In the light of these findings nested oscillations seem

to reflect processes connected both to the formation and subsequent reactivation of cell assemblies representing memory patterns. Here we will focus on the latter aspect and study see more the hypothesis that cortical memories manifest themselves as distributed cell assemblies oscillating at gamma-like frequencies with life-times in the range of a theta scale. To this end we use a previously developed biophysically detailed attractor network model of association cortex (Lundqvist et al., 2006), which has been observed to display nested delta/theta (2−5 Hz) and gamma oscillations (25−35 Hz) as a correlate of active memory retrieval (Lundqvist et al., 2011 and Lundqvist et al., 2012). Although we model association cortex, we hypothesize, taking into account the distributed nature of cortical memories, that similar dynamics might be observed in sensory cortex and hippocampus. Relative to our previous studies we are concentrated here on the neural mechanisms behind the emergent coupling between these oscillations and their distinct spatial synchronization profiles AZD4547 in the context of attractor memory function, which allows then for relating our findings to vast biological data on cortical memory retrieval and maintenance. We simulate our network in two functional modes where activation of a stored

attractor memory pattern can serve as a mechanistic model of neural processes underlying two different physiological phenomena: (i) sequential memory replay as part of ongoing working memory maintenance ( Fuentemilla et al., 2010), and (ii) so-called pattern completion allowing for retrieval of

memory from fragmented input ( Jo et al., 2007). In both cases, delta/theta waves with spatially distributed gamma-like oscillations on their ridge appear in the synthesized field potentials. The activity in delta/theta band reflects the activations of coding cell assemblies in the network and it is globally coherent. Nested gamma oscillations are on the other hand the substrate of local processing and exhibit spatially dependent coherence, similarly as in the experimental observations ( Jacobs et al., 2007 and Sirota triclocarban et al., 2008). In addition, we observe under some circumstances the emergence of ~10 Hz active alpha rhythm, which is part of a 1:3:9 phase locking hierarchy constituted by theta, alpha and gamma oscillations ( Ito et al., 2012). We demonstrate biological plausibility and functional advantages of nested theta/gamma oscillations in comparison with the non-oscillatory regime of the attractor network. The nested oscillations also lead to a significant increase of precise firing sequences revealing the presence of spatiotemporally structured firing patterns that reoccur with increased likelihood during assembly activations ( Abeles et al.

Dilutions of compounds were prepared with purified water (aqua bidest.). Controls and references are described below in the context of

the individual protocols. The conventional calculation method is a standard method in the EU to provide an estimate of the hazardous properties of a preparation based on the Alectinib manufacturer classification of its ingredients (EU, 1999). In the case that specific concentration limits have been assigned to substances, these must be used for the calculation; in all other instances generic limits are applied. A preparation is considered • corrosive, if ∑ (Pcor/Lcor) ⩾ 1 Pcor/irr are the percentages by weight or volume of each corrosive substance which is assigned to a corrosive (cor) or irritating (irr) classification in the preparation; Lcor/irr are the corresponding concentration limits. For eye effects, two separate calculations are performed to assess severe eye irritation and eye irritation. We refer to the calculation method and classification symbols of DPD and DSD which is still valid for the classification of products until June 2015. Also, since not for all product constituents GHS classifications were available at the time of the

study, a similar exercise with GHS provisions could not be conducted. GSI-IX The procedure was performed as described previously (Young et al., 1988). In brief, for liquids, the pH of the undiluted liquid was determined where possible. The acid/alkali reserve is usually determined by titration with 2 N sodium hydroxide for acid and with 2 N sulphuric acid for alkaline solutions. Acid/alkali reserve (AR) is expressed as NaOH/H2SO4 (equivalent) in [g] per 100 g liquid required to adjust the pH to pH 4 (for acids) or pH 10 (for alkaline substances or products). A sample is classified as • corrosive, if pH + 1/12 alkali reserve ⩾ 14.5 or pH − 1/12 acid reserve ⩽ −0.5 The EpiDerm™ skin model, produced by MatTek Corporation

(Ashland, MA, USA), consists of normal human keratinoctyes (NHEK) cultured to form a multilayered, highly differentiated many model of the human epidermis in vitro. The model consists of organized basal, spinous, granular and cornified layers analogous to those found in vivo. The EpiDerm™ Tissues (surface area 0.63 cm2) were cultured on specially prepared cell culture inserts and shipped as kits containing 24 tissues on agarose. Each batch was controlled by the manufacturer. Both the tissues and the provided culture media were tested for viral, bacterial, fungal, and mycoplasma contamination. The manufacturer also provides information on the ET50 (50% reduction in tissue viability at a given time) for the standard test chemical Triton X-100, and on tissue viability (tested with MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)) for each lot. All tests were performed according to GLP. The experiments were performed according to OECD guideline 431 (OECD, 2004a).

We assume that no significant changes in land cover occurred during this 5-year period. The catchment size was also included in the analysis to see if size is influencing nutrient loads and concentrations. Furthermore, TNC, TNL, TPC and TPL were excluded in the analysis to isolate climate- and land cover related factors. Gefitinib manufacturer In the factor analysis, no distinction was made between east and west due to the fact that the east contains too few catchments (28) for a reliable estimate of the factor loadings and scores. Therefore, east and west were grouped together. First, all land cover variables, discharge and the catchment size were log-transformed

(temperature and precipitation were already normally distributed) where after the factor loadings and factor scores of the first three factors were extracted from the analysis (a varimax rotation was used for the factor loadings). In the factorial analyses, the first three factors reflecting

the most important relationships among the variables were used in this study. The factor loadings were used to interpret the factors whereas the factor scores were used for Kendall’s rank correlation. Here, the scores and the original variables of TNC, TNL, TPC and TPL were put into the analysis to see if the factors were significantly correlated with the corresponding nutrients. The seasonal Mann–Kendall trend Epigenetics inhibitor test revealed a positive trend in temperature across almost the entire BSDB with an average increase of 0.04 °C yr−1 over the 31-year record (Table 2). Temperature increase was higher in catchments located at the coast of the Baltic Sea compared to catchments located further away from the Baltic Sea, as shown in Fig. 2a where for each catchment the yearly trend is plotted for the whole BSDB. A positive trend in precipitation was visible in 18% of total eastern area (AE) and in 39% of total western area (AW) with an average

increase of 3.2 mm yr−2 across the entire BSDB ( Table 2). The spatial map of precipitation trends for the BSDB shown in Fig. 2b does not have a clear spatial pattern although most of the trends are located in the more northern catchments. Fig. 2c shows that in general, discharge decreased however in the more southern catchments and increased in the more northern catchments with the average rate of increase being 2.8 mm yr−2 and the average rate of decrease being 0.9 mm yr−2. The Mann–Kendall trend test performed on annual time series confirmed significant trends for temperature (east and west) and discharge (west) ( Fig. 1a and c). Note that Fig. 1 shows all catchments while in Table 2 only catchments with significant trends are included. There were clear, significant, differences between east and west (Fig. 3a and b) in terms of the concentrations (TNC and TPC).

Actual evapotranspiration is however considerably smaller than potential evapotranspiration due to dry soils. This changes towards the end of the rainy season (February, March) when soils become wet and actual evapotranspiration is similar to potential evapotranspiration. www.selleckchem.com/hydroxysteroid-dehydrogenase-hsd.html During this period with wet soils runoff is eventually generated from precipitation, but the overall amounts of runoff are still an order of magnitude smaller than the other water balance components. After the end of the rainy season in April runoff is still significant due to base flow. Actual evapotranspiration becomes larger than precipitation

– which is basically zero during the dry season from May to September – resulting in drying up of soils indicated by negative storage change. The peak in potential evapotranspiration in September and October – caused by hot, dry and windy conditions – has no direct impact on actual evapotranspiration due to lack of water. In addition to the evaluation based on visual comparisons presented in the previous section, we also report on the model performance statistics for the calibration period (1961–1990) and the Talazoparib cost independent evaluation period (1931–1960). Table 4 lists the performance

statistics for discharge simulation at key locations. At some gauges data are available only in a limited number of years during the evaluation period, but time-series are mostly complete in the calibration period. In general the model performance is high in both periods, with a few exceptions as discussed further below. In most cases the correlation is above 0.90 and the Nash–Sutcliffe efficiency is above 0.80. This applies for the calibration period as well as the independent evaluation period. Even though performance statistics in Table 4 are also listed for the gauge Carnitine palmitoyltransferase II Tete, it has to be considered that the reported observed discharge data for this gauge are of limited accuracy. This mainly affects the computed bias ratio (β), but not so much temporal dynamics as measured by the computed correlation (r). In the calibration period the correlation is low (r = 0.74) because operation rules imposed on the model reflect the current situation

(as effective during the 2000s), whereas the actual historic operation of Kariba and Cahora Bassa reservoirs changed over time (see discussion in previous section). In contrast to the calibration period, the correlation between simulated and observed discharge is high (r = 0.95) in the independent evaluation period, with observed data at Tete available from 1952 to 1960. The first seven years represent undisturbed (pristine) conditions, whereas the last two years are affected by the filling of Kariba reservoir. Of greater interest than the poor bias ratio and correlation at Tete is the model performance for simulation of Zambezi discharge at Victoria Falls. Discharge data measured at this gauge are considered to be accurate – and are not affected by upstream reservoir operations.

Studies of esophageal precancers revealed that the degree of clonal diversity was found to increase the probability of progression from esophageal precancer

to adenocarcinoma [22]. Minor subpopulations of primary tumors were shown to be responsible for relapse after drug administration [34]. Intratumor heterogeneity of PTEN protein expression corresponded with loss of heterozygosity and shorter OS in glioblastoma [35]. Tumor heterogeneity of Ki-67 protein in prostate cancer correlated with more aggressive tumor characteristics [5]. In this study, we have demonstrated that heterogeneity of Obeticholic Acid individual proteins, namely PIK3CA, MYC, TOP2A, ESR1, PGR, RUNX1, RAD21, and CDKN2A, correlates with more aggressive tumor behavior and, in case of MYC, TOP2A, ESR1, and RAD21, also confers poor prognosis. Interestingly, prognostic significance of the studied proteins depends on whether the heterogeneity or the expression level is being analyzed. Apart from ESR1, PGR, and TOP2A, which were significantly correlated with prognosis in terms of both the heterogeneity and the expression level, there

were also proteins that were either informative in the context of tumor heterogeneity (PIK3CA, MYC, CDKN2A, RAD21, and RUNX1) or protein expression level (ERBB2, ERBB3, and TP53). Thus, protein heterogeneity and staining intensity might be two distinct phenomena, differently reflecting the course of the disease. Correlations between protein heterogeneity of ESR1 and PGR, ESR1 and RAD21, and ERBB1 and pAKT1 were especially strong. ESR1 and PGR1 expression was found to correlate

strongly in EC [36]. Investigation of ERBB1 and LY2109761 manufacturer pAKT1 expression revealed strong correlation between those two proteins in head and neck squamous cell carcinoma [37]. Similarly, we have found statistically significant correlations between ESR1 and PGR, ESR1 and RAD21, and ERBB1 and pAKT1 (data not shown). Mentioned proteins are functionally related. Perhaps if their expression is co-dependent, so could be the heterogeneity. Cumulative tumor heterogeneity of selected proteins’ heterogeneity proved to be an independent predictor oxyclozanide of survival and showed the strongest correlations with clinicopathologic data. Apparently, simultaneous analysis of a large number of protein markers gives more thorough image of clonal diversity present in the tumor. Therefore, we conclude that the larger the extent of intratumor heterogeneity in EC, the more aggressive the tumor behavior is and thus the worse the prognosis is. One of the limitations of the study was relatively short follow-up period. Furthermore, due to variable quality and sometimes small amount of collected material, reliable analysis of all four cores per patient not always could have been achieved. This issue was even greater in case of global protein heterogeneity determination. However, despite TMA limitations, there is an increasing number of publications based on tumor microarrays due to their convenience.

A systematic characterization of the lesions

phenotype, in particular the location, size, shape, and histology, is often lacking. Very few data are available about the occurrence of interval cancers during surveillance for IBD. The first paper dates back to 1982.16 In this surgical review of 676 patients with UC undergoing long-term follow-up, a total of 35 CRCs were identified. Twelve of these Selleckchem EPZ015666 were diagnosed because of symptoms, 10 as incidental findings at proctocolectomy, and 13 CRCs were diagnosed during the follow-up at least 1 year after the initial UC diagnosis. This latter subgroup was referred to as “interval CRCs.” In a St Mark’s study reviewing the UC surveillance program over approximately three decades, a total of 74 patients (12.3% of the total population) developed neoplasms, including 30 CRCs.17 The authors http://www.selleckchem.com/products/INCB18424.html defined interval CRCs as “cancers presenting after a negative index-colonoscopy or advanced (Dukes’ C/disseminated) cancers detected at surveillance.” During a median follow-up of 1.5 years,

nine patients were identified with Dukes’ C cancers and four patients with disseminated cancers (4 of these 13 cases were diagnosed within 12 months). In three cases, CRC was diagnosed at colonoscopy because of symptoms; one of these was attributable to noncompliance. Of note, more than half (16 out of the 30) of the CRCs identified with this program were interval cancers, raising concerns

about the effectiveness of colonoscopic cancer prevention. A statistically significant reduction in CRC rates over time was observed in this study (r = −0.40; P = .04), aminophylline especially in the proximal colon. From these data, we can conclude that there is sparse understanding of the magnitude and clinical significance of interval CRCs in patients with IBD. Indeed, a wide variation exists with regard to the terminology used in endoscopy and pathology diagnostic protocols across countries, IBD centers, and studies. Standardization of the nomenclature and clinical protocols, and uniformity in reporting on interval CRCs during IBD surveillance, would help to define quality targets. As a first step, a universal terminology is required for dysplasia and interval cancers. Previously used terms, such as flat dysplasia or dysplasia associated lesion or mass, need to be revisited. A rigorous description of the endoscopic shape and histologic features of the detected lesions is required, using international classifications (ie, Paris-Japanese endoscopic classifications18 and 19 and the World Health Organization histopathologic classifications20 and 21). Interval cancers should be considered those invasive cancers diagnosed after a negative screening examination, but before the next recommended follow-up colonoscopy, as endorsed by the current international IBD surveillance guidelines.

The spatial similarity between Etoposide concentration the submitted and reference expert prostate

contours was assessed using a Dice’s coefficient (9). The median prostate volume was 33.4 cm3 (range, 19.4–70.1 cm3). The median %V100, %D90, and %V150 were 91.1% (range, 45.5–99.8%), 101.7% (range, 59.6–145.9%), and 53.9% (range, 15.7–88.4%), respectively. Low gland coverage was observed in some patients: 27 (39%) were noted to have a D90 lower than 100% of PD; and of those, 12 (17%) had a D90 lower than 80% of the PD. For this data set, there was no correlation between D90 coverage and prostate volume, number of seeds, or total implanted activity. In addition, there were no apparent differences in D90 dose coverage according to the different institutional strata. The median V100 for the rectum was 0.3 cc Ku-0059436 chemical structure (range, 0–4.3 cc). The median D2cc rectum doses were 64.3% (range, 27.3–126.1%). No differences were observed in terms of dosimetric outcomes according to the institutional strata. The Dice’s coefficient was used to compare the submitted and reviewed prostate volumes, as shown in Fig. 1. The coefficient measures the intersection between the two volumes to be compared; thus a Dice’s coefficient of 1 means that the two volumes can be superimposed and are equal. The average Dice’s coefficient for the prostate volumes

in these patients was 0.83 (range, 0.75–0.92) with a standard deviation (SD) of 0.04. The median and SD of %D90 for the submitted and reviewed scans were 101.5% (SD, 17.6%) and 101.1% (SD, 18.5%), respectively ( Fig. 2). We define D90 concordance to be good if the D90 value reported by the treating institution is within 10% of the reevaluated D90. Good D90 concordance

was observed in 44 of the 69 cases. The median and SD of %V100 for the Cepharanthine submitted and reviewed scans were 88.1% (SD, 10.7%) and 87.9% (SD, 11.2%), respectively. For the submitted contours and calculated doses, there were 32 patients (46%) with D90 lower than 100% of the PD and 18 patients (26%) with D90 lower than 90% of the PD. When these contours were centrally reviewed and doses were recalculated, 28 patients (41%) were noted to have a D90 lower than 100% of the PD and 17 patients (25%) had a D90 lower than 90% of the PD. Figure 3 illustrates the similarities between the submitted and reviewer evaluations for %V150. As demonstrated in Fig. 3, 4% and 7% of patients had V150 greater than 80%, suggestive of a “hot implant” based on the submitted and centrally reviewed dose calculations. The average Dice’s coefficient for the rectal volumes in these patients was 0.8369 (range, 0.7533–0.9165) with an SD of 0.0431. The median and SD of rectal D2cc as a percentage of the PD was 62.7% (SD, 18.1) and 64.3% (SD, 20.3) for the submitted and reviewed scans, respectively ( Fig. 4). When all the above-mentioned analyses were performed excluding the 10 test cases, the findings were found to be not significantly different (data not shown).

1% citrate, 0.1% Triton X-100 and 50 μg/mL propidium iodide and fluorescence was measured afterwards. Cell membrane integrity was evaluated by the exclusion of propidium iodide. Briefly, 100 μL of treated and untreated cells were incubated with propidium iodide (50 μg/mL). The cells were then incubated for 5 min at 37 °C. Fluorescence was measured and cell morphology, granularity and membrane integrity were determined (Darzynkiewicz et al., 1992). PS externalization was analyzed by flow cytometry (Annexin V) according to Vermes and co-works (1995)

using Guava Nexin Assay Kit. Briefly, cells (3 × 105 cells/mL) were washed twice with cold PBS and then resuspended in 135 μL of PBS with 5 μL of 7-aminoactinomycin click here D (7-AAD) and 10 μL of Annexin V-PE. Cells were gently vortexed and incubated for 20 min at room temperature (22 ± 2 °C) in the dark. Afterwards, cells were analyzed by flow cytometry (EasyCyte from Guava® Technologies). Annexin V is a phospholipid-binding protein that has a high affinity for PS. 7-AAD, a cell impermeant dye, is used as an indicator of membrane structural integrity. Fluorescence of Annexin

V-PE was measured in yellow fluorescence-583 nm and 7-AAD in red fluorescence-680 nm. The percentage of early and late apoptotic cells and necrotic cells was then calculated. Birinapant Active catalytically caspases-3/7 were analyzed by flow cytometry using Guava® EasyCyte Caspase Kit after 24 h of incubation. HL-60 cells (3 × 105 cells/mL)

were incubated with Fluorescent Labeled Inhibitor of Caspases (FLICAs) and maintained for 1 h at 37 °C and 5% CO2. After incubation, 80 μL of washing buffer were added and cells were centrifuged at 2000 rpm for 5 min. The resulting pellet was resuspended in 200 μL of washing buffer and centrifuged again. Then, cells were resuspended in the working solution (propidium iodide 1:200 in 1× washing buffer) and analyzed immediately Tau-protein kinase by flow cytometry. Mitochondrial transmembrane potential was determined by rhodamine 123 dye retention using flow cytometry. Rhodamine 123 is a cell-permeable, cationic, fluorescent dye that is readily sequestered by lively mitochondria without inducing cytotoxic effects. Cells (3 × 105 cells/mL) were washed with PBS, incubated with rhodamine 123 at 37 °C for 15 min in the dark. Cells were incubated again in PBS at 37 °C for 30 min in the dark, and fluorescence was measured (Militão et al., 2006). Heparinized blood was collected from healthy, non-smoker donors who had not taken any medication for at least 15 days prior to sampling and with no history of recent exposure to potentially genotoxic substances (i.e., pesticides, drugs, alcohol, tobacco or ionizing radiation, such as X-rays). All studies were performed in accordance with Brazilian (Law 196/96, National Council of Health) and international (Declaration of Helsinki) guidelines.

The hot plate was pre-heated and kept at a temperature of 55±0.5 °C. All rats were acclimated to the hot plate for 5 min, 24 h prior to testing, as, again, the novelty of the apparatus itself can induce antinociception (Netto et al., 2004). Rats were placed in glass funnels on the heated surface and the nociceptive CH5424802 molecular weight threshold was assessed recording to the time taken to first response (foot licking, jumping, or rapidly removing paws), as described by Minami et al. (1994). Response was recorded in seconds (s) and a cutoff time of

20 s was used. After 11 weeks of chronic stress exposure, the rats of SN were subjected to a 20-min session of anodal tDCS every afternoon for 8 days. This period was established because tDCS has been shown to modify cortical excitability for up to 1 h after one session of stimulation (Nitsche and Paulus, 2000; Nitsche et al., 2003b). However, repetitive tDCS application has demonstrated better and longer-lasting effects on pain relief, and in recent study our group showed antihyperalgesic response in paw inflamed rats with this treatment period (Laste et al., 2012). The direct current was delivered from a battery-driven, Trametinib mw constant current stimulator using ECG electrodes with conductive adhesive hydrogel. Rats’ heads were shaved for better adherence and the electrodes were trimmed to 1.5 cm2 for better fit. After placement, electrodes were fixed onto the head with adhesive tape (Micropore™)

and covered with a protective mesh to prevent removal (Fig. 5A). The anodal electrode was positioned between the ears, from the neck of the rat (parietal cortex) (Fig. 5B) (Takano et al., 2011 with modifications), so as to mimic anodal placement in human pain studies (Mendonca et al., 2011 and Dasilva

et al., 2012). The cathodal electrode was positioned at the midpoint of the lateral angle of the eyes (supraorbital area). The electrodes were placed on the skin in a similar manner to that used in human studies of tDCS for pain (Nitsche et al., 2008, Antal and Paulus, 2011, Rosen et al., 2009 and Fregni et al., 2006c). A constant current of 0.5 mA intensity was applied for 20 min (Fregni et al., selleck chemicals 2006b, Dockery et al., 2011, Wachter et al., 2011 and Liebetanz et al., 2006). According to an earlier study (Liebetanz et al., 2009), a constant current of 1 mA intensity causes skin lesions, as current density is comparatively much higher than the traditional 1 mA tDCS using large pads in humans. We therefore chose to use 0.5 mA, an intensity that has also been used in other animal studies. In addition, in our study, electrodes were fixed onto the skin. We did not observe any lesions with montage and current intensity. An important point to consider was that this model required neither anesthesia nor surgery, unlike models used in the previous tDCS studies in rats (Dockery et al., 2011, Wachter et al., 2011 and Liebetanz et al., 2006).

Many scientists,

these days also rely upon a gel scanner to estimate protein in a given sample by running a SDS-PAGE. The few features of these methods are sometimes less clearly taken into account than desirable. 1. Most of the protein estimation methods rely upon the color-generating response of the protein during a chemical reaction (e.g. Biuret, Lowry or BCA methods) (Walker, 2002) or physical AG-014699 datasheet interaction with a compound (e.g. frequently used dye-binding assay) (Bradford, 1976). Different proteins respond in a quantitatively different way. In this respect, Biuret is an exception as it gives relatively uniform response for most of proteins. This is much less sensitive than other methods (Scopes, 1994). However, most of the industrial enzymes contain a good amount of protein/g, so Biuret actually may be a good option. Most

of the other methods give the relative protein concentration. For example, it is a general practice to say that a particular protein estimation method was employed and BSA was used for a standard curve. The color-generating Epigenetics Compound Library datasheet response by the protein can be very significantly different from BSA. This is not a cause of worry as we mostly track change in protein concentration during any operation/process. For example, during protein purification, we are only concerned with fold purification starting with a crude preparation. So, the relative protein concentration value should be good enough. However, when we calculate the amount of protein expressed and obtained as inclusion bodies ( Garcia-Fruitos et al., 2012), we tend to overlook that we are not talking of absolute protein concentration. The amounts of an enzyme present in a given sample, reaction system or bioreactor is obviously an important parameter. If the reaction condition cAMP obeys Michaelis–Menten kinetics, it is implied that [E]«[S]. Ideally, if the amount of enzyme is increased x times, the initial rate is expected to increase x times. In reality, it may not happen. The plot of velocity vs. [E] curve may have an increasing slope (display a lag period or a slow phase) if: (a) The oligomeric form of an enzyme has higher activity or

if the subunits of the enzyme dissociate in dilute solutions. On the other hand, the velocity vs. [E] curve may have a decreasing slope (i.e. the velocity slows down with increase in [E]) because: (i) The enzyme has a tendency to aggregate. These aggregates may be soluble. So, no visible precipitation is observed. Earlier, it was believed that extensive aggregation requires unfolding of the protein chain. Now, there is growing evidence that even “native-like structures” may aggregate (Bemporad et al., 2012). Intrinsically disordered proteins (IDP), of course, constitute an extreme case in this regard (Uversky, 2011). Aggregates are generally inactive although recently alpha chymotrypsin subjected to three-phase partitioning (TPP) (Rather et al.