The X-axis of Fig. 3A1 and A2 illustrates the overall changes in these markers, with the responses separated for selleck chemicals each treatment group.

Also shown in Fig. 3A are IP-10 and IL-6 data at 24 h, a time point of peak elevation, and relationship to ALC or CRP. As expected, there was a correlation between the observed decrease in ALC and the increase in IP-10 levels 24 h after Modulators immunization (r = −0.76) ( Fig. 3A). Increased CRP at 48 h was associated with increased IL-6 at 24 h (r = 0.59) ( Fig. 3A). Additionally, there was a significant association of Day 28 TNA NF50 values reported by Hopkins et al. [14] with IP-10, IL-6, ALC, and CRP. In addition, Day 28 IgG antibody levels directed against PA (reported below) correlated significantly with these early innate biomarkers ( Fig. 3B). Fig. 4A presents the sequence of steps by which PBMC ELISpot data in each of 6 treatment groups were analyzed for responder rates. Using criteria to include only those PBMC pairs (day 0 and day 21) having adequate positive responses to PHA or CEF-I, the IFN-γ ELISpot responder rate to PAp and/or rPA averaged 11% (1/9) in recipients of two full (0.5 mL) doses of AVA. In contrast, a significantly higher IFN-γ response rate was observed BTK inhibitor clinical trial for the subjects in treatment

groups that received the lower amount of CPG 7909 (0.25 mg), resulting in 5/11 and 7/12 positive responders for Formulations 2 and 4, respectively compared to those that received a higher amount of CPG 7909 (Suissa-Shuster, p = 0.03). There were no responders in the placebo group. Using the Suissa-Shuster unconditional

test [18], the IFN-γ responder rates of subjects immunized with AV7909 formulations containing half (formulations 3 and 4) compared to full (formulations 1 and 2) dose AVA were not statistically different (p = 0.57). Fig. 4B summarizes the IFN-γ T cell SFC cell count responses to PAp and/or rPA for each treatment group. ANOVA Statistics performed on the SFC counts in response to rPA (i.e. not on responder rate) demonstrated AV7909 F2 to be significantly different from AVA; this was not observed for the PAp mixture, however ( Fig. STK38 4B). The T cell IFN-γ response (reported as SFC) at Day 21 did not correlate with any of the other endpoints ( Fig. 3B). Of the investigated time points of Days 28, 42, and 70, IgG anti-PA content was highest in recipients of AV7909 compared to AVA, peaking at Day 28 (Fig. 5). IgG anti-PA content of 99 human serum samples obtained 14 days following the second immunization (study day 28) ranged from 21 to 160 μg/mL; this was a 5-fold or higher mean response for recipients of AV7909 compared to AVA. As expected, there was also an increase in mean serum content within AVA recipients (average 21 μg/mL on Day 28), compared to the saline (placebo) group. Significant correlations occurred between this parameter and the changes in both ALC and CRP (Fig. 3B).

In addition, electrical stimulation was applied to the ankle dorsiflexor muscles with the ankle in maximal dorsiflexion. This was done to maximise stretch and to strengthen the dorsiflexor muscles in their inner range, where they are often weakest.15 The induced muscle contractions were isometric. It is not clear whether different results would have been obtained if electrical stimulation had been applied in a different way or applied to the gastrocnemius muscles instead. Another possible

reason for not finding an effect is that many of the participants (64%) had severe weakness or no muscle activity (Grade 2 or less) in their ankle dorsiflexor muscles at baseline, and many also did not have the cognitive ability to contract their ankle selleck chemical muscles in synchronisation with the electrical stimulation. There is increasing evidence supporting the combination of electrical stimulation with volitional muscle contractions for motor training.29, 30, 31, 32, 33, 34, 35, 36 and 37 The potential value of electrical stimulation may be undermined if participants are unable to work voluntarily with

Dabrafenib supplier the electrical stimulation. Three other trials have investigated electrical stimulation in people with acquired brain injury and severe motor impairments, and the findings of all three were inconclusive.23, 38 and 39 It is possible that electrical stimulation is not effective for contracture management in people with severe traumatic brain injury. However, these findings may not be generalisable Rolziracetam to other clinical conditions or people with less-severe brain injury. Our study’s results indicate that there was no difference between a single modality treatment program of tilt table standing and a multimodal treatment program combining tilt table standing, electrical stimulation and ankle splinting. While it is always tempting to look at within-group changes in trials like this and use the data to conclude that both programs were equally effective (or ineffective), this is not a valid interpretation without a control group that had no intervention. No attempt was made to Modulators assess the effectiveness

of individual modalities in the present study. The findings, however, did suggest that the addition of splinting was not therapeutic; this is consistent with previous clinical trials on splinting that also failed to demonstrate treatment effects.27, 28 and 40 In summary, this study, along with the many others that have preceded it, does not provide a solution to contractures. Tilt table standing, electrical stimulation and ankle splinting were selected because they are commonly used in people with severe brain injury, and their effectiveness when used in combination has never been investigated. In addition, they are amongst the few modalities that can be used in people with severe brain injury who have a limited ability to actively participate in treatment.

Most cells recorded from direction-preferring domains exhibit directional

selectivity, while those recorded outside direction-preferring domains are mainly not directional selective. For example, five out of six cells in penetration 1 show strong direction selectivity. The preferred directions of these five direction cells (95.3° ± 13.4°) are close to the direction preference of the recording site revealed from optical imaging (82.9°; green arrow in Figure 5C). This indicates a columnar organization of direction-selective neurons in direction-preferring domains. There is also a certain Selleck Rucaparib degree of heterogeneity in the direction-preferring domains. For example, one cell did not show significant direction selectivity (cell

1, Direction Index [DI] = 0.33), while others are strongly (cell 3, DI = 0.99) or weakly (e.g., cell 5, DI = 0.71) directional. In non-direction-preferring domains, we also recorded a few direction-selective cells (e.g., cell 3 in penetration 4). However, direction-selective neurons were very rare in regions outside of the direction-preferring domains. In three cases, we recorded 32 cells from seven direction-preferring domains. Twenty-three (72%) of these were direction selective (p < 0.05, Rayleigh test for circular uniformity). Another 31 cells were recorded from nine locations outside of direction-preferring domains. Only two out of these 31 cells (6.5%) were direction selective (p < 0.05, Rayleigh test; DIs = 0.71 and 0.85, respectively). The distributions of direction selectivity and orientation selectivity of cells inside (black) versus outside (gray) direction-preferring ABT-199 ic50 domains are plotted in Figures 5D and 5E, respectively. Cells recorded from inside direction-preferring domains (DI, 0.63 ± 0.05, n = 32) have higher direction selectivity than cells recorded outside direction-preferring domains (DI, 0.28 ± 0.03, n = 31; p = 1.01 × 10−6, two-sample Kolmogorov-Smirnov test for equal distributions). In contrast, the orientation selectivity of these two groups of neurons

is not significantly different (p = 0.48, two-sample Kolmogorov-Smirnov test). These observations indicate that V4 directional neurons are concentrated in Electron transport chain direction-preferring domains and provide further support for the directional nature of these domains. In V2, direction-preferring domains tend to overlap with orientation-preferring domains but avoid color-preferring domains (Lu et al., 2010). In V4, orientation and color preference maps tend to segregate spatially (Tanigawa et al., 2010). This spatial segregation has been interpreted to indicate some degree of functional independence, while spatial overlap suggests a greater degree of modal integration. Here, we quantitatively evaluated the spatial relationship between direction-preferring domains and orientation- and color-preferring domains.

By examining retrograde flux, Erastin both groups found that the

disease mutations perturbed the ability of p150 to associate with microtubules and observed problems with the initiation of retrograde transport. Why then do they cause such different symptoms in humans? Both groups noted that protein aggregates formed when these alleles were expressed, but that this tendency, particularly in neurons, was more pronounced for the HMN7B mutations. This distinction correlates with the histopathology of affected individuals. Potentially more enlightening, however, were biochemical studies by Moughamian and Holzbaur (2012). Although both Perry and HMN7B mutations allow p150 to dimerize and incorporate into the dynactin complex, the HMN7B mutation alone prevents the dynactin complex from binding to dynein. Whereas the Perry syndrome mutations lie on the surface, in or very close to the site of microtubule and EB1 binding, the HMN7B mutation is in the core of the

domain and likely to interfere with Paclitaxel its folding. Thus, although CAP-Gly domain is far from the known dynein-interacting portion of p150, the likely severe misfolding of this domain may promote its aggregation and prevent proper incorporation into the motor. These biochemical changes are reflected in phenotypic differences observed in these studies. In DRG neurons, the HMN7B mutation seriously perturbed both anterograde and retrograde transport and decreased the processivity of through cargo, as might be expected if dynein was operating without its dynactin partner. This defect did not arise when the Perry syndrome allele was expressed. In Drosophila, only the HMN7B mutation caused dynein heavy chain to accumulate substantially in the terminal boutons, as might be expected if the dynein motor

is bereft of dynactin association. Thus, HMN7B may be understood as a dominant negative that compromises the entire function of the dynactin complex, while Perry syndrome selectively impairs retrograde initiation while leaving other functions of dynactin intact. Of course several questions remain unanswered. Most particularly, we do not yet know why the broader disruption of dynactin function is most manifest in the substantia nigra and brainstem while the motor neurons are most sensitive to the subtler impairment of retrograde initiation. That puzzle vexes most discussions of neurodegenerative disease. The specificities may arise from differences in the dependence of neuronal subtypes on retrograde transport of survival signals or in their sensitivity to protein aggregates.

Likewise, nose-touch-evoked calcium transients in FLP were significantly reduced, resembling in magnitude the responses in the RIH-ablated animals ( Figure S7); FLP harsh head touch responses, in contrast, were unaffected ( Figure S7). unc-7 loss-of-function

mutants showed partial defects in nose touch escape behavior ( Figures S7 and S8). These nose touch defects were rescued when a functional unc-7(+) transgene was expressed in the nose touch circuit using the cat-1 (expressed in the CEPs, RIH, and few other neurons) and egl-46 BIBF 1120 concentration (expressed in FLP and PVD) promoters ( Figure 6B; Figures S7 and S8). unc-7(+) expression using either promoter alone did not result in phenotypic rescue (data not shown), suggesting that gap junction formation requires production of the innexin protein in both connected neurons. In contrast, mutations in unc-13, which impair synaptic transmission, did not detectably impair RIH nose touch responses ( Figure 6B). Together, these results support the hypothesis that signaling in the RIH-centered nose touch circuit is predominantly, if not exclusively, mediated by gap junctions. If signaling in the nose touch circuit is mediated primarily by gap junctions, information flow through RIH might be bidirectional: just as activation of neurons such as OLQ can indirectly excite FLP,

FLP activation could be able to excite OLQ. We examined this possibility by imaging OLQ calcium dynamics in response to mechanical stimuli sensed by FLP. We observed that harsh touch applied to the side of the head led to robust calcium transients in OLQ as well as RIH selleck chemicals llc (Figures 8B and 8C; Figure S7E). Mutations in the mechanosensory channel mec-10 eliminated OLQ and RIH responses to harsh head touch, and these responses could be rescued by FLP-specific expression of mec-10 ( Figures 8B and 8C; Figure S7E). Moreover,

ablation of RIH eliminated the harsh head-touch-evoked calcium transients in OLQ ( Figures 8B and 8C), indicating that the FLPs indirectly activate the OLQs through the RIH-centered network. We also tested the effect of the network on nose touch responses in OLQ. Interestingly, a mec-10 mutation significantly impaired OLQ and RIH calcium responses to nose touch; second these defects were rescued by mec-10(+) expression in FLP ( Figures 8B and 8D). Furthermore, ablation of RIH significantly reduced the responses of the OLQ neurons to nose touch ( Figures 8B and 8D). These results indicate that just as the nose touch responses of the FLPs depend on a combination of RIH-mediated network activity and cell-autonomous MEC-10 function, OLQ nose touch responses depend on both RIH-mediated network activity and cell-autonomous OSM-9 function. We have shown here how a network of interacting mechanosensory neurons detects nose touch stimuli and in response evokes escape behavior.

Hypoglycemia in the brain is accompanied by an extracellular alkaline pH change (Bengtsson et al., 1990; Brown et al., 2001). Our results suggest that the alkaline shift during aglycemia leads to sAC activation NU7441 followed by the increased lactate production that we have observed. The sensitivity of the cAMP increase

and the glycogen breakdown to DIDS in aglycemia suggest that HCO3− entry via NBCs plays a predominant role in activating sAC during aglycemia as compared to intracellular HCO3− production. Our data expand upon a body of evidence showing the existence of an astrocyte-neuron lactate shuttle that is initiated by glutamate transport into astrocytes. Glutamate uptake is coupled to Na+, resulting in an intracellular Na+ load and enhanced Na+/K+-ATPase activity. The need for selleck chemical more ATP to drive Na+/K+ pumps increases glycolysis, leading to the production and release of lactate, which is subsequently taken up by neurons for fuel (Magistretti et al., 1999; Pellerin and Magistretti, 1994). The HCO3−-sensitive sAC mechanism described here may work in concert with this original shuttle model, whereby neural activity produces an elevation in extracellular glutamate and K+, both of which then act independently

through their respective mechanisms to augment lactate release for neurons. Finally, our results shed light on the importance of the astrocyte store of glycogen as an energy reserve. Previous data have shown that glycogen provides STK38 an important alternative energy source during ischemic-like conditions to prolong survival of neurons and integrity of axons (Brown and Ransom, 2007; Wender et al., 2000). Our data add to this concept, suggesting that glycogen stores can be recruited by moderate elevations in [K+]ext as well as more severe aglycemic challenges. Therefore, the unique presence of bicarbonate-responsive sAC in astrocytes and its critical role in controlling lactate levels through glycogenolysis demonstrate that this molecular pathway may be an essential process in the maintenance or optimization of total brain energy metabolism during both

physiological and pathophysiological conditions. Targeting this pathway may provide a site of intervention for the treatment of perturbed energy metabolism in the brain. Sprague-Dawley rats (postnatal days 18–28) were anaesthetized with halothane and decapitated according to protocols approved by the University of British Columbia committee on animal care. Brains were rapidly extracted and placed into ice-cold dissection medium containing the following: 87 mM NaCl, 2.5 mM KCl, 2 mM NaH2PO4, 7 mM MgCl2, 25 mM NaHCO3, 0.5 mM CaCl2, 25 mM d-glucose, and 75 mM sucrose saturated with 95% O2/5% CO2. Hippocampal slices (transverse, 400 μm thick) were cut using a vibrating tissue slicer (VT1000S, Leica) and recovered for 1 hr at 24°C in aCSF containing the following: 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 26 mM NaHCO3, 2.

elegans, we were able to induce long-lasting paralysis (>1 hr) with

480 nm light. Animals recovered movement when re-tested 24 hr later. We name this technique Inhibition of Synapses with CALI (InSynC). We believe InSynC is a powerful optogenetic technique for inhibiting neurotransmitter release with light and interrogating neurocircuitry in a spatially precise manner. To design a CALI-based synapse inhibition system, we chose candidate fusion proteins based on two criteria: (1) the protein is essential for vesicular synaptic release in synapses of the central nervous system; and (2) the engineered GSK J4 datasheet protein can achieve inhibition in a dominant-negative manner, without the need to eliminate endogenous protein expression. The SNARE protein synaptobrevin 2/VAMP2 is the core protein in the vesicular SNARE complex, with a cytosolic N-terminal α-helix capable of binding to the α helices of SNAP-25 and syntaxin during vesicle fusion. The C-terminal of VAMP2 consists of a transmembrane

domain that is anchored to the vesicular membrane. Both N and C termini of VAMP2 have previously been fused to fluorescent proteins without disrupting function (Deák et al., 2006). The second protein candidate that we chose was synaptophysin (SYP1), which is closely associated with the VAMP2 protein (Arthur and Stowell, 2007), although its role in vesicular release is still unclear. SYP1 has 4 proposed transmembrane domain helices transversing the vesicular membrane, with both N and C termini facing the cytosol. Nintedanib The C terminus of SYP1 has been previously tagged with fluorescent proteins without

affecting its function (Dreosti et al., 2009). We genetically fused miniSOG to the N terminus and C terminus of VAMP2 and SYP1, respectively (Figure 1A). To visualize expressing cells, mCherry was placed after the coding sequence of miniSOG-VAMP2 and SYP1-miniSOG, connected by a cotranslationally self-cleaving Thosea asigna virus 2A-like sequence (T2A) ( Osborn et al., 2005). The expression of the tagged synaptic proteins and the cytosolic red fluorescent protein were tightly linked genetically, even though the proteins were not fused first to each other. To assay the effects of miniSOG fused to VAMP2 and SYP1 on synaptic release, cultured hippocampal neurons were plated on microislands to induce autaptic synapse formation. The self-stimulated excitatory postsynaptic potential (EPSP) was typically observed as a prolonged depolarization after an action potential in current-clamp recording in response to a depolarizing current injection pulse (Wyart et al., 2005; Figure 1D). In voltage-clamp recording, a depolarizing voltage step can evoke a self-stimulated excitatory postsynaptic current (EPSC; Figure 1B). After establishing a stable baseline with repetitive stimulation, the recorded cell was illuminated for 2.5 min with 9.8 mW/mm2 of 480 nm light.

, 2003). Importantly, memory retrieval through these modified selleck products KC-output synapses was predicted to guide either odor avoidance or approach behavior. A KC synapse-specific representation of memories of opposing valence would dictate that it is not possible to functionally separate the retrieval of aversive and appetitive memories by disrupting KC-wide processes. We therefore tested these models by systematically blocking neurotransmission from subsets of the retrieval-relevant

αβ neurons. We found that aversive and appetitive memories can be distinguished in the αβ KC population, showing that opposing odor memories do not exclusively rely on overlapping KCs. Whereas output from the αβs neurons is required for aversive and appetitive memory retrieval, the αβ core (αβc) neurons are only critical for conditioned approach behavior. Higher-resolution anatomical analysis of the innervation

of reinforcing DA neurons suggests that valence-specific asymmetry may be established during training. Furthermore, dendrites of KC-output neurons differentially innervate the MB in a similarly stratified manner. We therefore propose that aversive memories are retrieved and avoidance behavior triggered only from the αβ surface (αβs) Ipatasertib manufacturer neurons, whereas appetitive memories are retrieved and approach behavior is driven by efferent neurons that integrate across the αβ ensemble. Several studies have reported the

importance of output from MB αβ neurons for the retrieval of aversive and appetitive olfactory memories (Dubnau et al., 2001, McGuire et al., 2001, Schwaerzel et al., 2003, Krashes et al., 2007, Krashes and Waddell, 2008 and Trannoy Phosphoprotein phosphatase et al., 2011). However, genetic labeling reveals further anatomical segregation of the ∼1,000 αβ neurons into at least αβ posterior (αβp or pioneer), αβ surface (αβs or early), and αβ core (αβc or late) subsets that are sequentially born during development (Ito et al., 1997, Lee et al., 1999 and Tanaka et al., 2008). We therefore investigated the role of these αβ subsets in memory retrieval. We first obtained, or identified, GAL4 lines with expression that was restricted to αβ subsets and verified their expression. Prior reports showed that the c739 GAL4 (McGuire et al., 2001) labels αβ neurons contributing to all three classes (Aso et al., 2009). In contrast, NP7175 expresses in αβc neurons and c708a in αβp neurons (Murthy et al., 2008, Tanaka et al., 2008 and Lin et al., 2007). Lastly, we identified the 0770 GAL4 line from the InSITE collection (Gohl et al., 2011) with strong expression in αβs neurons and weaker expression in αβp neurons. We expressed a membrane-tethered GFP (uas-mCD8::GFP) using the c739, 0770, NP7175, and c708a GAL4 drivers and localized expression within the overall MB neurons using a LexAop-rCD2::RFP transgene driven by 247-LexA::VP16 (Pitman et al., 2011).

It was expected that the predicted speed data would closely agree with the magnitude of calculated speed for each trial. However, it was expected that the phase lag that exists between cable force and linear hammer velocity, previously described above, would still be evident in the predicted speed data resulting in peaks in the predicted speeds not

coinciding with those in the calculated speeds. The calculated force and measured force data are in phase; therefore the phase lag described above is also present between the calculated speed and the measured force. To reduce the effect of the phase lag, all measured force data were also time shifted and trimmed so that the final peak in the measured force coincided

with release. As with the calculated force, the magnitude of the phase lag varies depending Osimertinib research buy on turn number, throw, and athlete, so it is not possible to apply the same time shift to every throw. It was hoped that using measured force data that are time shifted would result in predicted speed data that were more closely matched to both the magnitude and waveform of the calculated speeds than if the time shift were not applied. The predicted speed data were then compared with the SCR7 in vivo calculated speed data to ascertain the level of accuracy. The root mean square (RMS) of the differences was determined to compare the closeness in magnitude between the predicted and calculated speeds for each throw of each participant.8 click here These RMS values were then used to determine the average RMS values for the entire group. The average RMS difference between the calculated and predicted release speeds was also determined. The coefficient of multiple correlation (CMC) was determined to assess the closeness in the shapes between the predicted and calculated speed waveforms for each throw of each participant.8 and 9

The average CMC values was then determined for the entire group. A schematic of the process outlined here is shown in Fig. 1. The regression equations, CMC and RMS values of the two models are similar (Table 1). Both models give high CMC values (0.96 and 0.97). In addition, the reported RMS values of 1.27 m/s and 1.05 m/s are relatively low for the non-shifted and shifted models, respectively. In addition, the average percentage difference between the calculated speeds and the speeds determined via the non-shifted and shifted models were 6.6% and 4.7%, respectively. For the release speed, the RMS differences between the calculated and predicted values are 0.69 ± 0.49 m/s and 0.46 ± 0.34 m/s for the non-shifted and shifted models, respectively. The magnitudes of the predicted speeds found using the two regression models were similar to the magnitudes of the calculated speeds as the RMS values were both low (Table 1).

Assays of AMPAR surface expression in cultured hippocampal neurons

suggest that LRRTMs are required for the stabilization of AMPARs at synapses after LTP induction. These results reveal an unexpected role for LRRTMs in LTP at both young and mature synapses and are consistent with a model in which LRRTMs are required for maintaining or trapping AMPARs at synapses during the initial phase of LTP. To explore the role of LRRTMs in NMDAR-dependent LTP, we used well-characterized shRNAs that in dissociated cultured Pomalidomide in vitro neurons suppress endogenous mRNAs for LRRTM1 and LRRTM2, the two isoforms highly expressed in CA1 (Laurén et al., 2003), by ∼90% and ∼75%, respectively (Ko et al., 2011 and Soler-Llavina et al., 2011). A lentivirus expressing the shRNAs and GFP was injected into the hippocampus of P0 wild-type mice (Figure 1A).

Acute slices were prepared 14–18 days postinfection and whole-cell recordings from this website CA1 pyramidal neurons were made (Figures 1B and 1C). While control neurons in slices prepared from infected animals exhibited robust LTP (Figures 1D and 1E; 1.62 ± 0.23 of baseline 46–50 min after induction, n = 8), LTP was blocked in DKD neurons expressing the LRRTM1 and LRRTM2 shRNAs (Figures 1D and 1E; 0.99 ± 0.1, n = 19). Similar to other manipulations that block LTP (Malenka and Nicoll, 1993), DKD neurons exhibited an initial potentiation that returned to baseline over 40–50 min. To determine whether LRRTM1 and LRRTM2 have a specific role in LTP, we assessed the effect of the DKD on NMDAR-dependent long-term depression (LTD). LTD in DKD and uninfected control neurons was identical (Figures 1F and 1G; control = 0.49 ± 0.06, n = 9; DKD = 0.48 ± 0.04, n = 10), a result that is consistent with the lack of an effect of the DKD on NMDAR-mediated synaptic responses (Soler-Llavina et al., 2011). These results suggest that LRRTMs have a critical, requisite role in LTP and that the block of LTP by DKD is not due to an impairment in the induction of LTP. To test whether the

block of LTP by LRRTM Thiamine-diphosphate kinase DKD might be due to off-target effects of the shRNAs, we performed experiments in which we replaced LRRTM1 and LRRTM2 with an shRNA-resistant version of LRRTM2 (DKD-LRR2) (Ko et al., 2011 and Soler-Llavina et al., 2011). (We did not attempt rescue experiments with LRRTM1 because overexpressed recombinant LRRTM1 accumulates in the endoplasmic reticulum and traffics poorly to the plasma membrane; Francks et al., 2007 and Linhoff et al., 2009.) LTP was rescued by expression of shRNA-resistant LRRTM2 along with the shRNAs (Figures 2A and 2B; control = 1.57 ± 0.19, n = 10; DKD-LRR2 = 1.55 ± 0.15, n = 14). To interpret such rescue experiments, it is important to determine whether overexpression of the protein of interest alone has any measurable phenotype.