, 2012). Although in vivo siRNA delivery has continuously been improved over the last years ( Rettig and Behlke, 2012), it still represents a major challenge. In particular, targeted delivery into certain cell types or organs has proven tricky. In the past, viral vectors have frequently and successfully been employed for the delivery of protein-encoding DNA sequences into living organisms. Consequently, they have also been adopted

for the delivery of shRNAs and amiRNAs ( Liu and Berkhout, 2011, Mowa et al., 2010 and Raoul et al., 2006). Depending MEK inhibition on the type of target cell, organ, or delivery route, they may still outperform nonviral delivery systems in certain instances. Adenoviral vectors have been used for a long time to deliver DNA sequences into living organisms ( Goncalves and de Vries, 2006). Since they display the same cell tropism as wt

adenoviruses (when belonging to the same adenoviral species), they deliver transgenic DNA into exactly those cells that represent the main targets of their wt counterparts. Thus, adenoviral vectors may constitute a particularly attractive tool for the delivery of anti-adenoviral shRNAs or amiRNAs. Consequently, in the present study, we generated a series of replication-deficient adenoviral amiRNA expression vectors for the silencing of selected Ad5 genes and investigated whether these

amiRNAs are capable of efficiently MK-2206 datasheet inhibiting the replication of wt Ad5 upon transduction of a cell with the recombinant vector. The amiRNAs were designed to recognize those mRNAs that had been identified as candidate targets in our previous study (Kneidinger et al., 2012), i.e., mRNAs encoding the viral E1A protein, a key regulator of the infection cycle ( Pelka et al., 2008), the preterminal protein (pTP), NADPH-cytochrome-c2 reductase and the viral DNA polymerase, both essential for viral DNA replication ( de Jong et al., 2003). Here, we present data demonstrating the efficient silencing of the wt Ad5 pTP gene upon transduction with amiRNA expression vectors. Moreover, we demonstrate an increase in the knockdown rate upon concatemerization of amiRNA-encoding sequences, and we show that amiRNA expression is strongly boosted in wt Ad5-infected cells, a prerequisite for the efficient targeting of high numbers of viral transcripts. Taken together, our data indicate that amiRNA-mediated knockdown of wt Ad5 gene expression significantly inhibits viral DNA replication and efficiently decreases the output of infectious virus progeny in vitro.

CRL-1573), were obtained from American Tissue Culture Collection (Rockville, MD, USA). TANK (TRAF family member-associated NF-kappa-B activator)-binding kinase (TBK)1 and adaptor molecule [TIR-domain-containing adapter-inducing interferon-β (TRIF) or myeloid differentiation primary response gene 88 (MyD88)] were used as reported previously [16]. Fetal bovine serum and RPMI 1640 were purchased from Gibco (Grand Island, NY, USA), and phospho-specific

or total antibodies to c-Jun, c-Fos, ATF-2, IRF-3, extracellular signal-regulated kinase (ERK), p38, C-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), MKK3/6, transforming growth factor-β-activated kinase 1 (TAK1), TBK1, lamin A/C, and β-actin were purchased from Cell Signaling Selleck Epacadostat (Beverly, MA, USA). All other chemicals were purchased from Sigma Chemical Co. A stock solution (8 mg/mL) of PPD-SF was prepared with culture medium and diluted to 0–400 μg/mL: with media for in vitro, cellular assays, or suspended in 1% sodium carboxymethylcellulose for in vivo experiments. Male imprinting

Selleckchem Alpelisib control region (ICR) mice (6–8 weeks old, 17–21 g) were obtained from Daehan Biolink (Chungbuk, Korea) and maintained in plastic cages under standard conditions. Water and pelleted food (Samyang, Daejeon, Korea) were supplied ad libitum. Studies (approval ID: SKKUBBI 13-6-2) were performed in accordance with guidelines established by the Institutional Animal Care and Use Committee at Sungkyunkwan University, Suwon, Korea. RAW 264.7 and HEK293 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, glutamine, and antibiotics (penicillin and streptomycin)

at 37°C under 5% CO2. For experiments, cells were detached with a cell scraper. Under our experimental cell density (2 × 106 cells/mL), the proportion of dead cells was < 1% according to Trypan blue dye exclusion tests. After preincubation for 18 hours, RAW264.7 cells (1 × 106 cells/mL) were pretreated with PPD-SF (0–400 μg/mL) or the standard compounds (l-NAME, out SP600125, or BX795), and incubated with LPS (1 μg/mL) for 24 hours. The inhibitory effects of PPD-SF or standard compounds on NO, TNF-α, or PGE2 production were determined by analyzing the NO, PGE2, or TNF-α levels quantified with Griess reagent, enzyme immunoassay, or enzyme-linked immunosorbent assay, respectively, as described previously [17] and [18]. After preincubation for 18 hours, PPD-SF (0–400 μg/mL) was added to RAW264.7 cells (1 × 106 cells/mL) followed by incubation for 24 hours. The cytotoxic effects of PPD-SF were evaluated by MTT assay, as reported previously [19] and [20]. Phytochemical characteristics of PPD-SF with standard ginsenosides were identified by high performance liquid chromatography (HPLC) as reported previously [21] and [22].

This seasonal flow regulation largely favors water consumption in non-flood seasons, primarily for farming irrigation. In non-flood season, the difference between average daily water discharge at Huayuankou and Lijin

results mainly from water consumption loss. This value increased in a step-wise manner from 26 m3/s in 1950–1968 to 242 m3/s in 1969–1986 CH5424802 ic50 and 421 m3/s in 1987–1999, respectively, followed by a slight decrease of 384 m3/s in 2000–2011 (Table 2). This pattern can be explained by increasing water use favored by strengthening runoff regulations. The construction of large dams on the Huanghe has largely controlled the frequent floods on the lower reaches that are ded by monsoon rains. Long-term (1950–2011) RG7204 concentration observations of daily water discharge at Lijin reveal that peak flow > 6000 m3/s decreased dramatically from a total 155 days during 1950–1968 to 17 days during 1969–1986, and vanish completely since 1987 (Table 3). Even smaller flood peaks (4000–6000 m3/s) could not be observed after the construction of Xiaolangdi reservoir in 1999. Since 2000, low flow (<2000 m3/s) dominates the discharge pattern of the lower reaches most of the year, and flow >2000 m3/s is mainly concentrated within the annual WSM (often less than 20 days) when the released floodwater

is confined to <4000 m3/s. Huayuankou station recorded a similar trend, as shown in Table oxyclozanide 3. Here, we select representative years (1954, 1988, 2003) to show the stepwise

drops in the amplitude of flood peaks recorded at Lijin and Huayuankou over time (Fig. 2). Both the Lijin and Huayuankou records show a similar pattern, with the amplitudes of flood peaks dramatically decreasing. At Huayuankou station, pre-dam discharge levels (1950–1960) show several flood peaks during the flood season, with extreme peaks approaching ∼17,000 m3/s (e.g. 1954, Fig. 2A). In 1988 smaller flood peaks (<7000 m3/s) could be observed (Fig. 2B). In 2003 (after Xiaolangdi Reservoir was constructed), flood peaks >4000 m3/s become non-existent, e.g. in 2003 (Fig. 2C). Since 1950, no catastrophic flooding has occurred in the lower reaches of the Huanghe, owing to the effect of the dams. Sediment sequestration is a common problem in many large reservoirs. This problem is particularly severe for the Huanghe owing to the high suspended sediment concentration. Spatially, the Longyangxia and Liujiaxia reservoirs have a minor effect in trapping sediment, since only a small fraction of the Huanghe sediment is sourced from its upper reaches. The Liujiaxia and Longyangxia annually trap only 0.53 × 108 m3 (average 1968–1997 level) and 0.16 × 108 m3 (average 1986–1997 level) of sediment, respectively (Peng and Chen, 2009). The Sanmenxia and Xiaolangdi reservoirs in the lower middle reaches have trapped large amounts of sediment since their operation. The Sanmenxia Reservoir, in particular, had lost 45.

The bottom layer of the reference forest was characterized by over 70% cover of P. schreberi in the moss bottom layer and the shrub understory was over 50% cover of dwarf shrubs. In contrast the spruce-Cladina forest had less than 3% cover AZD2014 solubility dmso of P. schreberi and over 50% cover of Cladina in the bottom layer and about 18% cover of all dwarf shrubs in the understory. Soil characteristics in open spruce stands with Cladina understory were notably different than those found in neighboring spruce, pine, feathermoss forest stands within the

same area. Recurrent use of fire reduced the depth of O horizon by an average of 60% across all three forest sites. Both total N capital ( Fig. 1a) and total concentration ( Table 2) associated with the O horizon were significantly reduced by historical burning practices. Total N concentration in the O horizon decreased by about 50% where total N capital decreased by a factor of 10. Nitrogen capital values of greater than 800 kg N ha−1 exist on the reference forest stands as compared to less than 80 kg N ha−1 on the spruce-Cladina forests. Total C in the O horizon was also much lower in the spruce-Cladina forests ( Table mTOR inhibitor 2 and Table 3, Fig. 1b), but not to the extent of

N. Mineral soil total C and N were not significantly different between the spruce-Cladina and reference forest stands. Total P and extractable Mg are the only other nutrients in the mineral soil that have been significantly influenced by the years of periodic burning (Fig. 2 and Fig. 3). There were no differences in total Zn or exchangeable Ca concentrations in the mineral soil of the two forest types (Table 4). Total N:P (Fig. 4) of the O horizon were low for both forest types, but were significantly higher in the spruce-Cladina forests, likely as a result of reduced N2 fixation and increased net P loss from these soils. Ionic resins buried at the interface of the O horizon and mineral soil in both forest types revealed noted differences in N turnover between the spruce-Cladina forests

and the reference forests. Averaged across the three sites, NO3−-N accumulation on ionic resins was significantly greater in the degraded lichen-spruce Montelukast Sodium forest than that in the reference forest ( Fig. 5a). Resin adsorbed NH4+-N concentrations were notably greater in the reference forests ( Fig. 5b). Previous pollen analyses from the two sites Marrajåkkå and Marrajegge demonstrated a decline in the presence of Scots pine and juniper in conjunction with a great increase in the occurrence of fire approximately 500 and 3000 years BP, respectively (Hörnberg et al., 1999). The pollen record from Kartajauratj showed the same trend with a general decrease in the forest cover over time and the occurrence of charcoal indicates recurrent fires (Fig. 6).

, 2010). As we could expect it, the highest contamination levels (total 134+137Cs activities exceeding 100,000 Bq kg−1) Olaparib mouse were measured in sediment collected along the coastal rivers (i.e., Mano and Nitta Rivers) draining the main radioactive plume (Fig. 2). Contamination levels were logically much lower in sediment collected along the Abukuma River that drains less contaminated areas. The analyses conducted by the Japanese Ministry of Environment (MoE) provided an additional temporal insight into contaminated sediment exports in this area. Our samples were collected in November 2011, whereas samples provided by MoE showed that contamination of sediment was systematically the highest

in material collected in September 2011. The presence of contamination hotspots close to Fukushima City and behind a large dam located upstream of the city is likely due to the rapid wash-off of radionuclides on urban surfaces during the first series of rainfall events that followed the accident, to their concentration in urban sewers systems (Urso et al., 2013) and their subsequent export to the rivers. This rapid export of radionuclides learn more shortly after the accident along the Abukuma River is confirmed

by data collected by the MoE (Fig. 2) showing a peak of contamination in sediment collected in September 2011, and then a huge decrease to low activities even during snowmelt. Along the Hirose River, the snowmelt (in March 2012) led in contrast to an increase in sediment contamination. At the light of those first results outlining a very rapid wash-off of radionuclides obtained following the accident in the Abukuma River

basin, we decided to focus the next fieldwork campaigns on the coastal basins where radionuclide activities check in sediment were the highest. We extended sampling to the Ota River catchment, closer to FDNPP, where access was unauthorized during the first campaign (Fig. 1b). Whilst 137Cs and 134Cs gamma-emitting radioisotopes constitute by far the most problematic contaminants (with total activities in soils ranging from 50 to 1,110,000 Bq kg−1), 110mAg was also identified and measured in most samples (with activities ranging from 1 to 3150 Bq kg−1). Because of these low activities, contribution of 110mAg to the global dose rates was considered to be negligible. It appeared from the analysis of the MEXT soil database that the initial fallout pattern of 110mAg displayed significant spatial variations that were not observed for the radiocaesium fallout pattern at the scale of the entire Fukushima Prefecture. Soil activities in 110mAg were the highest within the main radiocaesium contamination plume as well as at several places along the coast located between 40 and 50 km to the north of the power plant (MEXT, 2011b). Most interestingly, the 345 values of 110mAg:137Cs ratio in MEXT soil samples strongly varied across the entire region (0.0004–0.15 with a mean of 0.006; Fig.

Subjective assessment of DES using a questionnaire was also conducted at each visit. The TBUT was identified following the procedure reported by Lemp [30]. check details A fluorescein strip (Haag-Streit AG, Köniz, Switzerland) was moistened with a drop of saline solution, and placed on the inferior palpebral conjunctiva. The patients were asked to blink several times to mix the fluorescein with the tear film. They were instructed to open their eyes and not blink, and the time between eye opening and the appearance of the first dry spot was measured in seconds. This procedure was repeated three times, and the mean

of the three measurements was recorded finally as TBUT. After the measurement of the TBUT, fluorescein staining on the ocular surface was evaluated using the standardized methods recommended by the National Institutes of Health Symposium on Dry Eye [30]. Briefly, corneal staining was scored 3 minutes after fluorescein instillation by observing the cornea through a cobalt blue light. It was graded using a scale of 0–3 (absent to diffuse) and recorded for the five corneal sections (central, superior, temporal, nasal, and inferior.). The maximum score for each area was 3. The scores of the five areas were summed to obtain a total score for each eye, producing a maximum score of 15. Conjunctival hyperemia

was evaluated by the investigator based on a visual inspection. A standard five-point scoring system was used with the following descriptors based on Nintedanib (BIBF 1120) photographic

standards: 0 (none) = normal, Selleck Autophagy inhibitor bulbar conjunctival vessels easily observed; +0.5 (trace) = trace flush, reddish-pink color; +1 (mild) = mild flush, reddish color; +2 (moderate) = bright red color; and +3 (severe) = deep, bright, diffuse redness. The Schirmer I test was performed under anesthesia. To obtain anesthetic conditions of all the ocular structures, more than three drops of topical anesthetic (proparacaine hydrochloride ophthalmic solution 0.5%) were applied to the conjunctiva and both lid margins. Then, Schirmer strip was placed on the lower lid 2 mm lateral to the lateral canthus. Patients sat in the dark with both eyes closed for 5 minutes. After the strip was removed, a length of the wet area of the strip was measured in millimeters. The quality and quantity of meibomian gland secretions were evaluated using manual expression. The quantity was graded using a three-point scale: 0 = normal; 1 = delay; 2 = partially blocked; and 3 = blocked. The quality was also scored similarly: 0 = clear; 1 = cloudy; 2 = granular; and 3 = opaque solid. To evaluate subjective symptoms of dry eye, the participants were asked to complete the Ocular Surface Disease Index (OSDI) prior to taking any clinical measurements.

A sedimentary record of about 1000 m of Pleistocene sand, silt, clay and peat underlays the lagoon. Within this record lies an altered layer, a few decimeters to a few meters thick, representing the last continental Pleistocene deposition, which marks the transition to the marine-lagoonal Holocene sedimentation. This layer shows traces of subaerial exposure (sovraconsolidation,

yellow mottlings) and other pedogenic features (solution and redeposition of Ca and Fe-Mn). It forms a paleosol, lying under the lagoonal sediments called caranto in the Venetian area ( Gatto and Previatello, 1974 and Donnici et al., 2011). The Holocene sedimentary record provides evidence of the different lagoonal find more environments, since various morphologies and hydrological regimes took place since the lagoon formation ( Canali et al., 2007, Tosi et al., 2009, Zecchin et al., 2008 and Zecchin et al., 2009). Starting from the 12th century, major rivers (e.g. the rivers Bacchiglione, Brenta, Piave and Sile) were diverted to the north and to the south of the lagoon to avoid its silting up. Since then, extensive engineering works were carried out (i.e. dredging of navigation channels, digging of new canals and modifications on the

inlets) ( Carbognin, 1992 and Bondesan and Furlanetto, 2012). All these BMS-387032 order anthropogenic actions have had and are still having a dramatic impact on the lagoon hydrodynamics and sediment budget ( Carniello Cell press et al., 2009, Molinaroli et al.,

2009, Sarretta et al., 2010 and Ghezzo et al., 2010). The survey area is the central part of the Venice Lagoon (Fig. 1a). The area of about 45 km2 is bounded by the mainland to the north and the west, from the Tessera Channel and the city of Venice and it extends for about 2 km to the south of the city reaching the Lido island to the east. In particular, we focus on the area that connects the mainland with the city of Venice (Fig. 1b). It is a submerged mudflat with a typical water depth outside the navigation canals below 2 m (Fig. 1c). This area has been the theatre of major anthropogenic changes since the 12th century. It is one of the proposed areas where the large cruise ship traffic could be diverted to. There are a number of proposed solutions to modify the cruise ship route that currently goes through the Lido inlet, the S. Marco’s basin and the Giudecca channel. One solution involves the shifting of the touristic harbor close to the industrial harbor from Tronchetto to Marghera, whereas another solution calls for the dredging of the Contorta S. Angelo Channel, to allow the arrival of the cruise ship to the Tronchetto from the Malamocco inlet. Both of these options could strongly impact the morphology and hydrodynamics of this part of the lagoon. The first archeological remains found in the lagoon area date back to the Paleolithic Period (50,000–10,000 years BC) (Fozzati, 2013).

The power (root mean square) of the filtered signal was calculated for each electrode and summed across electrodes designated as being in the CA1 pyramidal cell layer. The threshold for SWR detection was set to 7 SD above the background mean. The SWRs detection threshold was always set in the first sleep session, and the same threshold was used for all other sessions. The SWR firing rate histograms of pInt and nInt interneurons

were calculated during the sleep session before learning using 20 ms bin in reference to the SWR peak (i.e., peak SCH-900776 of ripple-band power) as previously described (Dupret et al., 2010; O’Neill et al., 2006). We thank P. Somogyi, P. Jonas, K. Allen, and D. Dickerson for their constructive comments on a previous version of the manuscript and K. Lamsa for helpful discussions; N. Campo-Urriza and L. Norman for their technical assistance. D.D. and J.C. were supported by a MRC Intramural Programme Grant (U138197111)

and J.C. by a European Research Council Starter Grant (281511). D.D. currently holds a Research Fellowship in Neuroscience from Saint Edmund Hall College, University CAL-101 price of Oxford. D.D. conducted the experiments. D.D. and J.O. carried out the data analysis. D.D. and J.C. wrote the manuscript. J.C. supervised the project. All authors discussed the results and commented on the manuscript. “
“The ventral striatum (VS) has been described as the “limbic-motor interface” because it is strategically poised to integrate emotional-motivational input and subsequently influence motor activity (Mogenson et al., 1980). The VS encompasses the nucleus accumbens and ventromedial aspects of the

dorsal striatum, as defined by the territories innervated by limbic inputs arriving from the hippocampus (HP) and medial prefrontal cortex (PFC) (Voorn et al., 2004), and integrates these and Thiamet G other afferent inputs to guide behavior. Individual medium spiny neurons (MSNs) of the VS receive afferents from the HP on proximal dendrites (Meredith et al., 1990), as well as the amygdala, thalamus, and PFC, in their more distal arbors (French and Totterdell, 2002, 2003; Moss and Bolam, 2008). VS MSNs must reconcile diverse and dynamic inputs into a cohesive efferent signal, and data suggest these inputs may interact in nonlinear ways (Goto and O’Donnell, 2002; O’Donnell and Grace, 1995). For example, HP inputs can drive VS MSNs into a depolarized up state, gating other inputs to the region (O’Donnell and Grace, 1995). This type of additive nonlinear interaction has been proposed to underlie the use of contextual information to guide motor plans. During goal-directed behaviors and in decision-making instances, however, interactions among inputs to the VS may assume a different profile.

Spaniol and colleagues (2009) analyzed 81 fMRI studies of episodic memory, a subset of which included contrasts of encoding success (subsequent hits greater than misses) and/or retrieval success (hits greater than CR). A quantitative meta-analytic procedure indicated that retrieval success consistently activated striatum across studies, including both dorsal striatum in the left caudate and ventral striatum in regions of caudate, putamen, and nucleus accumbens (also see Kim, 2011). Figure 2 shows this effect

selleck chemicals in an updated recoding and reanalysis of these data conducted for this review. Moreover, a contrast between retrieval success and encoding success showed that the ventral caudate was more reliably associated with retrieval success than encoding success across studies. Importantly, retrieval success in striatum is not dependent on an actual prior experience with an item. Rather, striatum shows greater activation for false

alarms (new items incorrectly judged as old) than CR or misses (Abe et al., 2008). Thus, like most regions showing retrieval success effects (Wagner et al., 2005), striatal activation tracks the perception of an item as being old during a recognition memory task, rather than it having been previously encountered on the study list. Thus, striatal retrieval success effects cannot be trivially explained based Enzalutamide ic50 on a prior association with positive reinforcement formed at encoding. Generally consistent with the neuroimaging data, deficits in patients with Parkinson’s disease (PD)—a disease arising from degeneration of cells in the substantia nigra that are a primary source of dopaminergic input into the striatum (Figure 1B)—indicate that the basal ganglia are broadly necessary for normal levels of recognition memory performance. In particular, though not suffering from the CTP synthase profound amnesias accompanying MTL damage, PD patients do demonstrate deficits in recognition memory relative to controls in studies with sufficient power (Whittington et al., 2000). Accounting for these recognition deficits in PD has proven difficult and multifaceted.

Across studies, deficits have been evident sometimes in recollection (Barnes et al., 2003; Edelstyn et al., 2007, 2010; Drag et al., 2009) and sometimes in familiarity (Davidson et al., 2006; Weiermann et al., 2010). Moreover, there seems ample evidence that at least a portion of memory deficits observed in PD arise from a failure to engage in effective encoding strategies (Knoke et al., 1998; Vingerhoets et al., 2005). However, a recent study has provided convincing evidence for a recollection deficit in PD when encoding strategy was controlled. Cohn et al. (2010) had PD patients and age-matched controls study word pairs under shallow and deep encoding conditions, and estimated familiarity and recollection using the process-dissociation procedure (Yonelinas et al., 1995).

Detailed classifications of playing positions (including detailed analysis of the goalkeeper position), fatigue development analysis during and after match-play and simultaneous Entinostat concentration analysis of physical, technical, and tactical game demands should also be considered in future research in this area. Investigations on the physiological demands of women’s football match-play involving simultaneous measurements

of heart rate (HR), oxygen consumption (VO2), and blood lactate (La) are still scarce (Table 4) mainly due to the difficulty, high cost, and laborious procedures required to conduct this type of studies. Even in the case of men’s football, they are also limited. To our knowledge, there is to date only one published study that has included simultaneous HR, VO2, La, and GPS measurements during find more a women’s football match.60 This investigation consisted of a full 90 min competitive friendly match (11 vs. 11), in which continuous HR and VO2 (via portable spirometry) and La assessment (every 15 min) was conducted simultaneously on 10 outfield players during the duration of the match ( Fig. 1). Similar to other authors, 46, 59 and 94 Martínez-Lagunas et al. 60 found a significant reduction in the players’ physical and physiological performance in the 2nd compared to the 1st half and a large individual variability of the results (mostly due

to the players’ positional role). However, the results of this latter study ( Table 4) are lower than published data on VO2 average values reported for male footballers collected via portable click here spirometry (57%–77% VO2max) 94 and 95

or by using Douglas bags (47%–60% VO2max) 96 during friendly games; average La values (2.4–10.0 mmol/L) 6 and 97 reported for male players during match-play; average HR (81%–87% HRmax), 46, 53 and 59 La (2.7–5.1 mmol/L), 22 and 98 and GPS (e.g., 9.1–9.6 km of total distance covered) 57 and 59 or computerized video-based (10.2–12.0 km of total distance covered) 46, 49, 53, 55, 61 and 99 physical data of female football players during competitive matches. The HR and VO2 results from Martínez-Lagunas et al. 60 are also lower than the average reported values based on indirect estimation via the HR-VO2 relationship (approximately 80%–90% of HRmax corresponding to ∼70%–77% VO2max), which may tend to overestimate actual VO2. 46, 53, 97 and 100 Possible reasons for the discrepancy of results may include gender, players’ characteristics and competitive level, game conditions, methodological differences, and movement impairment due to the measuring equipment. Further studies using a larger sample size (players and games) should be conducted in order to verify these results. Moreover, competitive level and positional role differences should also be evaluated in more detail in the future.