron morphological types.All experimental procedures were in accordance with guidelines of the Technion Institutional Animal Care and Use Committee.Cortical brain slices were prepared from the somatosensory cortex from 20–40 day old male Wistar rats. All experimental procedures were in accordance with guidelines of the Technion Institutional Animal Care and Use Committee. The neurons were visualized using a confocal scanning microscope (Olympus 1000) equipped with infrared illumination and Dot contrast optics combined with infrared video enhanced microscopy Full images were obtained with a temporal resolution of 1 Hz, and in the line scan mode with a temporal resolution of 512 Hz. Images were analyzed using Tiempo (Olympus), homemade software, and Igor software. Fluorescence changes were quantified as increase in fluorescence from baseline normalized by the baseline fluorescence (ΔF/F as a percentage). The background florescence was subtracted from all measurements before calculation of the ΔF/F. Calcium transients are reported as mean± SD.UV laser glutamate uncaging was used to deliver excitation in all except two experiments for somatic inhibition, in which case electrical stimulation was used. For the uncaging experiments, caged glutamate  was photolyzed with a 361 nm UV-laser beam  using point scan mode. The caged glutamate (5–10 mM) was delivered locally to a branch using pressure ejection (5–10 mbar) from an MNI-glutamate-containing electrode (2 micron diameter). Uncaging spots were selected on dendrites that did not have neighboring branches both in the XY plane and above or below them. Focal synaptic stimulation was performed with a theta patch pipette (3–10 MΩ resistance) located in close proximity (2–5 microns) to the selected basal dendritic segment. Stimulation duration was 0.1 ms, in a constant voltage mode. GABA was delivered by way of iontophoresis through a pipette  positioned adjacent to the cellular membrane. The effect of iontophoresis sensitively depended on the distance between the electrode to the cell, with about 2 fold decrease in IPSP amplitude with distances larger than 2 microns in vitro data set, while The peak membrane depolarization for a given level of excitatory stimulation  as a result of different binding and unbinding kinetics of NMDA channels ++ block that suppresses most current flow at negatively polarized potentials. The strength of excitation was controlled by the variable synN, representing the number of glutamate activated NMDA channels, with constant synN; three colored cases  are carried through p(t) is at its maximum value of 1. Capacitive currents were ignored based on the assumption that the NMDA conductance remains at or near its peak value long enough for the membrane to reach its equilibrium potential — an imperfect but reasonable assumption given existing data from pyramidal neurons This study concerns the effects of inhibition on NMDA spike generation. The NMDA channel behavior and the methods we used to study NMDA spike threshold and magnitude are shown in m(t)V numerically from the circuit equation given by Kirchhoff's current law:mV satisfied equation 2, we used the value closest to the resting potential The voltage-dependence of the NMDA channel prevented an algebraic solution of the circuit shown in Explicit traces of the time-varying membrane potentials, that is, the NMDA EPSPs, are shown in syn.p(t)N at different moments in time. Colored dashed arrows suggest the temporal evolution of an NMDA I–V curve as p(t) increases and decreases through time (shown explicitly in p(t) = 1) for one of the three levels of NMDA excitation highlighted in The mechanism of NMDA-based non-linearity ,E is illm(t)V to equation 2 at any given time corresponds graphically to the intersection between the NMDAI and −LI curves. These intersection points give the voltages at which the inward (NMDA) and outward (leak) currents are balanced at a stable equilibrium. As the NMDA conductance waxes and wanes through time (see dashed arrows), the equilibrium point slides back and forth along the green line, tracing the progress of the membrane potential in response to NMDA excitation. Colored dots indicate the peak voltages reached for the three levels of excitation highlighted in The solution peakt (orange dot) lay at a deeply polarized potential close to rest on the I–V plot in N at which the descending slope of the NMDA I–V curve skims below the green line to run entirely within the grey shaded region. When this occurs (red I–V curve), the balance point jumps to a far more depolarized value (red dot); this value corresponds to the height of the NMDA spike at threshold (black dashed line). Once past the spike threshold, further increases in NMDA excitation (burgundy case) produced only marginal increases in spike height (burgundy dot).When the excitation level was subthreshold for NMDA spike generation (e.g. orange case), the balance point at dLg as in dIg in cases involving dendritic inhibition. The reversal potential of the inhibitory conductance was equal to the resting potential IE = LE = −70 mV. The somatic compartment contained a leak conductance sLg and an inhibitory conductance sLg in cases involving somatic inhibition. The two compartments were coupled by an axial conductance ag, which was adjusted to achieve passive voltage attenuation between the dendritic and somatic compartment of at least a factor of 5 (depending on the level of somatic inhibition). As in the single-compartment model of somaV and dendV:somaV in equation 3. The resulting equation was solved numerically for dendV, then somaV was computed from equation 4.A 2-compartment model was used to study the effects of dendritic vs. somatic inhibition on NMDA spike threshold and height as seen at the soma . The denThe I–V curves from the perspective of the soma illustrate the differential effect of dendritic  vs. somaFigure S1A) Examples of IPSPs evoked by GABA iontophoresis at the soma. IPSPs evoked by GABA iontophoresis at the soma had only a fast component in 12 of the cells, with an average (±SD) amplitude of −0.65±4.5 mV at rest, rise time of 33.5±19.7 ms and decay time of 158.6±89.9 ms. In 10 cells the somatic IPSP had a slower component with amplitude of −1.74±3.22 mV, rise time of 172.4±231.1 ms and decay time of 558.6±242.3 ms. 3 cells responded with both slow and fast components. Dendritic IPSPs were smaller and on average faster; in 9 cells the dendritic IPSP had a fast component with amplitude of 0.96±1.31 mV, rise time of 35.6±17.1 ms and decay time of 101.7±58.4 ms. Two cells responded with additional slow component with amplitude of 1.5±0.7 mV, rise time of 151.5±111 ms and decay time of 550±353.5 ms. (B) The effect of somatic inhibition on input resistance was intensity dependent: at control, the recorded input resistance of the neuron was 61.7±12.6 MOhm. GABA iontophoresis at a current intensity of 3 nA reduced the input resistance by about half to 34.5±11.7 MOhm and at 5 nA to 14.6±5.03 MOhm. (C) Representative somatic EPSP recording without (red) and with GABA iontophoresis (black). The excitatory activation (uncaging in this case) was done 200 µm from the soma and was delayed with respect to the iontophoresis. The site of iontophoresis was 100 µm from the soma. Notice the delayed onset of excitation with respect to iontophoresis. (D) Overlayed traces for 4 different values  of delayed onset of excitation for the experiment in (C). The suppression of maximal NMDA spike amplitude depended on the onset delay.Characterizing the effect of GABA iontophoresis in slice preparation. ((PDF)Click here for additional data file.Figure S2Experiments in the detailed compartmental model to measure input resistance changes at the somatic and dendritic location of inhibition. Inhibitory conductances of increasing strength were activated under current clamp at the soma and the dendritic location. The peak input resistance was measured as the ratio of membrane potential trough and the clamp current. Note that both the X and Y-axes for the input resistance graphs on the bottom are dissimilar.(PDF)Click here for additional data file.Figure S3syn. Asterisk indicates time at which p(t) = 1. (C) I–V curves for leak (IL) and steady-state (i.e. p(t) = 1) NMDA conductance (INMDA) in the single compartment. Mathematical formulation of NMDA conductance shows a dependence on both membrane voltage and time-dynamics m. The I–V plots show the resulting Vm (colored dots) for the different values of Nsyn shown in B: it is the voltage for which net inward current (INMDA) is balanced by the outward current (IL). These I–V plots can also give us an idea of how the membrane voltage will change as p(t) changes in time, as shown in B. They can be thought of as instantaneous I–V curves giving an estimate for Vm (as described above) for different values of Nsyn×p(t). The “Time” arrow follows the I–V curves for INMDA as Nsyn×p(t) changes in time. The intersection of each of these time-changing I–V curves with the leak I–V curve  gives an estimate for Vm as a function of time. Notice that as Nsyn×p(t) changes follow the yellow curve in B, the intersection of resulting I–V curves with the leak I–V curve is a linear progression similar to the linear rise and fall of Vm in D (yellow curve). When Nsyn×p(t) changes follow the red curve in B, the intersection of resulting I–V curves with the leak I–V curve involves a non-linear jump (yellow dot and red dot) leading to the non-linear rise and fall of Vm in D (red curve). Any value of Nsyn larger than this will ensure a non-linear jump in the I–V domain  and consequently the time domain . Thus, spikes in the time domain correspond to all the NMDA I–V curves in C whose negative slope region lies completely below the -IL (green) l-V curve, as shown. Specifically, the smallest Nsyn (red curve) for which this is the case is a representative of the minimum conductance required, given the membrane leak, to generate a spike in time-domain. The exact Nsyn would depend on the exact time-course of the conductance and the membrane capacitance. (D) Vm at the single compartment in the presence of time-varying NMDA conductance, as described in C. Sufficient value of Nsyn (red) leads to a non-linear jump in peak Vm - the NMDA spike. (E). I/O curve for peak values of voltage traces shown in D. Notice the NMDA-based super-linear jump in the function.Model of NMDA channel and definitions of NMDA spike threshold and height. (A) A single compartment model of neural membrane with an NMDA and leak conductance. (B) Example time courses of peak NMDA conductance for different values of N(PDF)Click here for additional data file.Figure S4I–V curves at the soma in a 2-compartment model. Line colors are the same as in (PDF)Click here for additional data file.Figure S5Effect of reversal potential of the inhibitory conductance on the location effect. I/O curves for somatic inhibition  and dendritic inhibition  for 3 levels of inhibitory reversal potential: −60 mV (dashed), −70 mV(solid), −80 mV (dotted). The resting membrane potential was −70 mV in both the 2-compartment  and the detailed compartmental model .(PDF)Click here for additional data file.Figure S6A conceptual model for location effect of inhibition in the presence of dendritic NMDA spikes. Assuming firing rates are determined as an average of synaptic activity over a few hundred milliseconds, this can be modeled as an averaging/pooling operation (B) over the single pulse subthreshold response (A). Coupled with a threshold-linear function for the somatic spiking mechanism, we get the predictions for suprathreshold i/o curves for both somatic and dendritic location of inhibition (C). (D) shows firing rates as a function of excitatory synapses based on simulations in a detailed compartmental model. The inhibitory synapses were either co-localized with the dendritic excitation or placed at the soma.(PDF)Click here for additional data file.Figure S7in vitro data.Sensitivity of the location effect to the onset and duration of inhibition. (A) Representative location of excitatory (red square) and inhibitory (blue/dark green/light green circle) synapses in the morphologically detailed model. (B) I/O function between peak membrane potential and strength of excitation for the case of no inhibition (black), slow (blue), fast (dark green), and delayed fast (light green) inhibition in a detailed compartmental model. For the cases shown here, the peak inhibitory conductance was 4 nS for dendritic inhibition and 24 nS for somatic inhibition. (C) Summary of the effect of inhibition offset and duration at the somatic (triangles) and dendritic location (circles) in the detailed model, expressed as joint % change in spike height and threshold relative to no-inhibition control (black square at the origin). Peak conductance for dendritic inhibition cases shown here was 4, 8, 12, 16 and 20 nS, while that for somatic inhibition was 24, 48, 72, 96 and 120 nS. Each excitatory synapse in this experiment had 6 nS peak AMPA conductance. Excitatory synapses with 1.5 nS peak AMPA conductance with similar distribution of density along the dendrite gave similar results. Red and green shaded areas were carried over from (PDF)Click here for additional data file.Figure S8Comparison of divisive (green) and subtractive (blue) prediction for the computational effect of somatic inhibition on somatic membrane potential in the detailed model. Red squares are data points from simulation of the detailed compartmental model Click here for additional data file.Text S1Explanation for constancy of NMDA spike height with I–V plots.(PDF)Click here for additional data file.


Eurygaster integriceps Puton (Hemiptera: Scutelleridae) midgut amylases was examined. Biochemical studgawies showed that inhibitors from Triticale (a hybrid of wheat and rye) had inhibitiory effects on E. integriceps α-amylases. The effects of the triticale α-amylase inhibitor (T-αAI) on α-amylase of E. integriceps showed a dose dependent manner of inhibition, e.g. less inhibition of enzyme activity (around 10%) with a lower dose (0.25 mg protein) and high inhibition of enzyme activity (around 80%) when a high dose of inhibitor was used (1.5 mg protein). The enzyme kinetic studies using Michaelis-Menten and Lineweaver-Burk equations showed the Km remained constant (0.58%) but the maximum velocity (Vmax) decreased in the presence of a crude extract of Triticale inhibitors, indicating mixed inhibition. The temperature giving 50% inactivation of enzyme (T50) during a 30-min incubation at pH 7.0 was 73° C. The maximum inhibitory activity was achieved at 35° C and pH 5.0. Gel assays showed the meaningful inhibition of E. integriceps α-amylases by various concentrations of Triticale inhibitors. Based on the data presented in this study, it could be said that the T-αAI has good inhibitory activity on E. integriceps gut α-amylase.The effect of triticale α-amylases inhibitors on starch hydrolysis catalyzed by the Sunn pest,  Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is one of the most serious pests of wheat and barley in the wide area of the Near and Middle East, West Asia, and many of the newly independent states of central Asia. It also is found in Eastern and Southern Europe and North Africa  and rye  genomes resulting in hexaploid (AABBRR) or octaploid (AABBDDRR) triticales. However, less attention has been given to α-amylase inhibitors of triticale even though they have been isolated from parental wheat and rye .Triticale  is the product of an artificial cross between wheat  followed by centrifugation at 10,000 g for 20 min at 4° C. The 60% pellet containing the highest fraction of amylase inhibitors was dissolved in icecold sodium phosphate buffer (0.02 M and pH 7.0) and dialyzed overnight against the same buffer. This dialyzed solution was used as a source of amylase inhibitors in enzyme assays.T-αAI from seeds of Triticale was extracted according to Baker  and MeloM NaCl). The midgut was separated from the insect body, rinsed in ice-cold saline buffer, placed in a pre-cooled homogenizer, and ground in 1 ml of universal buffer containing succinate, glycine, 2-morpholinoethanesulfonic acid at pH 6.5. The homogenates from both preparations were separately transferred to 1.5 ml centrifuge tubes and centrifuged at 15,000 g for 20 min at 4° C. The supernatants were pooled and stored at -20° C for subsequent analyses.Enzyme samples from the midguts of adults were prepared. Adults were randomly selected, and midguts from these individuals were removed by dissection under a light microscope in ice-cold saline buffer (0.006 www.merck.de/) as substrate (-1) was constructed to enable calculation of the amount of maltose released during α-amylase assays. All assays were performed in duplicate, and each assay was repeated at least three times.The α-amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure , using 1ubstrate . One uniE. integriceps gut contains most of the α-amylase , succinate (0.02 M) and Mes (2-[morpholino]ethansulphonic acid) (0.02 M). Adjustment of pH was done by addition of NaOH (1.0 N). The amylase activityremaining after 30 min incubation at 30° C and in the presence of T-αAI (1.5 mg/ml) was determined by adding 0.4 ml of 1% starch solution. Controls were run at each pH value with midgut amylase alone as a control, and the percentages of inhibition were calculated from the controls vs. inhibited midgut amylase values measured at each pH.The extent to which T-αAI inhibits l buffer  with pH E. integriceps midgut α-amylase by T-αAI carried out by pre-incubation of enzyme extract with T-αAI in 2 mM sodium phosphate buffer (pH 5.0) at 25, 30, and 35° C for different times. To determine the time dependence of the thermal inactivation of the inhibitor, T-αAI was incubated in 2 mM sodium phosphate buffer at 50, 60, 70, and 80, ° C for 10, 20, 30, 40, 50, and 60 min, and then the remaining amylase inhibitory activity was assessed.Time course inhibition i) and dissociation constants of enzyme-substrate inhibitor complexes (K'i) were determined (The inhibition was measured with increasing concentrations of starch as a substrate (0.5– 2.0%) in the presence of different concentrations (0.2 to 1.5 mg/ml protein) of T-αAI. The type of inhibition was determined by Lineweaver-Burk plot analysis of the data, which was calculated from the results according to Michaelis-Menten kinetics. The inhibitory constants (Ktermined . All theE. integriceps. SDS-PAGE was performed in 10% (w/v) gel with 0.05% SDS for separating gel and 5% for stacking gel with 0.05% SDS.Enzyme extract was pre-incubated with different concentrations of T-αAI for 30 min at 30° C, and then the remaining amylase activity was determined by SDS-polyacrylamide gel electrophoresis (Native Page). SDS-PAGE was carried out using the procedures described by Lammli  and CampM Tris-Hcl, pH 6.8), 20% glycerol, 2% SDS, 0.005% (w/v) bromophenol blue, but without mercaptoethanol or heating. Electrophoresis was conducted at a voltage of 120V until the blue dye reached the bottom of the slab gel. To prepare gels for α-amylase assay, the gel was rinsed with distilled water and washed by shaking gently with 1% (v/v) Triton X-100 in phosphate buffer containing 2 mM CaCl2 and 10 mM NaCl for 30 min. Then, the gel was rinsed with distilled water and treated with a 1% starch solution for 1.5 h. Finally, after rinsing with distilled water, the gel treated with a solution of 1.3% 12, 3% KI to stop the reaction and to stain the un-reactedstarch background. Zones of α-amylase activities appeared as a light band against the dark background of the gel.The electrode buffer was prepared based on the method of , but SDSwww.bio-rad.com) as standard.Protein concentration was measured according to the method of Bradford , using bE. integriceps showed a dose dependent manner of inhibition e.g. the lower dose (0.25 mg) of inhibitor around 10% enzyme inhibition was achieved while the highest dose (1.5 mg) of inhibitor caused an 80% inhibition of amylase activity  caused an 80% inhibition of amylase activity. Also, Melo et al.  amylase inhibitor (αAI-1) from the common bean, P. vulgaris and Amaranthus  (The insect gut lumen is the place where the interaction between α-amylase and T-αAI occurs. It has been reported that pH (6.5) . TherefopH (6.5) .P. vulgaris amylase inhibitor at 37° C and LeBerre-Anton et al.  in a 30-min incubation time was determined to be 73° C. Oneda et al. (50 of α-amylase inhibitor from wheat kernel on the activity of porcine pancreas α-amylase (PPA) was 88° C in 30-min incubation time at pH 6.9.Thermal inactivation of T-αAI was observed at pH 5.0 in the temperature range of 50–80° C . E. intea et al.  reportedi, value of 45.5 mg , resulting from binding of the T-αAI compound to the active site, and the amylase/starch-T-αAI complex (ESI) inwhich T-αAI is bound at a secondary binding site other than the active centre, which is accessible only after starch binding has occurred at the active centre. This treatment assumes that enzyme-inhibitor interaction is not irreversible.A non-competitive inhibitor binds to an inhibitor site on the enzyme that is remote from the active site and brings about a conformational change in the active site. In this sense, it is very similar to one of the competitive inhibitor types. The difference is that the change in the active site is such that it does not prevent substrate binding but, rather, prevents the enzyme from converting the bound substrate to product. A classical noncompetitive inhibitor has absolutely no effect on substrate binding. In fact, a change to the shape of the active site is almost certain to alter the ability of the substrate to bind. It won't stop it altogether, but the affinity will be reduced . InhibitP. vulgaris seeds (P. vulgaris var. Great Northern against porcine pancreatic α-amylase (PPA).Mixed noncompetitive inhibition has been reported for other amylase inhibitors, i.e., inhibition of PPA by α-amylase inhibitor from is seeds  and inhiis seeds . Howeveris seeds  reportedE. integriceps amylases on T-αAI treatment was not due to the dissociation of the enzyme complex.Since colorimetric assay showed amylase activities were inactivated by T-αAI, it was of interest to see if the inactivation occurred due to the decomposition of the enzyme complex. The enzyme was subjected to a series of non-denaturing PAGE after the incubation of enzyme extract with different concentrations of T-αAI . AlthougSitophilus zeamais,Callosobruchus maculatus, Zabrotes subfasciatus and Acanthoscelides obtectus  it should inhibit the insect enzyme substantially at a low enough concentration and at the pH found in the insect gut and (b) it should be resistant to attack by insect gut proteases (Due to existence of more than one α-amylase isozyme in roteases .E. integriceps gut α-amylase. However, the present study is the first study of this kind, so more information, such as on the effect of gut protease on T-αAI, should be obtained.Based on the data presented here, it could be said that T-αAI has a strong inhibitory activity on


H-pyrazol-3(2H)-one (DMPO) was synthesized to be evaluated as a corrosion inhibitor. The corrosion inhibitory effects of DMPO on mild steel in 1.0 M HCl were investigated using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, open circuit potential (OCP) and electrochemical frequency modulation (EFM). The results showed that DMPO inhibited mild steel corrosion in acid solution and indicated that the inhibition efficiency increased with increasing inhibitor concentration. Changes in the impedance parameters suggested an adsorption of DMPO onto the mild steel surface, leading to the formation of protective films. The novel synthesized corrosion inhibitor was characterized using UV-Vis, FT-IR and NMR spectral analyses. Electronic properties such as highest occupied molecular orbital energy, lowest unoccupied molecular orbital energy  and dipole moment (μ) were calculated and discussed. The results showed that the corrosion inhibition efficiency increased with an increase in the EHOMO values but with a decrease in the ELUMO value.1,5-Dimethyl-4-((2-methylbenzylidene)amino)-2-phenyl-1 H-pyrazol-3(2H)-one, DMPO, and chemical structure elucidation using spectroscopic techniques . Recent studies have shown that organic compounds containing polar functional groups are quite efficient in minimizing the effect of corrosion in addition to heterocyclic compounds containing polar groups and π-electrons. The molecular design of the DMPO molecule is based on the fact that 4-aminoantipyrine consists of amine, methylamine, carbonyl and π-electron bonds, which would effectively contribute towards the inhibition of mild steel corrosion in acidic media. Moreover, Schiff bases containing imine groups would contribute more effectively to the inhibition of corrosion of mild steel in acid medium. The resonance effect of DMPO increases its inhibition activity. Structural parameters, including the frontier molecular orbital (MO) energies, specifically HOMO  and LUMO  energies and dipole moments, were calculated and correlated with corrosion inhibition efficiencies. The calculated values of inhibition efficiencies  were obtained from a quantitative structure-activity relationship (QSAR) and were subsequently correlated with the experimentally obtained values of inhibition efficiencies (IEexp%). The proposed structure of the synthesized novel corrosion inhibitor is shown in Corrosion inhibitors are of considerable practical importance, as they are extensively employed in reducing metallic waste during production and in minimizing the risk of material failure, both of which can result in the sudden shut-down of industrial processes, which in turn leads to added costs . It is aTo synthesize the new corrosion inhibitor DMPO, the reaction sequence outlined in −1. The high value of the C=N wavenumber was due to the high conjugation (resonance effect) of the substituted double bonds, whereas the aromatic carbon-carbon double bond stretching appeared at 1569.4 cm−1. However, two types of tautomers, i.e., amine and imine, could be expected from the DMPO structure  is usually a poor approximation, especially for systems in which an efficient inhibitor is present. The corroding surface of the mild steel in 1.0 M HCl is expected to be inhomogeneous because of its roughness; therefore, the capacitance is presented through a constant phase element (CPE).In these cases, the parallel network charge-transfer resistance double-layer capacitance , corrosion potential (Ecorr), anodic Tafel slope (βa), cathodic Tafel slope (βc), the degree of surface coverage (θ) and inhibition efficiency (IE%) with the concentrations of inhibitor DMPO are given in where icorr(uninh) and icorr(inh) are the corrosion current densities in the absence and presence of the inhibitor, respectively. The inhibition efficiency (IE%) is given byi.e., the so-called geometric blocking effect, and (ii) by modifying the activation energy of the cathodic and/or anodic reactions occurring in the inhibitor-free metal in the course of the inhibited corrosion process. It is a difficult task to determine which aspects of the inhibiting effect are connected to the geometric blocking action and which are connected to the energy effect. Theoretically, no shifts in Ecorr should be observed after the addition of the corrosion inhibitor if the geometric blocking effect is stronger than the energy effect [The results also indicate that the inhibition efficiencies increased with the concentration of inhibitor. Such behavior can be interpreted on the basis that the inhibitor acts by adsorbing onto the metal surface. In acidic solutions, the anodic reaction of corrosion is the passage of metal ions from the metal surface into the solution, and the cathodic reaction is the discharge of hydrogen ions to produce hydrogen gas or to reduce oxygen. The inhibitor may affect either the anodic or the cathodic reaction, or both . Becausey effect .The OCP of mild steel was monitored in the presence of 0 mM, 0.1 mM, 0.2 mM, 0.3 mM and 0.5 mM DMPO. Electrochemical frequency modulation (EFM) is a new electrochemical technique for determining the corrosion rate without preliminary knowledge of the Tafel constants. The main advantages of this technique include measuring the corrosion rate, Tafel parameters and causality factors in a single set of data. While using EFM, a potential perturbation signal composed of two sine waves is applied to any corroding specimen to obtain the current response. EFM has been used for different combinations of metals and electrolytes to accurately measure the corrosion parameters. EFM is related to the harmonic method of employing a lower amplitude (20 mV) sinusoidal perturbation signal but is composed of two sine waves instead of one. EFM is superior to the harmonic method in many aspects, including data validation, larger current response and insensitivity to harmonics in the perturbation signal.EFM%), the corrosion current density (μA/cm2), the Tafel constant and the causality factors (CF-2 and CF-3), with different concentrations of DMPO in 1.0 M HCl at a constant temperature of 30 °C are listed in The corrosion parameters, including the corrosion efficiency  has been used to analyze the characteristics of the inhibitor/surface mechanism and to describe the structural nature of the inhibitor in the corrosion process. Furthermore, DFT is considered a very useful technique to probe the inhibitor/surface interaction as well as to analyze the experimental data. This technique has been found to be successful in providing insights into the chemical reactivity and selectivity in terms of global parameters such as electronegativity (v), hardness (g) and softness (S), and local softness (sđ ~ r Þ) ,29.The design of the DMPO compound for use as a corrosion inhibitor was based on several factors. First, the molecule contains sulfur and nitrogen atoms (apart from the azomethine group) as active centers in addition to an azomethine group, which boasts biological functionality as an antibacterial and antifungal –32 agentThe calculated quantum parameters of the HOMO, LUMO, band gap and dipole moment for DMPO are presented in Frontier orbital theory was useful in predicting the adsorption centers of the inhibitor molecule DMPO responsible for its interaction with surface metal atoms . Terms iHOMO) and LUMO (ELUMO) as well as the dipole moment (μ) are shown in HOMO values along with a decrease in ELUMO values.A relationship between the corrosion inhibition efficiency of the synthesized compound with the orbital energies of the HOMO  to the appropriate acceptor molecule with low energy and an empty molecular orbital. Increasing values of EHOMO thus facilitate the adsorption of the inhibitor. Consequently, improving the transport process through the adsorbed layer would enhance the inhibition efficiency of the inhibitor. This finding can be explained as follows. ELUMO indicates the ability of the molecule to accept electrons; therefore, a lower value of ELUMO more clearly indicates that the molecule would accept electrons [The Electrons . The diplectrons ,39.The Mulliken charge distribution of DMPO is presented in LUMO – EHOMO) mean good inhibition efficiency [According to the description of frontier orbital theory, HOMO  is oftenficiency .s used were of reagent grade  and used as supplied without further purification. The FT-IR spectra were measured using a Thermo Scientific Model Nicolet 6700 spectrophotometer. NMR spectra were recorded on a Model AVANCE III 600 MHz spectrometer.All chemical1H-NMR (CDCl3): δ 9.712 , 6.983, 7.214, 7.307, 7.322, 7.418, 7.675, 7.711, 7.775, 7.876 , δ 2.017 , 2.964 , δ 3.128 ; IR: 3050.0, 3061.6 cm−1 , 2910.7, 2945.9 and 2970.0 cm−1 , 1646.6 cm−1 (C=O), 1569.4 cm−1 (C=C); 1588.6 cm−1 , 1484.4 cm−1 ; UV-Vis: 250 nm in acetonitrile.A solution of 2-methylbenzaldehyde (0.4 mmol) in 50 mL ethanol was refluxed with 4-aminoantipyrine (0.4 mmol) for 5 h. After cooling to room temperature, a solid mass was separated and recrystallized from ethanol in 87% yield. −4 to 5 × 10−4 M DMPO corrosion inhibitor. Solutions were freshly prepared from analytical grade chemical reagents using distilled water. All measurements were performed in triplicate, and the average values were reported. Measurements were performed using a Gamry Instrument Potentiostat/Galvanostat/ZRA model. The DC105 and EIS300 software by Gamry were used for potentiodynamic scans and electrochemical impedance spectroscopy (EIS). The potentiodynamic current-potential curves were swept from −0.25 to +0.25 Vsce at a scan rate of 0.5 mV·s−1. All impedance data were fitted to appropriate equivalent circuits (EC) using Gamry Echem Analyst software. Experiments for electrochemical measurements were initiated approximately 30 min after the working electrode was immersed in the solution to stabilize the steady state potential.Mild steel specimens obtained from the Metal Samples Company were used as the working electrodes throughout the study. The composition (wt %) of the mild steel was as follows: Fe, 99.21; C, 0.21; Si, 0.38; P, 0.09; S, 0.05; Al, 0.01. The specimens were cleaned according to ASTM standard G1-03 ,47. MeasHOMO), the energy of the lowest unoccupied molecular orbital (ELUMO) and dipole moment (μ).The molecular sketch of the synthesized compound was plotted using Gaussian 03, Revision C.01. All quantum chemical calculations were performed using Density Functional Theory (DFT). The following quantum chemical indices were calculated: the energy of the highest occupied molecular orbital amino)-2- phenyl-1H-pyrazol-3(2H)-one (DMPO), was sequentially synthesized and characterized using various spectroscopic methods. Changes in the electrochemical impedance spectroscopy (EIS), open circuit potential (OCP) and potentiodynamic polarization were used to study the corrosion inhibition of mild steel in 1.0 M HCl solutions at 30 °C using different concentrations of DMPO as an inhibitor. This compound exhibited excellent inhibition performance as a mixed-type inhibitor. In general, the acidic corrosion of mild steel was reduced upon the addition of an appropriate concentration of DMPO. The inhibition efficiencies obtained from the EIS data were comparable to those obtained from the polarization measurements in that they were greater for the inhibitory solution than those of the non-inhibitory solution. DMPO acts as an efficient corrosion inhibitor in 1.0 M hydrochloric acid and it exhibits a maximum inhibition efficiency of 87%. The adsorption of DMPO on a mild steel surface obeys the Langmuir adsorption isotherm. The correlation between the quantum chemical parameters and inhibition efficiencies of DMPO was investigated using DFT calculations. The inhibition efficiencies of the inhibitor are closely related to the quantum chemical parameters EHOMO, ELUMO and dipole moment. The fact that inhibition efficiency is increased with an increase in EHOMO values and with a decrease in ELUMO values has been established herein.In this study, a new 4-aminoantipyrine,


Combined targeting of MAPK and PI3K signalling pathways may be necessary for optimal therapeutic activity in cancer. This study evaluated the MEK inhibitors AZD6244 and PD0325901, alone and in combination with the dual mTOR/PI3K inhibitor NVP-BEZ235 or the PI3K inhibitor GDC-0941, in three colorectal cancer cell lines.Growth inhibition, survival and signal transduction were measured using the Sulforhodamine B assay, clonogenicity and western blotting, respectively, in HCT116, HT29 and DLD1 cell lines.All MEK/PI3K inhibitor combinations exhibited marked synergistic growth inhibition; however, GDC-0941 displayed greater synergy in combination with either MEK inhibitor. NVP-BEZ235 exhibited stronger inhibition of 4EBP1 phosphorylation, and similar inhibition of S6 and AKT phosphorylation, compared with GDC-0941. Both PD0325901 and AZD6244 inhibited ERK phosphorylation, and with MEK/PI3K inhibitor combinations inhibition of S6 phosphorylation was increased. The reduced synergy exhibited by NVP-BEZ235 in combination with MEK inhibitors, compared with GDC-0941, may be due to inhibition of mTOR, and the addition of the mTORC1/2 inhibitor KU0063794 compromised the synergy of GDC-0941:PD0325901 combinations.These studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition; however, the combination of specific PI3K inhibitors, rather than dual mTOR/PI3K inhibitors, with MEK inhibitors results in greater synergy. Targeted anticancer treatments are an important and evolving new approach to cancer therapy and, in order to treat tumours with defects in multiple oncogenic pathways, combinations of targeted agents are being actively investigated.via adaptor proteins. Ras then activates the Raf-MEK-ERK kinase cascade, and ERK phosphorylation leads to the activation of >100 downstream substrates involved in a myriad of cellular processes such as proliferation, survival, transformation, translational control and cytoskeletal rearrangements. This pathway can become constitutively activated by overexpression or mutation of RTKs, and mutations of Ras, especially the KRas isoform  activation causes phosphorylation of Ras  isoform , and Raf isoform . The benvia adaptor proteins, and PI3K then phosphorylates PIP2 to PIP3, causing AKT activation via two crucial phosphorylation events at threonine 308 catalysed by PDK1 and at serine 473, which may be catalysed by mTORC2 –streptomycin (50 mg ml−1) and 2 mM L-glutamine) and confirmed free of mycoplasma contamination by regular testing with Mycoalert .HCT116 , DLD1  and HT29  human colorectal cancer cells were obtained from the American Type Culture Collection, were grown in RPMI-1640 medium (supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin  to allow addition to cell cultures at a final concentration of 0.5% (v/v) DMSO, and were stored frozen under light-protected conditions at −20 °C.Growth inhibition was measured using the Sulforhodamine B (SRB) method as described previously (Clonogenic assays were performed as described previously (Western blots were prepared and developed as described in 50) of the five drugs after 72 h exposure. PD0325901 was the most potent MEK inhibitor with GI50 values of 21, 1460 and 6.5 nM, compared with 312, 4973 and 29 nM with AZD6244 in the HCT116, DLD1 and HT29 cell lines, respectively. The dual PI3K/mTOR inhibitor NVP-BEZ235 was a markedly more potent inhibitor of growth than either the PI3K inhibitor GDC-0941 or the mTOR inhibitor KU0063794; GI50 values of 21, 7.2 and 9.4 nM with NVP-BEZ235 compared with 1081, 1070 and 157 nM with GDC-0941 and 767, 568 and 402 nM with KU0063794 in the HCT116, DLD1 and HT29 cell lines, respectively.The growth inhibitory activity of the PI3K inhibitor GDC-0941, the PI3K/mTOR inhibitor NVP-BEZ235, the mTOR inhibitor KU0063794 and the MEK inhibitors AZD6244 and PD0325901, in HCT116, DLD1 and HT29 cells was measured using the SRB assay . All fiv50 concentration, and at equipotent concentrations at the same ratios in combination. Studies were then performed to determine the effect of combined PI3K and MEK inhibitor treatment on HCT116 and HT29 cell growth over 72 h. The PI3K and MEK inhibitors were used alone at 0.25 × , 0.5 × , 1 × , 2 × and 4 × their respective GI50 concentration compared with the compounds alone; however, despite NVP-BEZ235 being a more potent PI3K inhibitor, GDC-0941 displayed greater synergy when combined with either MEK inhibitor at a constant ratio of their respective GI50 values in the HCT116 and HT29 cell lines  to determine whether the greater growth inhibitory activity of the combinations reflected an additive or a synergistic effect. All combinations of PI3K and MEK inhibitors were synergistic when combined at the GIll lines . Similarll lines .μM, 97% cell kill in the HCT116 cell line and 82% in the HT29 cell line; however, the mean lethal concentration (LC50) of 0.5 μM NVP-BEZ235 in both cell lines was ⩾20-fold higher than the corresponding GI50 values. The three other compounds induced <50% cell death after 72 h treatment at 10 μM , and these mixed PI3K and mTOR inhibitor combinations were then combined with PD0325901, to determine the effect of dual PI3K/mTOR inhibition on MEK inhibitor synergy. A GITo confirm the impact of adding an mTOR inhibitor on the synergistic interaction of GDC-0941 and PD0325901, different molar ratios of GDC-0941 to KU0063794, from 0.01 : 1 to 10 000 : 1, were used in combination with PD0325901 and synergy was evaluated using median effect analysis. The data were then analysed using Spearman's Rank Correlation analysis, which demonstrated a statistically significant positive correlation between increasing ratios of GDC-0941 to KU0063794 in combination with PD0325901, with synergy in the HCT116 and DLD1 cell lines plateauing at ratios ⩾100 : 1 GDC-0941:KU0063794 . Data foNumerous targeted small molecule inhibitors are being developed as a new approach to cancer therapy; however, as tumours often have defects in multiple signalling pathways single agent antitumour activity is modest thus combination treatment is being investigated. The current study has shown that the PI3K inhibitors NVP-BEZ235 and GDC-0941 are synergistic in combination with the MEK inhibitors PD0325901 and AZD6244. However, despite NVP-BEZ235 being a more potent PI3K inhibitor, GDC-0941 showed greater synergy in combination with either MEK inhibitor.50 values determined here for the PI3K inhibitors NVP-BEZ235 and GDC-0941, and the MEK inhibitors AZD6244 and PD0325901 (Previous studies using a variety of cell lines report data consistent with the GID0325901 . In contin-vitro data in which, for example, PD0325901 exhibited synergy with the PI3K inhibitor LY294002 (All the MEK and PI3K inhibitor combinations tested in this study displayed marked synergistic growth inhibitory activity, which is consistent with other LY294002 , and the50 of 0.5 μM, which could be due to the inhibition of mTOR either alone or in combination with PI3K. These results are consistent with other studies, for example, NVP-BEZ235 was found to be cytotoxic to breast cancer cell lines (et al, who noted that cell death induction is not always observed with NVP-BEZ235 (Whereas all of the compounds were growth inhibitory, only NVP-BEZ235 was cytotoxic to HCT116 and HT29 cells, with an LCll lines . Howeverin-vitro study concluded that combined inhibition of PI3K and ERK, induced by PIK3CA downregulation and PD0325901, respectively, was at least in part due to the induction of cell death (in-vivo study in mice with tumours carrying PI3K and Ras mutations demonstrated synergistic tumour regression with NVP-BEZ235 and AZD6244 (in-vivo studies processes such as antiangiogenic effects may also be important determinants of response, as suggested by The marked synergy observed in the growth inhibition studies did not translate into cytotoxicity, as the effect of the combinations was no greater than that observed with the compounds alone in the HCT116 cell line, although some combinations involving GDC-0941 in combination with either MEK inhibitor and the lowest concentration of NVP-BEZ235 in combination with PD0325901 did show a small and statistically significant difference in cytotoxicity in HT29 cells. Where enhanced cytotoxicity was observed this could be due to increased inhibition of mTORC1 as combined inhibition of both pathways relieves the AKT- and ERK-mediated inhibition of TSC2 (M, whereas 312 and 29 nM AZD6244 were required to produce substantial inhibition of ERK phosphorylation in the HCT116 and HT29 cell lines, respectively, consistent with the relative growth inhibitory potencies of the compounds. These results are similar to previous reports that ERK phosphorylation was completely inhibited by 200 nM AZD6244 in all cell lines tested (In the cell signalling investigations, PD0325901 completely ablated ERK phosphorylation at 21 and 6.5 nThe dual PI3K/mTOR inhibitor NVP-BEZ235 potently inhibited S6 phosphorylation, a well-defined downstream target of both PI3K and mTOR in the PI3K/AKT signalling pathway, consistent with a study by 50 concentration of either PI3K inhibitor in the HT29 cell line, and only at 10 × GI50 concentration of GDC-0941 in the HCT116 cell line. This result is consistent with some previous studies where AKT phosphorylation had recovered by 24 h (In contrast to effects on S6 phosphorylation, inhibition of AKT phosphorylation was somewhat less pronounced with no effect after a 24-h exposure to 1 × or 10 × the GIM in the HCT116 cell line and 94 nM in the HT29 cell line, NVP-BEZ235 completely inhibited 4EBP1 phosphorylation consistent with the inhibition induced by 100 and 500 nM NVP-BEZ235 in the H929 and OPM-2 myeloma cell lines (In comparison with S6 phosphorylation, higher concentrations of NVP-BEZ235 were required to inhibit 4EBP1 phosphorylation. Nevertheless, at 210 nTo test the hypothesis that mTOR inhibition can impair the synergy of PI3K and MEK inhibitors, increasing concentrations of the mTORC1/2 inhibitor KU0063794 were added to the GDC-0941/PD0325901 combination. As predicted, the addition of KU0063794 was found to decrease the synergy of the GDC-0941 and PD0325901 combination; and these data suggest that by inhibiting 4EBP1 phosphorylation and thus preventing the synthesis of proteins that negatively regulate cell-cycle progression, the synergy seen with PI3K/MEK inhibitor combinations is compromised. In contrast, a more selective PI3K inhibitor, such as GDC-0941, is more strongly synergistic in combination with a MEK inhibitor. Additionally, the synergy in the combination of GDC-0941 with a MEK inhibitor may be due to the convergence of the MAPK and PI3K pathways at the level of TSC2, which leads to increased mTORC1 inhibition, whereas the dual PI3K/mTOR inhibitor NVP-BEZ235 is already potently inhibiting mTORC1; and thus, the synergy with a MEK inhibitor is not realised to the same extent.In conclusion, these studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition. However, despite the dual mTOR/PI3K inhibitor NVP-BEZ235 being more potent as a single agent than the specific PI3K inhibitor GDC-0941, the combination of GDC-0941 with either MEK inhibitor was more synergistic than the combination with NVP-BEZ235.


Background: Ferritin stores iron by ferroxidation to form a mineral core, enabling diatom blooms upon iron input.Results: Ferrous iron binds solely to ferroxidase site A anaerobically. Ferroxidation kinetics has two observed phases.Conclusion: Ferrous iron and dioxygen binding to the di-iron ferroxidase site is stepwise.Significance: Iron storage by ferritins requires a coordinated binding of iron and dioxygen. Pseudo-nitzschia multiseries (PmFTN), a marine pennate diatom that plays a major role in global primary production and carbon sequestration into the deep ocean. Crystals of recombinant PmFTN were soaked in iron and zinc solutions, and the structures were solved to 1.65–2.2-Å resolution. Three distinct iron binding sites were identified as determined from anomalous dispersion data from aerobically grown ferrous soaked crystals. Sites A and B comprise the conserved ferroxidase active site, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous soaked crystals revealed only one ferrous iron in the active site occupying site A. In the presence of dioxygen, zinc is observed bound to all three sites. Iron oxidation experiments using stopped-flow absorbance spectroscopy revealed an extremely rapid phase corresponding to Fe(II) oxidation at the ferroxidase site, which is saturated after adding 48 ferrous iron to apo-PmFTN (two ferrous iron per subunit), and a much slower phase due to iron core formation. These results suggest an ordered stepwise binding of ferrous iron and dioxygen to the ferroxidase site in preparation for catalysis and a partial mobilization of iron from the site following oxidation.A novel ferritin was recently found in  Ferritin is a ubiquitous, iron storage, and detoxifying protein found in mammals, plants, and many microorganisms. Ferritin is a 24-mer that forms a hollow sphere, takes up soluble ferrous iron, oxidizes it at di-iron ferroxidase centers, and stores the iron oxide mineral in its central cavity. Iron is subsequently released upon demand of the organism's metabolism , 2. Mamm2 uptake from the atmosphere is limited due to iron availability. These regions are sporadically pulsed with new iron inputs from dust or upwelling deep waters. Pennate diatoms readily bloom upon such iron additions and continue to grow and divide after iron levels return to a low and ambient level  was resolved at 1.95-Å resolution  cells transformed with the expression vector were inoculated into 2× YT medium  and grown at 37 °C to an optical density of ∼0.8 at 600 nm. Protein expression was induced with addition of 0.2 mm isopropyl β-d-thiogalactosidase. The cells were incubated at 25 °C overnight and afterward pelleted by centrifugation. The pellet was resuspended in 20 mm Tris-HCl, pH 8, 1 mm TCEP, and 5% glycerol (v/v), and the cells were lysed using an EmulsiFlex-C5 homogenizer (Avestin). Insoluble cell debris was removed by centrifugation. The supernatant was treated with DNase I type 2 and filtered through a 0.8-μm syringe filter.The construct used for protein expression was a pET28a(+) vector containing the coding region of PmFTN genomic DNA lacking the signal peptide and plastid-targeting sequences . The expet al.  resin. The buffer used for the Source 15Q purification was 20 mm Tris, pH 8 and 5% glycerol (v/v), and the salt gradient used was 0–50% 1 m NaCl. Purified PmFTN was dialyzed into 3% sodium dithionite (w/v), 1 m sodium acetate, pH 4.8, and 1 mm TCEP to remove bound iron to yield the apoprotein. Apo-PmFTN was further dialyzed into 50 mm MES, pH 6.5, 100 mm NaCl, and 1 mm TCEP (Buffer A). The cysteine residues were alkylated by first incubating PmFTN in Buffer A supplemented with 2 mm TCEP for 2 h at 37 °C with shaking followed by the addition of 10 mm iodoacetamide and incubation for 45 min at 37 °C with shaking.PmFTN was purified using a heat shock method as described by Marchetti et al. . Brieflym EDTA was included in the buffer used for Source 15Q chromatography.Some early preparations retained minor DNA contamination, which did not prevent crystallization but prevented accurate determination of kinetic parameters. Thus, subsequent preparations used an alternative DNA precipitation by the addition of 10 μl of 10% polyethyleneimine (w/v)  before flash freezing in liquid nitrogen.Diffraction quality crystals of PmFTN grew by vapor diffusion in a 1:1 mixture of 20 mg/ml protein in Buffer A supplemented with 2 mm sodium acetate, pH 5.5, 1.3–1.5 m ammonium sulfate, and 0.9–1 m NaCl. The crystals were soaked anaerobically in mother liquor supplemented with 2 mm ammonium ferrous sulfate hexahydrate for time periods of 75 min and 2 h. Thereafter, crystals were transferred into mother liquor supplemented with 30% glycerol (v/v) and immersed anaerobically in liquid nitrogen through a specialized port in the glove box.To obtain crystal structures from anaerobic crystals, a PmFTN protein solution was brought into a glove box (Belle Technology UK) containing a dinitrogen atmosphere maintained at less than 35 ppm dioxygen. Anaerobic diffraction quality crystals of PmFTN grew in 0.1 3E6S) as the search model after removal of the iron and solvent atoms  by the stopped-flow instrument with the various ferrous iron working solutions, resulting in a protein concentration of 0.5 μm during data acquisition. All stopped-flow experiments were carried out at 25 °C. Resulting data were fit to a single exponential function using the program Origin . Regeneration experiments were carried out using 1 μm PmFTN containing 48 iron ions added under aerobic conditions for a fixed period (30 min or 20 h) prior to the stopped-flow experiment.Rapid kinetic experiments were carried out using a stopped-flow instrument (Applied Photophysics DX17MV). Changes in absorption on addition of ferrous iron to aerobic apo-PmFTN were measured. A 50 mP23 with eight ferritin monomers in the asymmetric unit  or Zn(II) under various conditions diffracted to at least 2.2-Å resolution. Analysis of Ramachandran plots showed that in all structures more than 96% of the residues were in the preferred regions. All but one of the crystals analyzed were of space group ric unit . The refric unit : 24 subum ferrous iron. Two crystal structures were obtained with crystals soaked in ferrous iron for 75 min and 2 h to resolutions of 2 and 2.1 Å, respectively. The ferroxidase site of the higher resolution structure obtained from the crystal soaked for 75 min is shown in Fig. 2A. Ferrous iron ions are exclusively found in the ferroxidase sites of each monomer. Furthermore, ferrous iron is observed solely in site A  and is refined with an occupancy of ∼50% and B-factors similar to the coordinating residues. The iron in site A is coordinated in an approximate tetrahedral arrangement by the side chains of Glu-15 (∼2.2 Å) and Glu-48 (∼2.2 Å). His-51 forms a weaker interaction with ferrous iron (2.5–2.9 Å). The coordination sphere is completed by one or two solvent molecules (∼2.1 and ∼2.5 Å) depending on the monomer in the asymmetric unit. Ferroxidase site B is occupied by a solvent molecule.The first step in the ferritin reaction is binding of ferrous iron to the ferroxidase site. To observe the binding of ferrous iron, apo-PmFTN crystals were grown in an anaerobic environment, and the octahedral crystals were soaked in 2 mm ferrous sulfate. Structures of crystals exposed to ferrous iron for 4 h or longer revealed iron bound in all three sites as well as up to two additional sites at the inner surface of the ferritin sphere (B and 3A). The latter two sites may serve as the nucleation site for formation of the mineral core. The iron in site A is coordinated by the conserved residues Glu-15, Glu-48, and His-51. Coordination of the iron bound in site B is by 2–3 glutamate residues, depending on the subunit. A bridging oxygen atom is modeled between sites A and B and between sites B and C (B). A second solvent oxygen atom is coordinated to iron in site A. The bridging solvent molecule between sites A and B might be a bridging oxo group rather than a water molecule, building a diferric-oxo bridge as seen in other ferritins . In addition to Glu-130, the site C iron is coordinated by Glu-47 and Glu-44.In our previous work, apo-PmFTN crystals were soaked for 10 min in ferrous sulfate in the presence of dioxygen . The resn sphere B and 3A. B and C B. A secoerritins . Sample o site C , A and BIron occupancy of the sites A, B, and C varies depending on the incubation time of the crystals in ferrous iron in the presence of dioxygen . Iron isC). Glu-130 is predominately coordinated to the zinc ion in site C, although in three of the monomers of the asymmetric unit, Glu-130 adopts two equal occupancy conformations, one that coordinates to the zinc ion in site B, and the other that coordinates to the zinc ion in site C (C). Furthermore, the zinc ion at site A is modeled as coordinated by two water molecules, one of which is bridging to site B. This arrangement of coordinated solvent is similar to that observed in the iron-soaked structures. In the case of iron, the modeled oxygen atom may be an oxo bridge rather than water. An overlay of the zinc structure with the iron structures showed that the zinc atom in site B is slightly shifted toward site A and that the zinc atom in site C is closer toward the core as compared with the iron.Zinc ions are competitive inhibitors of ferritin and bacterioferritin . CrystalFig. 2n site C C. FurtheA shows the absorption changes following the mixing of 1 μm apo-PmFTN with a variable amount of ferrous iron as a function of time. Fe(II) oxidation occurred extremely rapidly with completion for all additions within 0.5 s. For the addition of ∼50 Fe(II) ions per protein (two Fe(II) ions per subunit), the t½ was <50 ms, demonstrating a rate of reaction that is an order of magnitude faster than those measured for EcFtnA and human H ferritin under comparable conditions , giving an observed (pseudo-first order) rate constant, k0, for each addition. Fig. 4B shows a plot of k0 as a function of Fe(II) concentration. Remarkably, there is a linear relationship, demonstrating a first order dependence of the rate of oxidation on the concentration of Fe(II). This observation is significant because it indicates that Fe(II) binding, rather than Fe(II) oxidation, is the rate-determining step of the reaction, consistent with the unprecedented overall rate of Fe(II) oxidation. The slope of the line gives an apparent second order rate constant, k, of 2.54 ± 0.10 × 105m−1 s−1.Stopped-flow experiments were performed to monitor kinetics of iron oxidation after ferrous iron addition to apo-PmFTN. Absorption was measured at 340 nm, which reports on the oxidation of Fe(II) to Fe(III). Fig. 4nditions , 16. EacA340 nm at 0.5 s versus Fe(II) added per PmFTN complex (C) reveals that the rapid oxidation phase is saturated at ∼50 iron ions per protein or two per subunit. Such behavior is characteristic of ferritins in which the ferroxidase center is the initial site of rapid Fe(II) oxidation. Clearly, Fe(II) added in excess of that required to saturate the ferroxidase center is not oxidized at a rate close to that of the initial oxidation phase, and so we conclude that Fe(II) does not undergo rapid oxidation at the third site (site C). This is demonstrated in Fig. 4D, which shows changes in A340 nm over 1000 s following the addition of 100 Fe(II) ions per protein. Although the first ∼50 Fe(II) ions are oxidized extremely rapidly, the subsequent 50 are not; in fact, oxidation is not complete at 1000 s. Thus, the kinetic distinction between these phases is unusually clear.A plot of Δ complex C revealsA340 nm was monitored over a longer time period (E). The data show that a small increase in absorption occurs during the few seconds following the rapid oxidation phase. This increase was observed at all levels of Fe(II) additions, including those that are substoichiometric, and so these changes are not due to oxidation of Fe(II) bound elsewhere. Thus, these absorption changes may be attributed to reorganization of iron in the ferroxidase center and possibly site C following the initial rapid oxidation.The partial occupancy of iron in the ferroxidase sites A and B after prolonged exposure to Fe(II) under aerobic conditions  indicatee period E. The daA). To determine whether the rapid oxidation phase is recovered, PmFTN was loaded with 48 Fe(II) ions per 24-mer and then subsequently incubated for 30 min or 20 h. Then, a further aliquot of Fe(II) (either 40 or 80 Fe(II) ions per protein) was added, and changes in A340 nm were measured. The data  show that no rapid phase of Fe(II) oxidation is observed. Two distinct phases are observed, although it is not yet clear what these correspond to. Data for 20 h are similar (not shown). Clearly, there is no full regeneration of the apo form of PmFTN following oxidation of the initial addition of Fe(II).Mobility of iron following oxidation of Fe(II) at the ferroxidase center could result in complete loss of Fe(III) from the center, leading to regeneration of the apo form. This form would be expected to exhibit the rapid oxidation phase observed above A. To detIron storage ferritins are the archetype of the ferritin-like superfamily of proteins, which includes the enzymes ribonucleotide reductase and methane monooxygenase. A common feature is a catalytic di-iron site where dioxygen is reduced. In ferritin, Fe(II) is the source of electrons, and the resulting oxidized Fe(III) is deposited in the mineral core. A key question in ferritin function is the mechanism of iron mobilization in and out of the ferroxidase site and the core. Iron was visualized in the ferroxidase site (sites A and B) and a third site C of PmFTN. The residues interacting with the iron in site A  are conserved with those of all characterized eukaryotic and prokaryotic ferritins. In addition to Glu-48, Glu-94 coordinates iron in site B and is also conserved.E. coli (EcFtnA)  , 17, Pyrfuriosus , and Arcfulgidus . Neverthfulgidus , and is   for over an hour (A). This observation is in contrast to short (∼1-min) aerobic Fe(II) soaking experiments with frog ferritin in which both sites A and B are occupied   demonstrates a close link between binding and oxidation events such that they cannot be distinguished. Thus, oxidation occurs immediately upon Fe(II) binding to PmFTN, and the binding event can be viewed as the slow step of the reaction. This is in contrast to previous reports of ferritins in which binding and oxidation are considered to be kinetically distinct events. Measurement of Fe(II) binding kinetics is not generally straightforward, although it was possible for EcBFR because Fe(II) binding caused a perturbation of absorbance due to the heme groups. In that case, Fe(II) binding occurred on a much shorter time scale than the subsequent Fe(II) oxidation. Interestingly, Fe(II) binding to EcBFR occurred with a second order rate constant of 2.5 × 10t 30 °C) , a value2  and a possible fourth iron at an unknown metal site, leading to reduction of O2 to water rather than hydrogen peroxide (2 stoichiometry is retained. The kinetic data reported here support the conclusion that only two Fe(II) ions are initially oxidized per subunit. A key structural difference is that site C in PmFTN is only 3.5–3.7 Å from site B, whereas site C in EcFtnA is 7–8 Å from the A/B pair. Third iron binding sites were observed in EcBFR as well as human mitochondrial ferritin (An oximetric assay previously showed that the ferroxidase reaction of PmFTN is associated with consumption of dioxygen in a ratio of 1.9 ± 0.2 Fe(II):O2 . Furthererritins  but not erritins  or Dps ferritins . In contperoxide . The ferperoxide . Site C,peroxide . In contferritin , 24. Howferritin .m) used for soaking experiments, a second Fe(II) ion is not observed at the center in the absence of the oxidant (dioxygen). We note that a similar model was proposed for the two Dps proteins from Bacillus anthracis  during catalysis. Crystal structures of Zn(II) complexes of EcBFR and human mitochondrial ferritin have Zn(II) bound at sites A and B of the ferroxidase center, consistent with a proposed model of Fe(II) binding (Zn(II) is an inhibitor of EcFtnA and is proposed to compete with ferrous iron for the dinuclear center and consequently inhibit oxidation at these sites . In the  binding , 28. How binding . Zn(II) P. furiosus ferritin  at the ferroxidase center are consistent with this conclusion. Nevertheless, for PmFTN, the rapid oxidation of ferrous iron was not regenerated upon up to 20 h incubation, indicating that iron remains present at least in part at the ferroxidase site. Thus, if Fe(III) is displaced, the subsequent iron oxidation is much slower than the initial oxidation. Thus, the mechanism of mineralization in PmFTN appears to be more complex with partial iron migration to the core.Two models have been proposed for the mechanism of ferroxidation by ferritins and bacterioferritins. In one model, the ferroxidase site functions as a substrate site as seen in human H ferritin , EcFtnA ferritin , the fer  under anaerobic conditions. The two distinct kinetic phases observed after the second addition of 48 Fe(II) ions may also be related to slow iron migration to the core likely involving the third iron binding site (site C) and perhaps other sites along a path to the cavity. In frog ferritin, the transit of Fe(III) from the ferroxidase center to the cavity has been shown to occur via a pathway through the subunit toward a 4-fold channel . Site C


The accumulation of fibroblasts and myofibroblasts contribute to inflammation, remodeling and fibrosis by production and release of mediators such as cytokines, profibrotic factors and extracellular matrix proteins. This study investigated the effects of the phosphodiesterase 4 inhibitor roflumilast, combined with the long acting βThe effects of roflumilast and indacaterol treatment were characterized on transforming growth factor β1 (TGFβ1)-treated normal human lung fibroblasts (NHLF). NHLF were evaluated for expression of the profibrotic mediators endothelin-1 (ET-1) and connective tissue growth factor (CTGF), expression of the myofibroblast marker alpha smooth muscle actin, and fibronectin (FN) secretion. Tumor necrosis factor-α (TNF-α) was used to induce secretion of chemokine C-X-C motif ligand 10 (CXCL10), chemokine C-C motif ligand 5 (CCL5) and granulocyte macrophage colony-stimulating factor (GM-CSF) from NHLF and drug inhibition was assessed.Evaluation of roflumilast (1-10 μM) showed no significant inhibition alone on TGFβ1-induced ET-1 and CTGF mRNA transcripts, ET-1 and FN protein production, alpha smooth muscle expression, or TNF-α-induced secretion of CXCL10, CCL5 and GM-CSF. A concentration-dependent inhibition of ET-1 and CTGF was shown with indacaterol treatment, and a submaximal concentration was chosen for combination studies. When indacaterol (0.1 nM) was added to roflumilast, significant inhibition was seen on all inflammatory and fibrotic mediators evaluated, which was superior to the inhibition seen with either drug alone. Roflumilast plus indacaterol combination treatment resulted in significantly elevated phosphorylation of the transcription factor cAMP response element-binding protein (CREB), an effect that was protein kinase A-dependent. Inhibition of protein kinase A was also found to reverse the inhibition of indacaterol and roflumilast on CTGF.These results demonstrate that addition of roflumilast to a LABA inhibits primary fibroblast/myofibroblast function and therapeutically this may impact lung fibroblast proinflammatory and profibrotic mediator release which contributes to small airway remodeling and airway obstruction in COPD. Fixed airway obstruction in chronic obstructive pulmonary disease (COPD) is characterized by small airway narrowing which can sometimes lead to airway occlusion. This has a profound influence on lung function by reducing the rate of emptying of air from the lungs . Recentl®; Daliresp™) has recently been approved in the US and EU for GOLD stage 3 and 4 COPD patients. In Europe, Daxas® is indicated for maintenance treatment in severe COPD patients with chronic bronchitis and a history of exacerbations as an add-on to bronchodilator treatment . Long acting β2 adrenergic agonists (LABA) have been used as the standard of care for asthma and COPD to provide bronchodilation and symptom relief [The novel anti-inflammatory phosphodiesterase-4 (PDE4) inhibitor roflumilast  expression, while inhibition was significantly augmented indirectly by endogenous PGE2 generation [PDE4 inhibitors have been shown to decrease early stage inflammation and fibrosis in a bleomycin-induced fibrosis model using preventative and therapeutic dosing regimens ,13. In vneration ,15.Transforming growth factor β1 (TGFβ1) is a well characterized profibrotic molecule that functions in normal wound healing, and can induce differentiation of fibroblasts to myofibroblasts . MyofibrReagents used were obtained from: RPMI and BSA , TGFβ1 and TNFα , H89 and DMSO . Compounds were synthesized in Gilead Sciences laboratories.2, 37°C, and used at passage 2-5 for experimentation. For experimentation, NHLF were cultured to subconfluence, starved in RPMI + 0.1% BSA overnight, pretreated with compound for 1 h, and stimulated with 10 ng/mL transforming growth factor-β1 (TGFβ1) for 6 h (RNA), 24 h (ET-1 production) or 48 h (FN production and αSMA expression). For experiments with H89, inhibitor was added 30 min before compound pretreatment. All experimental groups were performed in triplicate.Normal human lung fibroblasts  were routinely cultured in FGM-2 complete media (Lonza), 5% COCell lysates were generated at 55°C for 30 min , and run on a Luminex bead-based custom multiplex according to manufacturer's instructions. Median fluorescence intensity (MFI) were generated for each target and normalized to housekeeping genes, peptidylprolyl isomerase B (PPIB) or hypoxanthine phosphoribosyltransferase (HPRT1), which were chosen to match the target transcript abundance. Fold induction was calculated as compared to non-stimulated control.ET-1 production was quantitated by use of QuantiGlo ELISA kit , according to manufacturer's instructions. Results were calculated in pg/mL based on a standard curve, and normalized to cell number.FN production was quantitated by use of QuantiMatrix ELISA (Millipore), run according to manufacturer's instructions. Results were calculated in ng/mL based on a standard curve, and percentage of control calculated from TGFβ1 stimulated DMSO treated cells.Cells were trypsinized, fixed , permeabilized , stained with an anti-αSMA-FITC antibody  and visualized on a LSR II flow cytometer (BD Biosciences). MFI was determined and percentage of control calculated from TGFβ1 stimulated DMSO treated cells.Cells were serum starved and were pretreated for 30 min with H89 (10 uM) or DMSO (0.1%), followed by roflumilast and/or indacaterol treatment or DMSO (0.1%) for 30 min, 37°C, washed with cold PBS and then lysed on ice for 20 min . Lysates were analyzed for pCREB with Luminex bead-based assay (Milliplex MAP Phospho CREB (Ser133) MAPmate, Millipore), as measured by MFI. Results were calculated as percentage of DMSO control.Cells were serum starved, pretreated with compound or DMSO , stimulated with TNFα  and cell supernatants run in a Luminex bead-based assay, according to manufacturer's instructions (Millipore). Results were calculated in pg/mL based on a standard curve, and percentage inhibition calculated relative to TNFα-stimulated DMSO control.50 data for the effect of indacaterol alone on ET-1 and CTGF protein and transcript levels represents the arithmetic mean ± SEM. IC50 data for cytokine inhibition represents the geometric mean with 95% confidence intervals Statistical analysis used a paired two-tailed Student's t-test or repeated one way ANOVA with Tukey's post hoc test. Results where the p-value was < 0.05 were considered significant .ICA panel of candidate genes involved in fibrosis and wound healing was assessed in a time- and concentration-dependent manner after TGFβ1 treatment of NHLF. These studies showed that gene induction occurred at 2, 6 and 24 hours after TGFβ1 treatment, but that a 6 hour treatment with TGFβ1 was optimal for maximal gene induction (data not shown). Further characterization of TGFβ1-induced transcripts showed a robust upregulation for CTGF, ET-1 and VEGF . Induction of other genes examined was in the 1-3 fold range, with no induction of TNFα or collagen 1A2 (COL1A2) seen  alone led to a statistically significant inhibition of ET-1 transcript, although there was a trend for inhibition with indacaterol. When assessed together, indacaterol (0.1 nM) plus 1 or 10 uM roflumilast combination treatment demonstrated 71.7 ± 1.1% (p < 0.01) and 74.5 ± 1.1% (p < 0.01) inhibition respectively, which was greater than either agent alone Figure .Further characterization of ET-1 protein production demonstrated an increase from 2.2 ± 0.7 pg/mL to 20.5 ± 3.2 pg/mL for non-stimulated versus TGFβ1 stimulated NHLF. Roflumilast treatment alone (1 and 10 μM) inhibited ET-1 secretion by 11.1 ± 2.7% and 27.6 ± 3.2% of control, respectively, while indacaterol (0.1 nM) inhibited by 43.3 ± 1.5% (p < 0.05). Similar to the results observed with ET-1 transcript levels, when indacaterol (0.1 nM) was added to roflumilast treatment (1 and 10 μM), the inhibition of ET-1 protein secretion was more pronounced than with either agent alone, with inhibition of 78.3 ± 4.0% (p < 0.001) and 85.7 ± 3.6% (p < 0.001) of control, respectively Figure .50 of 57.4 pM and maximal inhibition of 73.5 ± 0.4%. Indacaterol at 0.1 nM caused 36.6 ± 2.2% inhibition, within the same range seen for ET-1 transcript and protein inhibition at this concentration . Indacaterol (0.1 nM) had no significant effect alone, but in combination with roflumilast at both 1 or 10 μM caused a significant decrease in transcript levels compared to control, with 47.8 ± 1.1% (p < 0.001) and 52.7 ± 1.1% (p < 0.001) inhibition, respectively Figure .To interrogate the mechanism of action for the superior inhibition seen with roflumilast plus indacaterol treatment, addition of the PKA inhibitor H89 was evaluated. As previously seen, roflumilast alone produced very little inhibition of CTGF transcript levels and the addition of H89 had not effect Figure . IndacatRoflumilast treatment (0.001-10 μM) resulted in no increased pCREB at any concentration tested Figure . IndacatAfter TGFβ1 treatment, fibroblasts acquire a more contractile phenotype, and this is characterized by expression of αSMA, accompanied by an increased potential to secrete ECM proteins. Treatment of NHLF with TGFβ1 caused a 10.4 ± 5.5 fold upregulation of αSMA expression. Roflumilast (1 and 10 μM) and indacaterol (0.1 nM) alone failed to inhibit αSMA expression Figure . In combEvaluation of the production of the ECM protein FN showed that after treatment of NHLF with TGFβ1, FN levels increased from 4580 ± 2029 ng/mL to 8700 ± 3693 ng/mL. Treatment with either roflumilast (1 or 10 μM) or indacaterol (0.1 nM) alone caused no significant inhibition of FN secretion Figure . Roflumi50 values of 0.1 nM (CI 0.03-0.4 nM), 0.02 nM (CI 0.007-0.06 nM), and 0.09 nM (CI 0.04-0.2 nM) for GM-CSF, CCL5 and CXCL10, respectively  treatment was 1.0 nM (CI 0.1 - 7.0 nM), 0.2 nM (CI 0.1 - 0.3 nM) and 0.4 nM (CI 0.07 - 2.2 nM) for GM-CSF, CCL5 and CXCL10, respectively.Conditioned media from TNFα- treated NHLF was assessed on a panel of cytokines, where GM-CSF, CCL5 and CXCL10 were found to be increased to 228 ± 12, 21042 ± 513, and 6220 ± 241 pg/mL at 24 h post-stimulation, respectively. Roflumilast or indacaterol alone, and in combination, were assessed for their ability to inhibit TNFα-induced production of these three cytokines from NHLF. Indacaterol caused a potent concentration-dependent inhibition of all three cytokines, with ICy Figure . A submay Figure . Roflumi2 agonist treatment in the context of small airway disease.The use of the PDE4 inhibitor roflumilast provides a novel therapeutic option that improves lung function in COPD patients, and preclinically has been shown to produce a variety of anti-inflammatory effects -23. WhilIn vivo, the intratracheal administration of TGFβ1 led to increased collagen deposition in the airways and airway hyperreactivity [TGFβ1 has been well characterized on fibroblasts for its effects in wound healing , but excactivity . TGFβ1-dactivity ,18,27,28activity ,30. Our 2 can lead to enhanced inhibition of PDE4 inhibitors on fibroblast function, due to either increased endogenous PGE2 production or exogenous addition [2 to point to this as a possible mechanism of action for enhanced inhibition (data not shown).Previous studies looking at the effects of roflumilast on anti-remodeling events have demonstrated a modest effect on collagen gel contraction and FN secretion from collagen embedded NHLF . While iaddition ,15, in oTGFβ1 can signal downstream of its receptor through activation of the transcription factor family of Smad proteins . Evaluat2 and forskolin, can reduce CCL5 production in a H89-dependent manner [Fibroblasts can both respond to, and secrete proinflammatory cytokines. The well characterized proinflammatory cytokine, TNFα, in our model system induced secretion of CXCL10, CCL5 and GM-CSF. CXCL10 and CCL5 are chemokines that are important for monocyte and T cell recruitment to the lung, and GM-CSF is responsible for myeloid survival, differentiation and activation ,36. CCL5t manner . While wWe are the first to report the characterization of the combination of a PDE4 inhibitor with a LABA on primary normal human lung fibroblasts, and demonstrate an enhanced effect by addition of a PDE4 inhibitor to a low concentration of beta-2 agonist on inhibition of NHLF cell function as they relate to proinflammatory and profibrotic mechanisms. It is possible therefore that therapeutically the addition of roflumilast to LABA treatment could impact lung fibroblast proinflammatory and profibrotic functions in the small airways, a process which contributes to fixed airway obstruction in COPD.2 adrenoceptor agonist; TGFβ1: transforming growth factor-β1; ET-1: endothelin-1; CTGF: connective tissue growth factor; αSMA: alpha smooth muscle actin; FN: fibronectin; TNFα: tumor necrosis factor-α; pCREB: phosphorylated cAMP response element-binding protein; CCL5: chemokine (C-C motif) ligand 5; CXCL10: chemokine (C-X-C motif) ligand 10; GM-CSF: granulocyte macrophage colony-stimulating factor; PKA: protein kinase A.PDE4: phosphodiesterase 4; COPD: chronic obstructive pulmonary disease; LABA: long-acting βThe authors declare that they have no competing interests.ST and MS developed the hypothesis, ST conducted the study design, performance of experiments, analysis and interpretation of the data and manuscript preparation. CW and MS helped in the study design, interpretation of the data and manuscript preparation. All authors have read and approved the final manuscript.