Attention affects neuronal processing and improves behavioural performance. In extrastriate visual cortex these effects have been explained by normalization models, which assume that attention influences the circuit that mediates surround suppression. While normalization models have been able to explain attentional effects, their validity has rarely been tested against alternative models. Here we investigate how attention and surround/mask stimuli affect neuronal firing rates and orientation tuning in macaque V1. Surround/mask stimuli provide an estimate to what extent V1 neurons are affected by normalization, which was compared against effects of spatial top down attention. For some attention/surround effect comparisons, the strength of attentional modulation was correlated with the strength of surround modulation, suggesting that attention and surround/mask stimulation  might use a common mechanism. To explore this in detail, we fitted multiplicative and additive models of attention to our data. In one class of models, attention contributed to normalization mechanisms, whereas in a different class of models it did not. Model selection based on Akaike's and on Bayesian information criteria demonstrated that in most cells the effects of attention were best described by models where attention did not contribute to normalization mechanisms. This demonstrates that attentional influences on neuronal responses in primary visual cortex often bypass normalization mechanisms. The neuronal underpinnings of this have been studied in detail, whereby some studies have reported that attention changes tuning curves, whereas others have argued that it changes neuronal gain in either a multiplicative manner, an additive manner or that it mostly affects contrast gain  were used.All procedures were carried out in accordance with the European Communities Council Directive RL 2010/63/EC, the US National Institutes of Health Guidelines for the Care and Use of Animals for Experimental Procedures, and the UK Animals Scientific Procedures Act. The study received institutional review board committee approval . In the present investigation two adult awake male macaques  was presented at pseudorandom locations on a 1° by 1° sized grid, spaced into 10 × 10 squares of 0.1° , while monkeys kept fixation on a central fixation point. Details of the RF mapping were published previously  presented centrally on a 20-inch analogue cathode ray tube monitor . Eye position was monitored with an infrared-based system  with a fixation window of ±0.7–1.1°. Each trial was initiated when the monkey held the touch bar and fixated the central point . Stimulus orientation varied pseudo-randomly from trial to trial in steps of 15°, ranging from 0 to 165° (i.e. 12 different orientations). After 500–800 ms (randomly assigned in steps of 1 ms) a brighter patch  appeared at the centre of one of the bars. The patch had 50% probability of being at the cued (target) or un-cued (distracter) location. If the patch occurred in the cued location monkeys had to release the touch bar within 500 ms to receive a juice reward. If the patch occurred in the un-cued location, the monkeys had to wait for the second patch to appear at the cued location. The bars were presented either alone or centred on the RF of the neuron , or the centre bars were surrounded by multiple orientated bars. The surround bars were identical in size and luminance to the bar at the centre, but varied in orientation. The surround bars were placed equidistantly on two concentric circles  around the centre bar location  on a grey background , whereby R indicates ‘response’ and C indicates ‘RF centre’], as well as the conditions when a surround stimulus was present . This was done separately for the attend RF [R(Catt) and R] and for the attend away [R(C) and R] conditions. Additionally we determined the response in the ‘surround only’ condition for attend RF [R(Matt)] and for attend away trials [R(M)].We analysed neuronal responses for the different stimulus conditions in a response window from 200 to 500 ms after stimulus onset. This period captured the sustained response where attentional modulation is usually more profound (Roelfsema R(Catt)] condition was presented. To establish the time course of attentional modulation for the different stimulus conditions we calculated the population peri-stimulus-time histogram (PSTH) for the population of cells when the preferred orientation was present. This was done based on normalized single-cell activity, whereby the maximum of the response in the attend RF – no surround condition [R(Catt)] was taken as the denominator in the normalization process.To determine the orientation tuning at the population level for the different stimulus and behavioural conditions, we normalized the activity of each cell by dividing all stimulus-driven responses by the response obtained when the preferred orientation in the ‘attend RF –no surround’ [R(M)] with the spontaneous activity (300 to 0 ms before stimulus onset) and determined whether the two differed significantly (t-test). Cells where the ‘surround only’ stimulus induced significant responses were labelled as such and could be excluded from analysis as necessary, to control for surround intrusion effects (for details see Results section and explanation therein). As an additional control we subtracted the response during the ‘surround only’ condition response from the responses when the surround and the centre stimulus was present. For those controls we then also subtracted the spontaneous activity from the centre only responses (i.e. when no surround stimuli were presented) to control for offset effects.For each cell we determined whether the ‘surround only’ stimulus affected neuronal activity by comparing the surround-driven response (if any) . The single parameter additive model (as described above for the multiplicative case) was tested by setting β = 7.39 , for conditions when attention was directed to the RF, and β = 0 (or 1 as described above) otherwise.Finally, an additive attention model, where attention does not contribute to normalization was implemented as:2, as it should not affect the second half of the numerator for conditions when the surround stimulus had 0% contrast. The effect under those conditions would still be additive as the product (β * c2) is added to the R(M) response, rather than multiplying it.Note, that in Eqns 2 error minimization) aiming to predict the following responses:We used unconstrained non-linear optimization to determine the parameters α, and β  from R(C) and R(M) responses  for each of the 12 different centre orientations [here β was set to 1 for the multiplicative models and 0 (but see above) for additive models].Effect of normalization: R from R(Catt) and R(Matt) responses, i.e. another determination of normalization effects for each of the 12 different centre orientations [β was set to 1 for the multiplicative models and 0 (but see above) for additive models for this condition again].Effect of normalization: R(Catt) from R(C) responses (i.e. the effect of attention on centre only responses from centre only responses (attend away condition) for each of the 12 different centre orientations.Effect of attention: R from R(C) and R(M) responses (effects of attention on centre and surround/mask responses predicted from attend away – no surround and from attend away surround only responses) for each of the 12 different centre orientations.Effect of attention: R from R responses (effects of attention on centre and surround/mask responses predicted from attend away centre/surround responses) for each of the 12 different centre orientations.Effect of attention: 2 error minimization for the 60 different responses (five predictions as outlined above for 12 different centre stimulus orientations each). The models were able to accommodate the hypothesis that the focus of attention was narrow for all stimulus conditions (i.e. affecting only the drive centre stimuli yield), or that the focus of attention was wide for all stimulus conditions (i.e. affecting the drive that centre stimuli yield and the drive that surround stimuli yield). However, it might be assumed that the animals would alter their focus of attention depending on the stimulus condition, whereby they use a wide focus of attention when centre only stimuli were presented, while they use a narrow focus of attention when centre and surround stimuli were present. This hypothesis was also accommodated by one set of models, whereby it required us to alter the value for c2, when no surround stimulus was present as the narrow focus of attention and the variable focus of attention models would otherwise be identical. To test for the validity of the variable attention focus model it was set to 0.01 when no surround was present. Overall, this approach yielded 18 different models .We performed χTo determine which model best explained our data we calculated Akaike's information criterion (AIC). Under the assumption of independent normally distributed errors, AIC corresponds to :where χi corresponded to AICi – AICmin, where AICmin was the smallest AIC obtained for the 18 model fits. The larger the wi, the more evidence existed in favour of model i.Here, δThe Bayesian information criterion (BIC) is often used instead of AIC. BIC applies a larger penalty on free parameters in the model and is calculated according to2 is the summed squared error across the fitted data for each neuron and model, k corresponds to the number of free parameters in the model, i.e. 1 and 2, respectively, and n corresponds to the number of data points . The BIC weights are calculated as in Eqn x and y eye position for each stimulus  and attention condition [attend away/attend RF for the period of interest ]. For each neuron recorded (i.e. for each recording session) we calculated a grand mean x-position and a grand mean y-position  and subtracted these grand mean values from the mean single trial x- and y-position. This procedure eliminates potential differences in eye-position calibration between recording sessions, and thus allows comparison across sessions. We performed separate two-factor anovas on the x- and y-position eye-movement data to determine whether mean eye-position in x or in y differed between the attention and stimulus condition. We found no significant difference in mean x-position between different attention conditions . Similar results were obtained for the y-position eye data . This suggests that differences in mean eye position did not contaminate our analysis.We calculated the mean We recorded 105 neurons in two monkeys . Post-mortem histological analysis confirmed that recordings from intended V1 craniotomies were indeed in V1 in both animals.Neuronal tuning width, tuning amplitude and tuning baseline were determined by fitting the neuronal responses with a wrapped Gaussian. The investigation of fitted data was restricted to neurons that were reasonably well described by the wrapped Gaussian function as assessed by our exclusion criterion . After exclusion of poorly fitted responses we were able to analyse 70 neurons  in this part of the study, while we used all 105 cells to investigate whether normalization models account for attention effects in our study.An example cell that was well fitted with the wrapped Gaussian in the four conditions  is shown in Fig.Surround stimuli reduced the tuning amplitude for the example cell shown in Fig.et al., et al., et al., The surround stimulus used was identical for all cells. The specific distribution of orientations present in the surround might therefore yield different effects on neurons, depending on their preferred orientation. This argument is based on the established differential effects of iso- and cross-orientation inhibition  influences the effects of attention/normalization for area MT neurons .To determine whether any of the surround- and attention-induced changes seen in Fig.P < 0.001), and there was a trend towards a reduction of the tuning amplitude (P = 0.083). Attention significantly increased the tuning amplitude (P < 0.05), and it showed a trend towards increasing the tuning baseline (P = 0.095). The changes in significance are probably accounted for by the reduced sample size. Thus, the effects described are not a consequence of direct surround intrusion.It could be argued that the effects described above were a consequence of ‘direct’ surround intrusion, whereby the RFs of the cells extended into the area that was covered by the surround stimulus. To account for that possibility we restricted our analysis to cells that were not directly affected by the ‘surround only’ stimulus condition. We also performed the surround control measures as described in the Methods (i.e. surround only activity subtracted). This control reduced our cell sample to 36 cells, but it did not change the basic results. For this reduced sample size, the surround stimulus reduced the tuning baseline , but the MI distribution for the attend away condition was significantly larger than zero. Surround stimuli significantly reduced tuning baseline for both attention conditions . Surround stimuli did not have systematic effects on the tuning width . The effects described were also found when the ‘surround only’ stimulus-induced activity was subtracted from the surround plus centre stimulus conditions, and when neurons that exhibited significant activity changes upon ‘surround only’ stimulus presentation were excluded from the sample (see Methods). Thus, the main effect of surround stimulus presentation was an overall reduction in stimulus-driven activity, irrespective of the stimulus orientation, i.e. an offset effect, which was reflected in the overall reduced tuning baseline.The above analysis directly used the values of the fitted wrapped Gaussian parameters as input variables. A corresponding picture emerges when analysing modulation indices (MIs of fitted parameters) instead. The MI distributions are plotted in Fig.et al., et al., P < 0.05, Spearman correlation). Baseline MI and tuning width MI were also significantly negatively correlated . The negative correlations were present in each monkey individually . The above described significant negative correlations were equally found when the ‘surround only’ stimulus-induced activity was subtracted from the surround plus centre stimulus conditions and neurons with significant activity changes upon ‘surround only’ stimulus presentation were excluded from the sample. Thus, if surround stimuli induced a reduction in tuning amplitude (or baseline) then they usually also caused a widening of the tuning curve. Conversely, if surround stimuli induced an increase in tuning amplitude (or baseline), then they usually also caused a sharpening of the tuning curve. Note that an increase in tuning amplitude does not necessarily imply an increase in neuronal activity with surround presentation, as the tuning amplitude can increase if the presence of a surround reduces the tuning baseline more profoundly than it reduces the tuning amplitude.Previous studies have reported that surround-dependent suppression of tuning amplitude was positively correlated with surround effects on tuning bandwidth . We used two approaches to calculate the MIs. For the first approach we only used the preferred orientation responses, while for the second we used the average response to all 12 orientations. From these responses attention MIs and normalization MIs were obtained. We found that the correlation for one of our comparisons depended on the approach taken to calculate MIs. Using MIs calculated from preferred orientation responses only, we found a trend for a negative correlation between attention MI – no surround and surround – attend away MIs . This is contrary to predictions from attentional normalization models. However, using the same MI types calculated from responses to all bar orientation yielded a trend for a positive correlation between the surround MI – attend away condition and the attention MI – no surround , results somewhat reminiscent of those described previously for MT neurons . Moreover, surround MIs (measured when attention was directed to the RF) were positively correlated with attention MIs measured in the absence of surround stimuli . No correlation between surround MIs measured in the attend RF condition and attention MIs measured when surround stimuli were present was found . To summarize, correlations between surround suppression indices and attentional modulation are at best weak to moderate. Some of our data support a normalization model of attention, but the latter would have predicted positive correlations between attentional MIs and surround MIs for all comparisons, which we did not find, and notably not for a critical comparison as shown in Fig.So far we have investigated how the different tuning parameters are affected by surround stimulation and attention, and whether changes in a given parameter were correlated with changes in a different parameter. We now turn to the question of whether the effects of attention and of surround stimulation on the neuronal activity were correlated. This might suggest that attention and surround suppression use a similar mechanism, as proposed by normalization models of attention. A recent study reported that strength of normalization was correlated with the strength of attentional modulation, when attention was shifted between preferred and anti-preferred stimuli that were placed within the RF of MT neurons . We decided to use two main types of models (additive vs. multiplicative) based on previous reports about the effects of attention on neuronal activity for single stimuli inside the RF in striate and extrastriate cortex  weights] than models assuming either variable or wide fields of attention. Specifically, 60/105 neurons were best fit with a narrow focus of attention model, 32/105 were best fit with a variable focus of attention model and 13/105 neurons were best fit with a wide focus of attention model. Using models for all three focal attention sizes, we found that the majority of cells were best described by an additive model which assumes that attention does not contribute to normalization (59/105), with a further ten cells which were best described by a multiplicative model which assumes that attention does not contribute to normalization mechanisms, Thus, 69/105 cells were best described by models where attention does not contribute to normalization. While it is possible that monkeys used different attentional strategies in different sessions (and thus different neurons would need to be fit with different basic models), we assume it is more parsimonious that monkeys used the same approach throughout, and therefore applied a single fitting approach (narrow focus of attention which yielded the best fit for the majority of cells) for model comparison. We thus excluded wide and variable attentional focus models from further analysis . Importantly, this reduction in model numbers has no effect on our main conclusions that we draw from the data in the next few sections  weights based on these six models only (see Methods). We counted how often a given model had the largest evidence (when compared with the other models). The number of cells best described by a particular model (using AIC and BIC weights) is listed in TableTo test whether the above reported data would also hold for conditions where fitting was entirely based on attend RF vs. attend away conditions (i.e. ignoring the fits where the effects of surround/mask stimuli were estimated under constant attention conditions), we refitted our data for just three conditions, namely attend RF rates predicted from attend away – no surround conditions, and attend RF rates predicted from attend away – with surround/mask conditions (two versions as outlined in Methods). These fits yielded similar results with respect to the effect of attention on normalization, Under these more constrained fitting conditions, 60/105 cells were best supported by the two-parameter attention additive non-normalization model, while 23/105 were best described by a multiplicative non-normalization model . Fifteen cells were best described by a two-parameter additive normalization model (i.e. 71.4% of cells were best described by an additive model under conditions when the effect of surround stimulations is not part of the fitting routine). Finally, we restricted our fitting to the conditions where we predict attend RF – no surround responses from attend away – no surround responses (i.e. excluding any surround modulation). Under these conditions, 60/105 cells were best supported by a two-parameter attention additive non-normalization model, 19/105 cells were best supported by the two-parameter multiplicative non-normalization model and 17/105 cells were best described by a two-parameter additive normalization model . These controls demonstrate that the results do not simply arise from specific fitting selections.n = 70, see above), it may include neurons where a genuine preferred orientation cannot be delineated. But given the results shown in Fig.While the above data show the relative quality of fits when compared among the different models, they do not provide insight into the absolute quality of the fits. It might be that none of the models describe the data well, and therefore account for little of the variance. Figurer = –0.020, P = 0.843), whether we only used the cells that were best fitted by the additive non-normalizing model  or whether the parameters were used obtained from whichever model best fitted a given cell . The absence of a correlation between β and α has previously been used as an argument against such a two- (independent) parameter model  and the surround modulation term (α) for the additive non-normalization model were correlated. We found no correlation between these parameters, irrespective of whether the parameters of all cells were used  were best described by multiplicative and additive attention models which did not assume attention to contribute to normalization.et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Attention increases the amplitude of neuronal tuning functions in striate , but rather a form of un-tuned normalization. Ni et al.  for our data. We emphasize the fact of ‘relatively’ best model, as Akaike weights, while taking the number of model comparisons into account, do not assess the quality of the model in an absolute sense. There may well be other models, which we did not explore, that would give yet better accounts of our data, even though the fits shown in Fig.Normalization models have successfully explained visual responses under a variety of stimulus conditions , while in our study attention was either directed to the RF centre , or into the opposite hemifield. Shifting attention between different parts of the RF (Ni et al., et al.'s (and with the suppressive drive (Reynolds & Heeger, et al. (et al. (et al. (et al., The discrepancy between our data and previously published data (Ni et al.'s  study. G, et al.  suggest  (et al.  for area (et al. , where t (et al. . Ghose & (et al.  reported et al., , either  et al., . These h


For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq.The α Crude membranes were prepared from transduced or transfected HEK-293/EBNA cells as published -prazosin or [125I]-CYP binding. Reactions were set up as described previously  supplemented with protease inhibitor cocktail III , 1 mM PMSF, and phosphatase inhibitors . Equal amounts of proteins (0.5 mg) were precleared by incubation for 30 min at 4°C with 30 μL of protein A/G Agarose beads (Santa Cruz Biotechnology). After brief centrifugation, supernatants were removed and incubated overnight at 4°C with 50 μL of streptavidin-agarose beads . Samples were then centrifuged and washed three times with 1 mL of Triton lysis buffer. Proteins were eluted from the beads using Laemmli sample buffer, followed by analysis via SDS-PAGE and Western blotting.HEK293 cells transiently transfected with α2+ mobilization experiments were plotted as ΔF/F0 vs. agonist concentration and fit to a sigmoidal concentration-response equation using the GraphPad Prism software package. In cases where biphasic concentration-response curves were observed data were fit to a two-site concentration-response model:0 and Rmax are respectively the response minimum and maximum; Fr1 is the response fraction attributable to receptor characterized by half effective concentration of the first response phase , with the reminder response attributable to the second half effective concentration . For radioligand binding experiments, Bmax values were calculated from maximum binding using the equation Bmax = (SB × (IC50 + [L])/[L]), where SB is the specific binding expressed as fmol per mg of membrane protein, and [L] is the ligand concentration.Experiments were carried out in independent replicates (n indicated in figure legends). Graphs shown reflect either pooled or representative data (see figure legends). For concentration-response analysis, results from Ca1A-AR close to the ones observed in primary cells . This suggested the presence of two distinct mechanisms mediating the observed Ca2+ response. The half-maximal amplitude of the high potency phase was observed at 4.0 nM Iso, whereas the half-maximal amplitude of the lower potency phase occurred at 2.6 μM Iso (Table 1). Since HEK-293/EBNA cells are known to express β2-AR endogenously (∼100–300 fmol/mg protein [2+ mobilization with EC50 = 4 ± 1 nM (Table 1), equivalent to the EC50 determined for the high potency phase of the biphasic dose-response curve in α1A-AR_HEK-293/EBNA cells. Furthermore, the maximal response (ΔF/F0 = 1.3) matched that of the first, high potency phase observed in α1A-AR-expressing cells, derived from fitting data to a biphasic concentration-response equation (ΔF/F0 = 1.3). This finding indicates that Iso occupancy of endogenous β2-AR leads to a small magnitude Ca2+ transient response in untransduced HEK-293/EBNA cells and potentially in the α1A-AR-transduced ones. Since the Iso EC50 in non-transduced cells corresponds to the higher potency phase of the biphasic Iso response observed in α1A-AR_HEK-293/EBNA cells, we used receptor-selective antagonists to further characterize the observed Ca2+ response. The lower potency phase of response to Iso was sensitive to the highly selective α1A-AR antagonist, RS100329 (pKB = 9.6 for α1A-AR)  [0 = 1.2) matched the magnitude of the high potency response phase observed in vehicle pretreated cells, as determined using the two-site model (ΔF/F0 = 1.2). Consistent with this, the potency of Iso in the presence of RS100329 (EC50 = 4.6 nM) corresponded well to the potency calculated for the high potency phase of biphasic dose-response curves in cells pretreated with vehicle . This finding indicates virtually complete blocking of the low potency phase by RS100329 with no effect on the high potency phase. On the other hand, pretreatment of α1A-AR_HEK-293/EBNA cells with the β2-AR selective antagonist ICI 118,551 (10 nM) resulted in monophasic concentration-response to Iso . The calculated potency of Iso in the presence of this β2-AR selective antagonist (EC50 = 2.4 μM) matched the EC50 of 2.6 μM determined for the low potency phase of the bipasic response to Iso in vehicle-treated cells. Interestingly, the maximal response amplitude for Iso in the presence of ICI 118,551  was lower than the amplitude of the lower potency phase observed in vehicle treated cells . This suggests some level of synergy when Iso simultaneously occupies α1A-AR and β2-AR. The β1-AR selective antagonist atenolol (1 μM) had no effect on the Iso concentration-response relationship . In untransduced HEK-293/EBNA cells, response to Iso was blocked by 10 nM ICI118551, a β2-selective antagonist, but not by the α1A-AR-selective antagonist RS100329 . Thus, the biphasic Ca2+ transient concentration-response relationship observed with Iso seems to require co-expression of both the α1A- and β2-adrenoceptors.In baculovirus-transduced HEK-293/EBNA cells homogenously expressing low levels of αprotein, ), the β- protein ,31), we 2+ accumulation mediated by α vs. β2-AR are very different, due to the distinct sources of Ca2+ involved with each pathway downstream from the receptor. The Gαq-mediated α1-AR transient is very fast in onset relative to the Gαs-mediated β2-AR transient [2+ responses to Iso and norepinephrine (NE) in α1A-AR_HEK-293/EBNA transduced and untransduced cells . Stimulation of α1A-AR_HEK-293/EBNA cells with 100 μM Iso (a concentration that produces high agonist occupancy at both α1A-AR and β2-ARs) elicited a slow onset Ca2+ transient response (5–10 second delay from addition of agonist to the rise in Ca2+) that return towards baseline (i.e. decline from peak amplitude >75%) within 100 sec and lacked the sustained phase . A smaller magnitude response with similar slow onset and no sustained elevated phase was observed in response to 100 nM Iso, a concentration that would achieve high fractional occupancy of β2-AR, yet insignificant at α1A-AR . Responses to application of 100 nM or 100 μM Iso in untransduced negative control cells are shown by the gray dashed and solid traces, respectively. By contrast, treatment of α1A-AR_HEK-293/EBNA transduced cells with 100 nM NE resulted in an almost immediate Ca2+ transient onset, with peak response amplitude for the population average attained within 10 seconds post-agonist addition . This response was characterized by a sustained plateau phase of elevated cytosolic Ca2+, not found in untransduced control cells . Because NE possesses ∼ 10-fold higher affinity at α1A-AR relative to β2-AR , the observed response to 100 nM NE is likely driven mostly through occupancy at α1A-AR . Thus, the rapid and sustained Ca2+ elevation in response to 100 nM NE reveals an ability of this agonist to elicit Ca2+ transient responses through a mechanism distinct from the slow onset and transient mobilization response induced by Iso. Responses to 100 μM Iso in cells that were preincubated with the α1A-AR selective antagonist RS100329 or the β2-AR-selective antagonist ICI 118551 revealed the functional contributions of both receptors : both antagonists significantly diminished responses and delayed their onset when compared to the vehicle control . Interestingly, the biggest delay was observed in cells pretreated with the β2-AR-selective antagonist ICI 118551, a condition that isolates the α1A-AR component of Iso-mediated Ca2+ transients .It has been shown previously that the kinetics of intracellular Caransient ,33–35. T2+ were due to influx of extracellular Ca2+ by measuring Ca2+ mobilization in HEK-293/EBNA cells upon exposure to Iso in the presence or absence of external Ca2+. Omitting Ca2+ from the extracellular buffer eliminated Iso-evoked Ca2+ responses in untransduced HEK-293/EBNA cells . In α1A-AR_ HEK-293/EBNA cells, Iso-mediated responses were greatly reduced in amplitude in the absence of extracellular Ca2+ . However, the low potency phase of the concentration-response relationship was only partially affected, unlike responses to Iso < 100 nM, which were almost fully inhibited. Fitting of those data to a biphasic concentration-response model yielded EC50 values of 8.6 nM and 4.5 μM for the high and low potency phases, respectively, close to the EC50 values of 6.3 nM and 2.5 μM measured in the same cells responding to Iso in the presence of extracellular Ca2+. Moreover, the amplitude of the Iso-induced maximal responses under nominally Ca2+-free conditions  was lower than that determined in the same cells in the presence of extacellular Ca2+ (ΔF/F0 = 1.5 and 1.4). Thus, under near-zero Ca2+ concentration both the Iso response of untransduced cells and the high potency phase of the response in α1A-AR transduced cells were largely absent. This finding suggests that the observed β2-AR dependent Ca2+ mobilization requires an influx of Ca2+ from the extracellular compartment, likely via activation of cAMP nucleotide gated-channels. As described in the previous section, the time course of intracellular Ca2+ mobilization in response to 100 μM Iso (a concentration that produces high occupancy of α1A-ARs) was very similar to that observed for β2-AR response and dramatically different from typical Gαq-initiated signaling canonical for α1A-AR. Remarkably, Ca2+ mobilization in response to Iso occupancy of both β2-AR and α1A-AR was found to be less sensitive to removal of extracellular Ca2+ , indicating intracellular stores as the source of Ca2+.We next addressed whether observed rises in cytosolic Cai, the most abundant G protein α subunit, has been shown to lead to activation of PLC activity via the Gβ/γ subunits and thus, can lead to IP accumulation and release of intracellular Ca2+  resulted in a slight increase in the maximum amplitude of the Ca2+ transient response at saturating concentrations of Iso (ΔF/F0 = 1.5 vs. 1.7), with a negligible increase in EC50 from 6 to 10 nM . A similar increase was observed in the β2-AR–dependent phase of the Iso concentration-response curve (from ΔF/F0 = 1.3 to 1.5) in α1A-AR expressing cells, also with no change in observed potency (EC50 = 6.6 vs. 5.6 nM) . Since in these cells induction of cAMP leads to Ca2+ mobilization [2+ mobilization following PTX pretreatment may be a result of an increase in intracellular cAMP. This observation would be consistent with previous findings indicating that β2-AR can couple to both Gαs and Gαi in HEK-293 [1A-AR transduced HEK-293/EBNA cells, pertussis toxin treatment did not significantly affect Ca2+ mobilization responses to Iso (EC50 = 2.8 vs. 2.7 μM with PTX and ΔF/F0 = 1.6 vs. 1.5 with PTX), suggesting that coupling to Gαi is not necessary for this Iso-mediated function of α1A-AR .Receptor coupling to Gαiewed in . Since tlization , increas HEK-293 . On the 1A-AR-dependent phase of the Iso concentration-response relationship was found to be only partially dependent on the extracellular Ca2+ and insensitive to pertussis toxin treatment, we asked whether Iso-occupancy at α1A-AR results in Gαq activation. In α1A-AR transduced HEK-293/EBNA cells, the selective α1A-AR agonist A-61603 elicited concentration-dependent IP accumulation . A-61603-stimulated IP accumulation was best described by a single phase sigmoidal equation yielding an EC50 of 40 nM (Table 1). Furthermore, preincubation of those cells for 5 minutes with 10 nM of the α1A-AR selective antagonist RS100329 significantly attenuated IP accumulation and produced a large, rightward shift in A-61603 potency. In contrast, when replicate cells were treated with Iso (up to 10 mM) no significant accumulation of IP was detected. Furthermore, IP formation in untransduced cells upon treatment with either A-61603 or Iso was not detectable (data not shown). We employed an LC-MS method to increase our detection sensitivity and to measure individual inositol phosphates rather than total IPx accumulation . In α1A-AR transduced HEK-293/EBNA both NE and the selective α1A-AR agonist A-61603, produced concentration-dependent increases in cellular levels of inositol phosphates IP1, IP2 and IP3 that were best described by a monophasic sigmoidal equation . On the other hand, when replicate cells were treated with Iso (up to 10 mM), no significant changes in intracellular inositol phosphates were detected. Thus, it appears that Iso occupancy at α1A-AR biases receptor signaling toward a Gαq-independent pathway.Given that the αe.g. in cardiomyocyte physiology, see [1A-AR transduced HEK-293/EBNA cells with Iso induced a dose-dependent increase of p-ERK formation . The resulting concentration-response relationship was best fit by a biphasic model. The calculated EC50 values were 5 nM and 6 μM for the high and low potency phases, respectively. In untransduced cells a monophasic relationship was observed . Interestingly, in α1A-AR transduced HEK-293/EBNA cells, A-61603 or NE yielded concentration-dependent activation of ERK that was best fit by a monophasic sigmoidal model. The measured EC50 values were 0.8 and 90 nM for A-61603 and NE, respectively . In those cells, A-61603 appeared to be almost 4 times more potent at inducing p-ERK formation than in effecting Ca2+ mobilization , while NE had about the same potency for both response modes.Significant efforts have been placed in understanding the role of the mitogen-activated protein kinase (MAPK) cascade in adrenoceptor signaling . We dec1A-AR _ HEK-293/EBNA cells resulted in a biphasic concentration-response relationship for both p-ERK formation and Ca2+ mobilization transients. The high potency phase of response to Iso (via occupancy of β2-AR) and the low potency phase (via occupancy of both β2-AR and α1A-AR) were quite similar for the two different readouts (Table 1).Iso stimulation of α1A– AR internalizes upon stimulation with A-61603 or ISO. In several prior studies α1A– AR has been found to undergo constitutive, ligand independent trafficking and to internalize rather modestly (∼20%) upon agonist stimulation [1A– AR, we employed constitutively active and dominant-negative forms of Rab5 and Rab11, respectively, to the analysis of α1A– AR endocytosis using confocal microscopy. Constitutively active Rab5 Q79L enhances endocytosis and early endosome fusion, causing the formation of enlarged early endosomes, and its overexpression has been shown to inhibit transferrin recycling. Rab11 S25N is defective in GTP binding and impairs recycling by inhibiting exit from sorting endosomes (early endosomes) to the recycling endosomes and/or plasma membrane. In HEK-293/EBNA cells transfected with α1A – AR only and stimulated with A-61603 or ISO, α1A – AR was mostly localized at the plasma membrane, as was the case for the vehicle control . Overexpression of Rab5Q79L caused some intracellular α1A – AR accumulation in vehicle control cells, indicative of constitutive trafficking, as well as significant α1A – AR accumulation upon A-61603 or ISO treatment. Overexpression of Rab11 S25N led to some accumulation in A-61603 or ISO-treated cells, not as pronounced as the one observed upon Rab5Q79L overexpression. In vehicle-treated cells, α1A – AR appeared predominantly at the plasma membrane. To confirm the observation that stimulation with Iso led to internalization of α1A – AR, we took advantage of a cleavable biotin labeling reagent to discern levels of surface versus internalized α1A – AR by means of immunoprecipitation . Intracellular α1A – AR could be detected at low levels in control cells  indicative of constitutive trafficking. Treatment with A-61603 caused an increase in intracellular α1A – AR levels after 5, 30, and 60 min of treatment  relative to control cells . Similarly, treatment with ISO caused an increase in the intracellular α1A – AR , as compared to control cells . We next examined whether Iso-induced internalization of α1A – AR in HEK-293 cells involved the recruitment of β-arrestins. The ability of the N-terminally FLAG-tagged α1A-ARs to trigger the translocation of GFP-tagged β-arrestins in those cells was investigated by confocal microscopy. As shown in Fig. 8, in vehicle treated cells, the α1A-AR was localized mainly at the plasma membrane while both β-arrestins 1 (left panels) and 2 (right panels) were homogenously distributed throughout the cytoplasmic compartment with no visible co-localization with the surface receptor. In cells expressing the α1A-AR, stimulation with A-61603 for 30 min did not induce significant translocation of either β-arrestin-1 nor β-arrestin-2, to the plasma membrane. Similarly exposure of the same cells to Iso did not cause visible β-arrestin rearrangement. Internalization of the α1A-AR was not detected with either agonist by this method.We next investigated whether or not αmulation –41. Furtmulation . To elim2-AR as well as transduced α1A-AR, we next tested the activity of this β–agonist at α1A-AR in CHO cells stably expressing moderate levels of the receptor (∼1 pmol/mg of membrane proteins) [2+ transient response in α1A-AR_CHO cells . The peak amplitude of Ca2+ transient response, ΔF/F0, as a function of agonist concentration yielded a monophasic concentration-response curve with the half-maximal amplitude occurring at 20 μM Iso . That response to Iso was blocked by preatreatment with 100 nM RO100329, but was insensitive to propranolol at 100 nM. We next examined whether Iso-mediated Ca2+ mobilization involved formation of inositol phosphates. Stimulation of α1A-AR_CHO cells with NE led to concentration-dependent IP accumulation with an EC50 of 0.6 μM . On the other hand, no IP accumulation could be detected in the same cells stimulated with Iso at concentrations up to 1 mM. Similarly, in α1A-AR CHO cells both NE, and the selective α1A-AR agonist A-61603, produced concentration-dependent increases in cellular concentration of inositol phosphates; IP1, IP2 and IP3  that were best described by a single-site sigmoidal equation. In contrast, when replicate cells were treated with Iso (up to 1 mM) no significant changes in intracellular levels of inositol phosphates could be detected. Thus, it appears that Iso occupancy at α1A-AR biases receptor signaling to a Gαq-independent pathway. Yet, similarly to the observations made with α1A-AR_HEK-293/EBNA cells, stimulation of α1A-AR_CHO cells with Iso resulted in a concentration dependent increase in phospho-ERK formation . The amount of p-ERK formed at the maximal concentration of Iso represented 35% of the level observed in these cells when stimulated with saturating concentrations of NE or A-61603. Iso was less potent than NE and A61603 at inducing ERK activation in α1A-AR_CHO cells . These results indicate that Iso is ineffective at inducing α1A-AR-mediated Gαq coupling and PLC activation, yet shows partial agonist activity at mediating α1A-AR induced activation of the MAPK signaling cascade.Since in HEK-293 cells Iso can stimulate both endogenous βroteins) . The β-A1- AR subtypes are expressed  that are highly efficacious for IPx formation responses and considered typical for Gαq-initiated signaling. Responses to Iso showed delayed onset by 5 to 10 seconds and were much slower to rise in amplitude. Moreover, Iso was a partial agonist at α1A- ARs as recorded by this readout. Finally, the observed Ca2+ transients exhibited almost complete desensitization, unlike responses to NE. These data taken together indicate a mechanism of action distinct from classical Gαq coupling for Iso at the α1A-AR, and suggest functional selectivity  for the activity of this agonist.Our studies of HEK-293/EBNA cells with transient low expression levels of α1A-AR is widely known to couple to phospholipase C. However, this receptor also reportedly mediates activation of several other effectors such as phospholipase D, phospholipase A2, adenyl cyclase and several members of the MAPK family . Certaiiewed in –48. Withand MAPK ,50.1A-AR caused Ca2+ transient responses in HEK-293/EBNA cells without observable PLC activation: no formation of individual inositol phosphates or IP accumulation was detected in these cells under the same conditions. Unexpectedly, Iso occupancy of α1A-AR also mediated ERK1/2 activation, suggesting that Gαq coupling is not required for ERK activation by Iso. Adding to the surprise, A-61603 appeared to be significantly more potent at activating ERK as compared to activation of Gαq, whereas NE displayed equivalent potencies for both responses. Taken together, the signaling patterns observed with these agonists appear to reveal a spectrum of partially overlapping effector coupling mechanisms. Iso, and the imidazoline A-61603, appear to have a bias toward MAPK activation. Moreover, for A-61603 there seems to be greater receptor reserve or response amplification for the pERK formation response.We observed that Iso occupancy of α1A-AR has also been found to regulate mitogenic responses, but it is not clear if this activity is downstream of Gαq engagement [1A-AR-mediated MAPK phosphorylation in rat PC12 cells [q-induced Ca2+ mobilization and PKC activity [q, but also abolishes activation of the MAPK pathway [1A-AR-mediated MAPK activation may not be associated with activation of Gαq pathway [1A-AR /α1B-AR double-knockout mice also developed heart failure after transverse aortic constriction, and reconstitution of α1A-AR signaling in cardiomyocytes from those animals rescued them from NE-induced apoptosis [1A-AR function or in wild-type mouse cardiomyocytes, pointing at a non-Gq signaling mode for this receptor in myocardial function. More importantly, Huang et al. (2007) reported that α1A-AR stimulation of an ERK-mediated pathway is critical for cardiomyocyte survival. Our data support those findings as well as provide further evidence indicating that Iso occupancy of α1A-AR induces an active receptor “state” that leads to MAPK pathway activation without activating the canonical Gαq pathway.The αgagement ,51. Minn12 cells , indepenactivity . The sam pathway . It was  pathway ,55. α1A-poptosis . The expq coupling defective variants of α1A-AR combined with examination of the effects of Ca(II) channel blockers uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation [1A-AR_HEK293/EBNA cells. Interestingly, in the presence of a β-AR-selective antagonist, the amplitude of the α1A-AR-mediated Ca2+ transient response to Iso (i.e. the lower potency phase of the Iso concentration-effect relationship) was not only lower than expected, but also occurred with a significant delay in onset. This may indicate a synergy occurs between these receptors in generating the lower potency phase of response. Depletion of extracellular Ca(II) did not affect Iso induced activation of α1A-AR indicating that intracellular stores were the source of Ca(II) released. Finally, this signaling cascade did not involve coupling to Gαq, Gαs or Gαi but led to receptor internalization and activation of ERK1/2. An alternate mechanism for the control of Ca2+ release from the ER involves ryanodine receptors (RyRs). They have been shown to exist in several non-excitable cell lines, although their functional expression in HEK-293 cells is controversial. RT-PCR analysis of HEK-293/EBNA cells found that significant levels of RyR2 mRNA are present in these cells.Our previous studies of Gαtivation . Moreove1A-AR-mediated activation of endocytic pathway that leads to phosphorylation of ERK1/2, independent of Gαq /PLC/PKC signaling [1A-AR induced activation of ERK1/2, but not p38 MAPK, was dependent on cytoskeleton and actin organization. On the other hand, α1A-AR-induced activation of PKC and C-Raf was not affected by endocytosis disruption. Neither PKC nor PLC inhibition blocked α1A-AR induced activation of ERK1/2 [1A-AR in HEK293 were investigated in combination with various inhibitors of PKC, receptor internalization as well as β-arrestin 2 silencing and differential patterns were discovered for Gq/PKC dependent as compared to the Gαq-independent signaling cascade. The rapid phosphorylation of ERK1/2 was dependent on activation of PKC downstream of Gαq and resulted in pERK1/2 translocation into the nucleus while sustained activation of ERK1/2 that was limited to cytoplasmic compartment, was independent of PKC but dependent on receptor internalization into acidified endosomes and was mediated by β-arrestin 2[1A-AR can induce activation of ERK1/2 in a manner that is independent of the canonical Gαq /PLC/PKC signaling cascade but involves receptor internalization. We also found that Iso is a biased agonist that can preferentially stimulate α1A-AR mediated activation of MAPK signaling pathway through receptor internalization and without induction of Gαq coupling. Although, our study did not conclusively detect Iso induced α1A-AR association with β-arrestins after agonist application, involvement of β-arrestin 2 in mediating this response cannot be ruled out. Several groups have demonstrated that agonist-mediated GPCR association with β-arrestin leads to G protein-independent activation of the MAPK pathway [1A-AR associates although weakly with both β-arrestin-1 and 2, but coupling to the MAPK pathway was not investigated in this study [Recent reports provide an independent line of evidence for αignaling ,58. In of ERK1/2 . More re pathway . In factis study . Further1A-AR transactivates EGFR via a β-arrestin1-dependent mechanism similarly to the β1-AR mediated EGFR transactivation and leads to ERK1/2 activation downstream of EGFR [1A-subtype. We have examined this potential mechanism in mediating the Iso induced α1A-AR stimulation of ERK1/2 using EGFR inhibitor AG1478 and found that activation of ERK was not affected by the inhibition of EGFR (data not shown). Further studies may be needed to fully explore this mechanism.On the other hand, it has been recently shown that a genetic variant of the α of EGFR –62. Tran of EGFR ,62 suggeet al., who reported that Iso evoked biphasic dose-effect relations in MAPK activation [2-AR/Gαs, or by formation of a macromolecular complex containing β2-AR and c-Src which directly activates c-Src. The conclusion that β2-AR was the sole mediator of both phases of the Iso response was based on their complete blockade by 1 μM ICI118551, as well as lack of detectable MAPK activation in MEF cells derived from β1-AR−/− and β2-AR−/− mice. Although these findings offer strong evidence for β2-AR involvement in mediating that response, they do not necessarily rule out a synergy between β2-AR and α1-ARs, if present in those cells.Our observations differ from those of Sun tivation . The obs1A-AR-mediated Ca(II) transient response was much slower and with the greatest delay , and the the amplitude of the α1A-AR-mediated Ca(II) transient  was lower than expected, indicating synergy between these receptors in generating the low potency response phase. This is consistent with previously published results with wild-type α1A AR and Gαq signaling deficient variants expressed in HEK EBNA cells with endogenous expression of β2 AR that showed that β2 AR signaling actually potentiates α1A AR- mediated Ca(II) response and the potentiation was most striking in cells expressing Gαq defective mutants [Interestingly, we observed that in the presence of a β-AR-selective antagonist, the kinetics of Iso-evoked α mutants . Further2+ mobilization (as well as ERK activation) in CHO cells lacking β2-AR expression, upon stable expression of α1A-ARs  and in α1A-AR_ HEK-293/EBNA cells in presence of β-AR antagonists indicating that this signaling is not dependent on β2-AR. The potency of Iso at mediating the Ca(II) response in α1A-AR_ CHO cells (EC50 = 18 ± 8 μM) was somewhat lower than the potency of the second phase observed in HEK-293/EBNA cells (EC50 = 2.6 μM). This response was blocked by an α1A-AR-selective antagonist, but was not sensitive to the β-AR selective antagonist propranolol. On the other hand, in untransduced HEK-293/EBNA cells expressing β2-ARs which lack α1A-AR expression, we observed only a high potency, monophasic response to Iso for both intracellular Ca2+ mobilization and p-ERK formation.Nonetheless, Iso can also induce intracellular Ca1A-AR was transiently expressed at low, physiological levels together with endogenous β2-AR in HEK293(EBNA), evidence already exist suggesting that similar mechanism may be utilized in physiologically relevant settings such as e.g. cardiomyocytes where both receptors are co-expressed. Sabri et al observed that in rat cardiomyocytes ERK activation in response to NE was mostly mediated by α1-AR receptor and only a minor component of ERK was activated by β-AR[in vitro and in vivo have been reported. The cardiotoxic effect was found to be mediated by PKA and sarcoplasmic Ca(II) overload downstream of β-AR since the effect could be blocked by PKA inhibitory peptide and β-AR antagonist. On the other hand the cardioprotective effect was attributed to ERK activation mediated by EPAC downstream of β-AR [1A/B-AR KO mice reported that α1A-AR induced ERK signaling pathway is cardioprotective and required for cardiomycocyte’s survival[1A –AR but not α1B-AR and required activation of ERK1/2. Similarly, in neonatal rat myocytes, α1-AR stimulation inhibited apoptosis caused by cAMP, and was abolished by a MEK inhibitor suggesting a role for ERK1/2[1A –AR signaling in the CNS . Chroni1A-ARs, and appears to manifest effector bias when bound at α1A-AR, inducing activation of a Gαq-independent signaling cascade. This may explain decades-old observations of unusual properties reported for this ligand. As discussed earlier, in cardiomyocytes, induction of the MAPK pathway via α1-ARs has been shown to protect those cells from apoptosis [q signaling induced cardiac hypertrophy and loss of β-AR inotropic responsiveness [1A-AR of the agonist ISO, adds pharmacological and chemical support for the potential to identify a novel class of therapeutics for treatment of heart failure [In conclusion, we have found that isoproterenol is a low potency agonist at αpoptosis . In contsiveness , ultimatsiveness . Thus, o failure ,69. AlthS1 Fig1A-AR transduced HEK-293/EBNA cells after pre-treatment with vehicle (filled symbols) or antagonist (empty symbols). HEK-293/EBNA cells were exposed to baculovirus encoding α1A-AR for 3–4 hours and then cultured in fresh media containing 4 mM NaBu for 18 hours prior to use in experiments as described in 1A-AR antagonist RS-100329 and then exposed to agonist for 10 minutes. IP concentration was determined by homogenous time-resolved immunofluorescence detection method. The plot is representative of two independent experiments with each data point being an average of quadruplicate.Inositol phosphate accumulation responses are shown for A-61603 (squares) or Iso (circles) in α(TIF)Click here for additional data file.S2 Fig1A-AR (●), were pre-treated with NaBu for 18 h to induce receptor expression. Cells were stimulated for 5 min with increasing concentration of Iso. Agonist treatment was terminated by addition of of SureFire lysis solution. Samples were analyzed for levels of phospho-ERK using an AlphaScreen SureFire p-ERK assay kit. Plots are representative of two independent experiments, with each data point being the average of triplicates.HEK-293/EBNA cells that were transduced with baculovirus bearing aldehyde oxidase(as negative control vector) (■) or α(EPS)Click here for additional data file.S3 Fig1A – AR. After serum deprivation for 24h, cells were pre-treated with a membrane impermeable, disulfide-cleavable biotin reagent to label plasma membrane α1A – AR. Cells were then left untreated, or stimulated 1 μM A-61603(A) or 1mM ISO(I) for 5, 30, or 60 min. After treatment, one dish of control cells was harvested without any further manipulations . The remaining seven dishes were divided into one control (C+GSH), three treated with A-61603 (A-61603+GSH) and three treated with ISO (ISO+GSH). They were stripped of surface biotin label using a reducing agent, in order to reveal internalized, labeled α1A – AR. Samples were then analyzed by immunoprecipitation (IP) with streptavidin followed by immunoblotting (IB) with an anti-FLAG antibody. Bands were quantified by densiometry, normalized to control. Plots are representative of three independent experiments.HEK293 cells were transiently transfected with α(EPS)Click here for additional data file.S4 Fig2+ transients (expressed as ΔF/F0) were measured as a function of Iso concentration (■) in fluo3-loaded cells by fluorometric plate imaging (FLIPR). Cells were stimulated with CCR5 agonist MIP1α  at 1 μM concentration to confirm their responsiveness. Plots are representative of two independent experiments with each data point being the average of triplicates.Ca(EPS)Click here for additional data file.


Dermatophagoides pteronissinus and Dermatophagoides farinae. In normal and AR subjects, neutrophil apoptosis was inhibited by extract of Dermatophagoides pteronissinus (DP), but not by extract of Dermatophagoides farina (DF). Aprotinin (serine protease inhibitor) and E64 (cysteine protease inhibitor) have no effect on neutrophil apoptosis due to DP. The anti-apoptotic effect of DP was blocked by TLR4i, an inhibitor of TLR4, rottlerin, an inhibitor of PKCδ, PD98059, an inhibitor of ERK, and BAY-11-7085, an inhibitor of NF-κB. DP induced PKCδ, ERK, and NF-κB activation in a time-dependent manner. DP inhibited the cleavage of procaspase 3 and procaspase 9. The expression of IL-6, IL-8, TNF-α, G-CSF, GM-CSF, and CCL2 increased in the supernatant collected from the normal and AR neutrophils after DP treatment and the supernatant inhibited the apoptosis of normal and AR neutrophils. In summary, DP has anti-apoptotic effects on neutrophils of normal and AR subjects through the TLR4/PKCδ/ERK/NF-κB pathway, and this finding may contribute to solution of the pathogenic mechanism of allergic diseases triggered by DP.House dust mite (HDM) is a primary allergen in allergic rhinitis (AR) and asthma. Neutrophil apoptosis is associated with allergic diseases and innate immunity to infection. The present study examined how HDM affects constitutive neutrophil apoptosis in normal and AR subjects. Total IgE increased in AR subjects when compared to normal subjects, and patients with AR were HDM-specific IgE positive (+), which is specific IgE to  Dermatophagoides pteronissinus and Dermatophagoides farinae, plays an important role in the onset and aggravation of allergic diseases, including AR Rhinitis is classified as allergic or non-allergic based on allergen sensitization Neutrophils are an abundant leukocyte in the peripheral blood that function as the first line of defense against infection RPMI 1640 and fetal bovine serum (FBS) were purchased from Life Technologies Inc. . CLI-095, an inhibitor of Toll-like receptor (TLR) 4 (TLR4i) was purchased from Invivogen . PKC δ inhibitor (rottlerin) MEK inhibitor (PD98059), and NF-κB inhibitor (BAY-11-7085) were purchased from Calbiochem . GM-CSF was purchased from R&D Systems . FITC-conjugated rabbit anti-mouse IgG, and FITC-conjugated rat anti-rabbit IgG were purchased from Molecular Probes . Antibodies against phospho-tyrosine and phospho-ERK1/2 were purchased from Cell Signaling Technology . Antibodies against ERK2, procaspase 3, and procaspase 9 were obtained from Santa Cruz Biotechnology . Anti-TLR2 antibodies were obtained from Biolegend . The protease inhibitors aprotinin and E64 were obtained from Sigma–Aldrich . DP and DF were obtained from the Korea National Arthropods of Medical Importance Resource Bank  and Cosmo Bio . Der p1 and Der p2 were purchased from INDOOR biotechnologies .Twenty-eight AR patients between 8 and 17 years of age (average  = 12.7 years) were recruited from the Department of Pediatrics at Eulji University Hospital. AR patients had general nasal symptoms for more than 4 days a week during more than 4 consecutive weeks. Allergic status was based on the presence of positive results of a skin prick test (≥2+) or multiple allergen simultaneous test (MAST) (≥ class 2) to common allergens. AR subjects suffering from asthma had mild to severe symptoms of the disease. Seventeen normal subjects between the ages of 16 and 20 years (average age  = 16.8 years) were recruited as controls. The normal subjects had no history of allergic diseases such as AR, asthma, and atopic dermatitis and did not require medication. This study was approved by the Institutional Review Board of Eulji University for normal volunteers and by the Institutional Review Board of Eulji University Hospital for AR patients. Written informed consent was obtained from study participants and from parents of children in this study. In addition, we met all the participants, informed them about the purpose of our study, and answered their questions about the project.Serum total IgE concentration was analyzed using an ADVIA Centaur immunoassay . The ADVIA Centaur IgE assay is a reverse sandwich immunoassay that employs direct chemiluminescent technology. In this system, the monoclonal mouse anti-human IgE antibodies are covalently bound to paramagnetic particles (PMP) in a solid phase. The anti-IgE coupled to the PMP captures the sample IgE, after which biotinylated allergen is added in excess and binds to the allergens-IgE antibody captured on the solid phase. Streptavidin labeled with acridinium ester is then added and binds to the biotin-labeled allergen. A direct relationship has been shown to exist between the amount of allergen-IgE present in the sample and the amount of relative light units emitted by the acridinium ester and detected by the apparatus.Blood samples were obtained by venipuncture and analyzed in duplicate on an ADVIA 2120 hematology system . The results for all samples were verified by microscopic analysis of a blood smear.6/ml in RPMI 1640 medium with 1% penicillin-streptomycin and 10% FBS. This method routinely yielded greater than 97% neutrophil purity as assessed by counting the cells using a cytospin system.Human neutrophils were isolated from the heparinized peripheral blood of healthy persons and AR subjects using Ficoll-Hypaque gradient centrifugation and a CD16 microbeads magnetic cell sorting kit . The cells were washed after hypotonic lysis to remove erythrocytes and then resuspended at 3×10An annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit  was used for detection of neutrophil apoptosis. Isolated neutrophils were incubated with FITC-labeled annexin V and propidium iodide (PI) for 15 min at room temperature. Apoptotic neutrophils were analyzed using a FACSCalibur flow cytometer with the CellQuest software (BD bioscience) and reported as the percentage of cells showing annexin V+/PI- and annexin V+/PI+.After being treated with DP, cells were harvested and lysed in a cytosolic extraction buffer. The homogenate was then centrifuged at 10,000 g for 1 min at 4°C, after which the supernatant was collected as a cytosolic fraction. The pellet was subsequently resuspended in 50 µl of nuclear extraction buffer and centrifuged at 12,000 g for 15 min at 4°C, after which the supernatant was collected as the nuclear fraction. The protein samples (50 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis, after which they were transferred to membranes and incubated with anti-phospho ERK, anti-procaspase 3 or anti-procaspase 9 antibodies and developed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The same blot was stripped and reprobed with anti-β-actin or anti-ERK2 antibodies for use as an internal control.After neutrophils were incubated with DP or medium at 37°C, the cells was lysed in lysis buffer . Following incubation on ice for 20 minutes, the detergent-insoluble materials were sedimented by centrifugation at 12,000 g for 15 min at 4°C. The supernatants were then pre-cleared by incubation with 10 µl of Protein A/G Plus agarose (Santa Cruz Biotechnology). For immunoprecipitation, samples were incubated with 1 µg/ml of anti-PKCδ antibodies (Santa Cruz Biotechnology) for 2 hrs at 4°C, followed by incubation with 20 µl of Protein A/G Plus agarose for 1 hr at 4°C. The immunoprecipitates were then washed three times with ice-cold lysis buffer and boiled with 20 µl of Laemmli buffer for 4 minutes, after which Western blotting was performed using anti-phospho-PKCδ or anti-PKCδ antibodies.The DNA-binding activity of NF-κB was assessed using EZ-Detect transcription factor kits for NF-κB p65 , following the manufacturer's instructions. DNA binding specificity was assessed using wild type or mutant NF-κB oligonucleotides. chemiluminescent detection was performed using a luminometer.The concentrations of IL-6, IL-8, GM-CSF, G-CSF, TNF-α, and CCL2 in cell supernatant were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) using OptEIA Set human IL-6, IL-8, GM-CSF, TNF-α, and CCL2  and G-CSF  according to the manufacturer's instructions.p value <0.05 was considered to indicate statistical significance.Data were expressed as the means ± SD. Statistical differences were analyzed using a paired t-test for two-group comparisons and one-way ANOVA for comparison of more than two groups. All analyses were conducted using the SPSS statistical software package , and a Prior to evaluating the effects of HDM in neutrophil apoptosis of normal and AR subjects, we investigated the association of HDM with AR. Exposure to allergens was detected by evaluating total IgE and allergen-specific IgE. Total IgE was significantly increased in the serum of AR subjects when compared to normal serum . As showp<0.01), whereas DF had no effect on apoptosis Click here for additional data file.Figure S2Neutrophil apoptosis and IgE are not different between AR and AR/BA subjects. (A) Neutrophils were isolated from peripheral blood of AR (n = 10) and AR/BA subjects (n = 18). The cells were incubated for 24 h in the absence (Con) and presence of 10 µg/ml of DP. Data are presented relative to the control, which was set at 100% as the means ± SD. (B) Total IgE in serum of AR (n = 10) and AR/BA subjects (n = 18) was measured by ADVIA Centaur immunoassay (C) AR (n = 10) and AR/BA subjects (n = 18) were grouped by absence or presence of DP IgE and DF IgE measured by skin prick test or MAST.(TIF)Click here for additional data file.Figure S3Differential leukocyte count is not different between AR and AR/BA subjects. Blood samples were obtained by venipuncture and were analyzed in duplicate on the ADVIA 2120. Total cells were set at 100% and data are expressed as the means ± SD.(TIF)Click here for additional data file.


Synechococcus elongatus PCC7942 has been the least successful; the FFA-excreting mutants constructed from this strain could attain lower rates of FFA excretion and lower final FFA concentrations than the mutants constructed from Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002. It has been suggested that S. elongatus PCC7942 cells suffer from toxicity of FFA, but the cause of the low productivity has remained to be determined.Among the three model cyanobacterial species that have been used for engineering a system for photosynthetic production of free fatty acids (FFAs), Synechococcus elongatus mutant (dAS1T). The final FFA concentration in the external medium was also higher than previously reported for other S. elongatus mutants. However, about 85 % of the total FFA in the culture was found to remain in the cells, causing severe photoinhibition. Targeted inactivation of the wzt gene in dAS1T, which gene manipulation was previously shown to result in loss of the hydrophilic O-antigen layer on the cell surface, increased FFA secretion, alleviated photoinhibition, and lead to 50 and 45 % increase in the final cell density and the total amount of FFA in the culture , respectively. The average rate of production of total FFA by the culture of the ∆wzt strain was 2.7 mg L−1 h−1, being five times higher than those reported for Synechocystis sp. PCC 6803 and comparable to the rates of triacylglycerol production in green algae.By modulating the expression level of the acyl–acyl carrier protein thioesterase and raising the light intensity during cultivation, FFA secretion rates comparable to those obtained with the other cyanobacterial species were attained with an engineered Synechococcus elongatus PCC7942 has larger capacity of FFA production than Synechocystis sp. PCC6803 but accumulates most of the product in the cell because of the imbalance of the rates of FFA production and secretion. This causes severe photoinhibition and exerts adverse effects on cell growth and FFA productivity. Enhancement of FFA secretion would be required to fully exploiting the capacity of FFA production for the purpose of biofuel production.The online version of this article (doi:10.1186/s13068-016-0506-1) contains supplementary material, which is available to authorized users. PCC 6803  and 18:0[d3]), extracted with a modified Folch method [For analysis of cellular and extracellular FFA, 5–30 ml aliquots of the cultures were centrifuged at 1700h method , 30, andh method , 30. To 8 of the cells.Chlorophyll (Chl) was determined as described by Mackinney . For the−2 s−1, using a Clark-type oxygen electrode . The photosynthetic yield of PSII was measured using an AquaPen-C fluorometer .Photosynthetic oxygen-evolving activity of the cyanobacterial cultures was measured at 30 °C under illumination at 1000 μmol photons m−2 s−1) at t = 48 h, using a combination of the TRIzol® Reagent (Life technologies) and the SV total RNA isolation system (Promega) as described by Kato et al. [‘tesA and rnpB (Additional file Total RNA was extracted from dAS1T cells grown with either nitrate or ammonium under high light (180 μE mo et al. . The iso


In this paper we present a review of the most current avenues of research into Kinect-based elderly care and stroke rehabilitation systems to provide an overview of the state of the art, limitations, and issues of concern as well as suggestions for future work in this direction. The central purpose of this review was to collect all relevant study information into one place in order to support and guide current research as well as inform researchers planning to embark on similar studies or applications. The paper is structured into three main sections, each one presenting a review of the literature for a specific topic. Elderly Care section is comprised of two subsections: Fall detection and Fall risk reduction. Stroke Rehabilitation section contains studies grouped under Evaluation of Kinect’s spatial accuracy, and Kinect-based rehabilitation methods. The third section, Serious and exercise games, contains studies that are indirectly related to the first two sections and present a complete system for elderly care or stroke rehabilitation in a Kinect-based game format. Each of the three main sections conclude with a discussion of limitations of Kinect in its respective applications. The paper concludes with overall remarks regarding use of Kinect in elderly care and stroke rehabilitation applications and suggestions for future work. A concise summary with significant findings and subject demographics (when applicable) of each study included in the review is also provided in table format. Elderly Care is comprised of two subsections: 1) Fall detection and 2) Fall risk reduction. Stroke Rehabilitation contains: 1) Evaluation of Kinect’s Spatial Accuracy, and 2) Kinect-based Rehabilitation Methods. We have allocated a third section titled ‘Serious and exercise games’ for studies that are indirectly related to the first two sections and present a complete system for elderly care or stroke rehabilitation in a Kinect-based game format. A concise summary with significant findings and subject demographics (if applicable) of each study included in the review is also provided in a table format Tables The median age of the general population is projected to significantly rise in the upcoming years . As the elderly care applications and medical alert support systems. These alert systems focus on reducing the probability a fall incident will occur; a leading cause of injury, emotional distress, and financial burden to the elderly [Users of the Kinect are able to intuitively interact with a computer through various gestures and postures. This natural method of human-computer interaction allows for the development of specialized forms of  elderly -17. Curr elderly -22 and a elderly -26.stroke rehabilitation applications have the potential to reduce this impracticality through guided interactive rehabilitation and virtualized therapists. The accuracy of the Kinect for clinical use to this end is strong [Ideally, all stroke rehabilitation exercises would be performed with therapist-assisted daily practice; however, the demand this would create for therapists make it logistically impractical and quite expensive. Kinect-based s strong -32, supps strong . The vars strong , but alss strong .game-based applications in both elderly care and stroke rehabilitation. Serious games offer patients rehabilitation environments which help motivate successful completion of otherwise dreary or demanding rehabilitation regimens [The Kinect also provides a platform for the development of stimulating regimens , whereasregimens -46.All peer-reviewed journal and conference proceedings articles published in English, directly  or indirectly  related to elderly care and stroke rehabilitation and conducted within one of the subfields presented in the previous section, and also in Figure As the volume of literature regarding the usage of the Kinect in the fields of interest was not anticipated to be extraordinarily large, but instead not yet aggregated, exclusion criteria for this review was minimal: studies which did not go through a peer-review process for publication or were published in a non-English language, were not directly or indirectly related to elderly care or stroke rehabilitation, or were out of the scope of the subfields mentioned previously, and also in Figure kinect and derived combination sets of the following: stroke, rehabilitation, gesture, posture, clinical, geriatrics, elderly, ageing, aged, alert, fall, gait, exergame, and serious game. No date range or other limits were imposed during the search. Titles and abstracts of all articles were scanned for relevance and a complete list of possible inclusions was compiled with citation information retained in a LaTeX bibliography file. All relevant papers were then closely examined and if a tagged journal paper was deemed a more complete study of a tagged conference paper, the journal version was included and the conference version was discarded. The literature review was concluded on August 1st, 2013.The following electronic bibliographic databases were searched: IEEE/IET Electronic Library, PubMed, ACM Digital Library, Computer Science Index, Safari Tech Books Online, and ISI Web of Science. Articles were located using the keyword As the topic of this review spans two overarching categories including multiple, not directly inter-related subcategories, data was collected, and is presented, on a by-topic basis through included charts and graphs as well as in-line text.Initially, we planned to assess study quality with the Downs and Black check-list ; howeverFigure fall detection and risk reduction alert systems is needed and there is significant interest in applications of Kinect to address this need. We refer the reader to Table In this section we provide a review of applications of Kinect in elderly care grouped under two categories: 1) Fall Detection and 2) Fall Risk Reduction. Technologically advanced alert support systems are a potential avenue of assistance for the independently living elderly person, and these systems could also then be leveraged to produce affordable in-home telerehabilitation methods of care . With faFall detection has traditionally relied on one or more technologies of panic buttons, audio sensors, physically worn accelerometers or gyroscopes, and/or 2D video capture. Each of these systems comes with inherent limitations: patients with various cognitive deficiencies may be unable to successfully utilize panic buttons, audio sensors are easily overloaded with background noise interference of televisions or music, physically worn devices are cumbersome and wearing them is easily forgotten, and performance of 2D video capture systems significantly degrade in shadow filled or light-less environments . The creAs fall detection systems are typically employed in living environments, variables such as distance from the sensor and illumination are major challenges for current video-based strategies to overcome. Nevertheless, Zhang et al.  presenteAnother current trend of research in RGBD camera-based fall detection support systems is the development of robust systems that can perform unthwarted by occlusion caused by static or mobile scenery. The following three methods aim to overcome this pitfall by utilizing a 3D bounding box, pre-occlusion velocity data analysis for falls which end in an occluded state, and the orientation of a participant’s derived “spine” in respect to a ground plane.First, Mastorakis and Makris’s  boundingThe second method was developed by Rougier et al.  and utilThe third method, presented by Planinc et al. , is a faCreation of successful algorithms to directly analyze and predict fall potential through real-time Kinect-based systems is a tantalizing, and potentially possible, idea; however, to date, no direct fall prevention algorithms have yet been demonstrated. The existing studies along this line of research are comprised mainly of gait assessment and pre-emptive in-home fall-prevention training exercises, and while gait-based and pre-emptive exercise methods are, at best, only indirectly related to true fall prevention methodologies, they nonetheless have strong potential to contribute to the enabling of accurate fall detection and prevention in the future.The studies in this section represent differing approaches in current Kinect-based automated gait assessment methodology research. Stone and Skubic ,24 conduStone and Skubic  collecteGabel et al.  developeTranslating Kinect-based gait assessment application from only system accuracy and biomechanic parameter readings to real-world applications, Stone and Skubic  developeAs the Kinect is a relatively new piece of hardware, establishing the limitations of the sensor within specific application scenarios is an ongoing process. Nevertheless, we provide a list of current limitations of Kinect that we noted based on our review of applications in elderly care systems. 1. Current Kinect-based fall risk reduction strategies are derived from gait-based, early intervention methodologies and thus are only indirectly related to true fall prevention which would require some form of feedback prior to a detected potential fall event.2. Occlusion in fall detection algorithms, while partially accounted for through the methodologies of the various systems discussed, is still a major challenge inherent in Kinect-based fall detection systems. Current strategies focus on a subject who stands, sits, and falls in an ideal location of the Kinect’s field of vision, while authentic falls in realistic home environment conditions are more varied, therefore the current results should not be taken as normative.3. The Kinect sensor must be fixed to a specific location and has a range of capture of roughly ten meters. This limitation dictates that fall events must occur directly in front of the sensor’s physical location. While it has been noted that a strategically placed array of Kinect sensors could mitigate this limitation ,54, a sy4. Without careful consideration of the opinions of a system’s proposed user base, concerns regarding ubiquitous always-on video capture systems, such as the Kinect, may inhibit wide-scale system adoption. During the review, it was noted that research related to the reception of alert support systems is at an early phase, likely due to in-home hardware previously being cumbersome and expensive. With the Kinect having the potential to be widely disbursed in in-home setting monitoring systems, this avenue of research has become more viable and relevant -57.In this section we provide a review of applications of Kinect in stroke rehabilitation grouped under 2 categories: 1) Evaluation of Kinect’s Spatial Accuracy, and 2) Kinect-based Rehabilitation Methods. These categories follow the trend of the literature to first evaluate the Kinect sensor as a clinically viable tool for rehabilitation. Motor function rehabilitation for stroke patients typically aims to strengthen and retrain muscles to rejuvenate debilitated functions, but inadequate completion of rehabilitation exercises drastically reduces the potential outcome of overall motor recovery. These exercises are often unpleasant and/or painful leading to patients’ tolerance for exercise to decrease as indicated by Dobkin et al. . Lange eAdvances in the field of gesture controlled user interfaces have only recently erupted in popularity and functionality due to the development of new, affordable computer vision technology. Historically, a majority of research in gesture controlled virtual reality interfaces has been focused on upper limb rehabilitation , usuallyThe following studies focus on evaluating the accuracy of the Kinect, and when placed under scrutiny, the Kinect has been found, in general, to carry significant potential for a cost-effective motion capture system for rehabilitation. Chang et al.  and LocoResearch related to the Kinect’s ability to accurately capture upper extremity movements is consistently reported as sufficient for clinical use with regards to the elbow and wrist joint tracking; however, mixed results have been reported for the shoulder. Loconsole et al.  leverageClark et al. , ObdrzálIn order to simplify the method of verifying the Kinect’s accuracy for rehabilitation and to avoid the influence of various errors introduced by human biomechanics, Pedro et al.  utilizedIn an attempt to further improve accuracy of Kinect, including lessening occlusion error or enhancing fine motor control capture, the use of the Kinect together with various inertial sensors has sparked interest. Hussain et al.  made useAs noted by Obdrzálek et al. , the KinThe ultimate goals of validating Kinect’s accuracy for rehabilitation are diagnostics (quantifying motor function improvement level of patients) and development of home-based rehabilitation protocols. In this section, we provide a summary of studies that focused on Kinect’s applications to pursue these goals, and their results on provisionary physical and mental benefits.Virtual reality-based rehabilitation offers a a highly interactive system with many documented benefits specifically to stroke patients , and a lPromoting proper form/posture, repetition, and enjoyability of stroke-based impairment rehabilitation exercises support and foster motor recovery. Toward enabling proper form, Da Gama et al.  developeHints of various multidisciplinary directions Kinect-based research is expanding toward can be seen in the more intricate applications of the Kinect presented by Frisoli et al.  and ShirThese initial benefits, system ideas, and hints toward future research demonstrate a strong potential for fruitful Kinect development, as well as enhancing previously developed widely used out-patient rehabilitation services . These iThe current stage of Kinect-based rehabilitation literature is lacking in reported functional and validation data because a majority of systems are only at a proof of concept stage. The following over-arching limitations have been derived from the current state of the literature: 1. While initial Kinect-based comparisons with research grade motion capture systems demonstrated highly correlated trends and reasonable accuracy, a majority of evaluation studies focused only on sets of gross movements that are advantageous for Kinect. Evaluation of more realistic and/or specific diagnostic movement sets are still needed.2. The Kinect is unable to accurately assess internal joint rotations of the shoulder and instead utilizes a much less clinically viable single-point estimation. Use of the Kinect for specific shoulder-based functionality requirements have yet to be shown to be clinically viable.3. Rehabilitation goals which include fine motor skills can not be captured by the Kinect alone; however initial studies suggest fusion systems of Kinect and inertial sensors can be a feasible alternative.4. Kinect systems are usually not suitable for severely disabled patients, as gross movements that remain extremely small in their entirety are difficult for the Kinect to accurately capture.Historically, virtual reality rehabilitation has always been a promising field with an infeasibly high price tag for mass implementation . Tanaka The benefits of focused physical tasks and exercise to stroke victims and the elderly have a rich and well documented history -46. GrowIn the past, game development has focused on utilizing a player’s cognitive abilities to create an enjoyable experience. The physical dimension of serious and exergames has added an extra challenge to game development while simultaneously enabling video games to find alternative applications in facilitating general and rehabilitation-based exercises. The majority of current design considerations focus on accessibility challenges caused by software development decisions  and handIn their comparison of elderly preferences between button-based, mixed button/gesture-based, and gesture-based controllers for game play, Pham et al.  observedArntzen et al.  examinedMcNiell et al.  offered Jiang et al.  suggesteThe Kinect is not unique in its ability to provide vision-based data capture capable of supporting gaming paradigms; however, current research grade multiple camera motion capture systems are typically expensive, difficult to set up, and require a knowledgeable operator. The Kinect does not suffer from these challenges, and with its low-cost, leading the emergence of natural gaming paradigms and development of targeted exercise games  in a variety of areas.Pham and Theng  demonstrGerling et al.  conducteEach of these systems concluded with overall positive results and demonstrated that Kinect-based gaming can significantly improve quality of life using a variety of measures, such as participant’s emotional state , physicaThe physical changes that accompany ageing affect a wide range of functions, including sensory-perceptual processes, motor abilities, response speed and cognitive processes . ResearcFor many stroke patients, balance and weight shift management constitute a risk of secondary injury . Lange eCrosbie et al.  noted thConcerning lower-limb rehabilitation, Llorens et al.  developeThroughout all of these developed systems, one thread of consistency is the positive reception by players and initial rehabilitation results. The overall view in the literature related to serious games is positive; however, the level of current confidence is almost unanimously recognized as tentative and needing further study.The limitations specific to Kinect-based serious and exercise gaming applications, considering the requirement of clinical data capture for specific limb movements; specific player-base-desired design considerations; varying levels of limb impairment, and previously defined serious and exergame-based requirements, can be summarized as the following: 1. Any games designed specifically for diagnostic usage are limited to non-occluding movements. This implies that standard stroke impairment level tests requiring extensive occluding movement sets may be untenable for a Kinect-based system to capture.2. Diagnostic potential for extremities is limited to gross movements, as fine movements of the hand and foot are currently outside the Kinect’s capture sensitivity.3. Games targeted at rehabilitation may be prone to “cheating” .4. Appropriate response to failure and poor performance, if not accounted for during game design, can inherently limit positive outcomes due to demotivation/discouragement resulting in less frequent/consistent use of the system.5. The current benefits of Kinect-based gaming have only tentatively been studied with mainly short term and small sample sized studies. Data to date should be seen as initial results, and not normative.As Kinect-based elderly care and stroke rehabilitation research is in its infancy, a majority of the data acquired is qualitative with a focus on self-report and personal opinion. Compounded with this observation is the fact that the data is derived only from small groups of participants, it is anything but normative and should be viewed as tentative initial results. Filling the deficiencies of quantitative and large population based research thus remains a potentially fruitful and necessary avenue of future work. The current applications of Kinect in elderly care seem to be at a more mature state than those of stroke rehabilitation; however, even with the current deficiency, we believe that the Kinect carries the potential to become a future cornerstone of widely dispersed care and rehabilitation systems.With regards to fall detection and fall risk reduction applications, each of the current technologies of panic buttons, audio sensors, body-fixed systems, 2D and 3D video capture systems comes with inherent physical and financial limitations. While the Kinect does not render any of these technologies obsolete, and comes with its own limitations, its unique aspects may enhance current systems, or potentially be the foundation for newly developed functionalities. 1. The autonomous nature of the Kinect allows for fall detection without requiring a user to physically trigger a panic button system, or the wearing of cumbersome physical devices, which can often be forgotten.2. The Kinect comes with built in directional sound capture capabilities, possibly enabling it to be used as, or in unison with, current audio sensor-based systems. This is a functionality of the Kinect not yet studied.3. The added third dimension depth field measurement offered by the Kinect requires less overhead than current methodologies, and enables the development of more accurate methods of fall-related image processing to be developed.4. The low-cost, marker-less, and widely dispersible nature of an already commercially available gaming system immediately enables the current clinical virtual reality-based rehabilitation methodologies to be rapidly relocated to individual home settings.While questions such as participants with what level of impairment most strongly benefit from using the Kinect, how to build an ideal exercise routine for preventative or rehabilitation needs, which is the most performant fall detection algorithm, or how to most efficiently leverage all the various capabilities of the Kinect are still unanswered, the overarching conclusions of this review point toward the Kinect as a promising technology for a wide range of elderly care and stroke rehabilitation applications and need for studies involving larger participant pools to establish reliability and validity.We have compiled the following list based on our review of the current state of Kinect-related elderly care and stroke rehabilitation literature. It contains our remarks as well as suggestions for relevant future research. 1. A majority of applications in elderly care and stroke rehabilitation require a robust and easily manipulated user interface which at present cannot be readily found in current commercial systems. When developing exercise games, serious games, and applications of Kinect-based rehabilitation, it is vitally important to remember that repurposing a technology initially intended for a younger and healthier audience requires careful adherence to new design strategies focused on both the physiological and psychological requirements of aging and injured users. Therefore we suggest that multidisciplinary research teams involving engineers as well as clinicians, human factors experts and cognitive psychologists would be best positioned to tackle the challenges that such game development efforts would entail.2. A critical validation step for Kinect-based applications to both fields is a focused experimental evaluation of the accuracy and latency of the motion capture data obtained from Kinect in comparison with a research grade motion capture device and statistical evaluation of this data for specific diagnostic potential. The effects of distance from the Kinect sensor on gathered data is another important consideration. In order to verify that the Kinect has the potential to make therapy financially accessible and medically beneficial to a large population of elderly and stroke patients, more targeted studies involving relevant rehabilitation and preventative exercise movements, are needed.3. Kinect applications may have the potential to simultaneously achieve care or stroke related goals while capturing real-time, clinically viable data for injury risk evaluation. The real-time data gathering aspect of the Kinect has yet to be satisfactorily examined as a majority of documented work focuses solely only providing assistance or motivation to the user while ignoring this important potential function.4. As alert systems potentially gather data in an always-on fashion even what might normally be considered mundane activities can then turn into potential privacy infringements. Because of this potential problem, the reception and concerns of the elderly related to always-on systems require a thorough and careful examination.5. Game content specifically designed for aging and/or injured users must simultaneously allow for high standards of captivating game play and long-term enjoyment potential while maintaining seamless methodologies of adaptation and monitoring of players needs, which are critical characteristics of a successful low-cost home-based rehabilitation paradigm.6. Generic game play may be unsuitable for many patients with secondary disabilities not solely defined by motor function. Strategic adaptation schemas for game play are necessary traits as complex demands for speech, memory, or cognition patterns significantly reduces the potential for games with large and diverse player bases.7. The stimulating aspect of exergames and serious games, while being beneficial in patient motivation, should be closely monitored as to provide an exercise or rehabilitation environment that discourages overexertion injuries in both individual movements and length of play.8. Assumptions of rehabilitation success should not be based on in-game score improvement as arbitrary scores do not necessarily correlate with actual functional improvement, and evaluation methods based specifically on player experience can result in notably different outcomes, therefore using player experiences or in-game scores as metrics should be avoided.In this paper, various applications of the Kinect in the fields of elderly care and stroke rehabilitation have been examined. We have classified these applications into the groups of (1) Fall Detection, (2) Fall Risk Reduction, (3) Evaluation of Kinect’s Spatial Accuracy, (4) Kinect-based Rehabilitation Methods, and (5) Serious and Exercise Games - serious games for stroke rehabilitation and exercise games for the elderly. While only in its initial stages of development, the Kinect already shows notable potential in making therapy and alert systems financially accessible and medically beneficial to a large population of elderly and stroke patients; however, some significant technological limitations still present are: a fixed location sensor with a range of capture of only roughly ten meters; a difficulty in fine movement capture; shoulder joint biomechanical accuracy, and fall risk reduction methodologies that only utilize indirect, gait-based pre-emptive training. The directions for future work are vast and have promise to enhance elderly care; stroke patient motivation to accurately complete rehabilitation exercises; rehabilitation record keeping, and future medical diagnostic and rehabilitation methods. Based on our review of the literature, we have reported a summary of critical issues and suggestions for future work in this domain.CVA: Cerebrovascular accident; RGBD: Red-green-blue-depth; FAAST: Flexible action and articulated skeleton toolkit.No financial or non-financial competing interest exists for either of this paper’s authors.OC conceived the review paper idea. Both DW and OC participated in designing and structuring the literature search and resulting review paper structure. DW conducted the literature search, as well as the drafting of the manuscript. OC guided the literature search and drafting of the manuscript. Both authors read and approved the final manuscript prior to submission.OC received his B.S. and M.S. degrees in Mechanical Engineering in 2004 and 2006 respectively, from Istanbul Technical University, Turkey. He completed his Ph.D. in Mechanical Engineering in 2011 at Rice University and was a Research Assistant at the Mechatronics and Haptic Interfaces (MAHI) Lab from 2006 to 2011. He was an Assistant Professor at San Francisco State University from 2011 to 2013. He is currently an Assistant Professor at Colorado School of Mines and Director of the Biomechatronics Research Laboratory. He was a recipient of the best paper award at the IEEE World Haptics Conference in 2011. His research interests include robotic rehabilitation, biomechatronics, robotics, haptics, human sensorimotor control system, motor adaptation and learning.DW received his B.A. in Music from the University of Georgia in 2009 and his M.S. in Computer Science from San Francisco State University in 2013. Between 2012–2013, he was a Research Assistant in the School of Engineering’s Biomechatronics Research Laboratory. His research interests include computer vision, human-computer interaction, biomechatronics, robotic rehabilitation, and kinematic modeling.