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It was found that the concentrations of lactic acid, acetic acid, and butyric acid in Y2 were higher than those of X2. The effect of the SCT on lactic acid and acetic acid production was not significant (p > .05). However, a significant difference was observed in butyric acid concentration (p < .05). Similarly, Donkor, Henriksson, Vasiljevic, and Shah (2006) reported that lactic acid concentration in yogurts containing probiotic culture was higher than the control containing standard yogurt culture. They also stated that the differences in lactic acid concentration were not statistically significant (p > .05). In contrast to the concentrations of other acids, it can be stated that X2 has higher amount of citric acid than Y2 (Figure 5c). Adhikari et al. (2002) noted that the citric acid content of the yogurts containing probiotic strains was lower than that of the control containing standard starter cultures. They related this result to the higher utilization of citric acid by the starter cultures during fermentation in the presence of bifid bacteria. As expected, the concentrations of organic acids significantly increased in both Y2 and X2 samples during cold storage with an exception of butyric acid (p < .05). Similarly, Adhikari et al. (2002) reported that there was no significant effect of the storage period on the concentration of butyric acid in set and stirred types of cow milk yogurt (p > .05). The SCT significantly affected the production of lactic acid, acetic acid, and citric acid during cold storage (Figure 5). The increases in the amounts of all organic acids presented in Figure 5 were found to be linear for X2 during 20 days of cold storage. However, for Y2, the amounts of lactic acid, acetic acid, and citric acid increased more rapidly in the first 10 days of cold storage. Then, the rate of increase slowed down gradually and further slight increases were observed throughout the storage period.
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The changes in concentrations of fatty acids in X2 and Y2 during cold storage are presented in Table 5. In both Y2 and X2 samples, the major fatty acids were myristic (C14:0), palmitic (C16:0), stearic (C18:0), and oleic (C18:1) acids. The ratio of total saturated fatty acids to total unsaturated fatty acids was calculated to be 58:42. Also, the effect of the SCT on the proportions of saturated and unsaturated fatty acids was not significantly important (p > .05). Yadav, Jain, and Sinha (2007) analyzed the control and probiotic dahi and reported the ratio of 65:35 and 55:45, respectively. The increase in unsaturated fatty acids of the buffalo milk yogurt samples might be mainly due to the increase in the amount of oleic acid (18:1) during fermentation. It was also easily noted that 20 days of cold storage did not significantly (p > .05) change the ratio of saturated fatty acids to unsaturated fatty acids. In general, the amounts of fatty acids in X2 and Y2 did not follow a regular trend during cold storage. Similar results were reported by Güler (2007), while Yadav et al. (2007) observed an increase in the amounts of fatty acids throughout the storage. Yadav et al. (2007) indicated that butyric acid was formed principally by lipolytic activity of lactic acid bacteria, and the lactobacilli having probiotic activity can produce conjugated linoleic acid (CLA) in milk during fermentation by lipolysis of natural milk fat. Alternatively, distinct changes in the butyric acid and linoleic acid contents of X2 and Y2 were not observed in this study, which can be attributed to the pH levels of the both Y2 and X2 samples. Kim and Liu (2002) also indicated that pH below 4.6 has more effect on CLA synthesis than SCT. Only, the decrease in linoleic acid content of X2 and Y2 samples was observed after 20 days of cold storage and it might be explained that the starter cultures used in this study utilized the linoleic acid as a substrate for CLA synthesis as stated by Yadav et al. (2007). Beshkova et al. (1998) stated that the hydrolytic activity of S. thermophilus toward milk fat is low, and the most important precursors of volatile fatty acids were amino acids. They also indicated that formation of volatile fatty acids (C2–C10) was more active in mixed cultures than that of in pure ones. However, SCT did not significantly affect the amounts of volatile fatty acids in this study (p > .05).
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The results of the presented study highlighted that the processing conditions such as the SCT and the IFpH altered the physicochemical and organoleptic properties of buffalo milk yogurt samples containing 3 g/100 g of milk fat. The IFpH mainly affected the activities of the strains in the mixed starter cultures. The probiotic cultures prolonged the fermentation time and decreased the reaction rate constant of postacidification during cold storage. Moreover, the extent of decrease in reaction rate constant varied depending on the IFpH. The absence of L. delbrueckii subsp. bulgaricus in the starter culture increased the viscosity for IFpH of 4.80. It also increased the syneresis for both IFpH 4.80 and 4.55. The IFpH did not affect the viscosity of T1 and T2, while it was vice versa for the other samples. Twenty days cold storage decreased the syneresis of the buffalo milk yogurt samples. L*, a*, and b* values of all the buffalo milk yogurt samples were not significantly affected by the IFpH and the SCT. The sensory results showed that a more acceptable buffalo milk yogurt was probiotic ABY‐2 culture added to yogurt samples. The organic acid and fatty acid profiles of the buffalo milk yogurt samples containing ABY‐2 culture were further analyzed and compared to the control yogurt containing YC‐X11 culture. The SCT significantly affected the production of lactic acid, acetic acid, and citric acid during cold storage. However, there was no significant effect of the SCT on the proportions of saturated and unsaturated fatty acids in the buffalo milk yogurt samples.
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As the only connecting term between water and energy balance, evapotranspiration is the best indicator for the response behavior of transport of water vapor and latent heat as well as hydrological course and the terrestrial ecosystems associated with climate change12. Along with significantly increasing near-surface air temperatures, decreasing pan evaporation (ETpan), potential evapotranspiration (ETp) and reference evapotranspiration (ET0) have been reported in many regions worldwide and continuously documented by many studies since the first publication by Peterson et al. in 19953. This phenomenon has been denoted as the “evaporation paradox”4. However, the interpretations for this phenomenon by different scholars are various in different regions, mainly including the following: (1) decreasing ETp or ET0 may actually be a strong indication of increasing terrestrial evaporation due to the complementary relationship between the actual evaporation and potential evaporation56789; (2) positive effect on evapotranspiration of increasing temperature could be offset by widespread “dimming” and “stilling” phenomena due to the fact that evaporative process is also primarily driven by radiative and aerodynamic components41011.
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Decreases in ETp, ETpan, and ET0 have also been reported to be occurring simultaneously in China or some regions of China with increasing trends of air temperature during the past decades1213141516171819. Reduced wind speed and shortened sunshine duration are generally considered to be responsible for these phenomena9202122. However, some of conclusions drawn from previous studies on evapotranspiration trends are based on the time series data ended around 2000. Whether the deceasing trends in potential evaporation are still true when looking at the longer record with updated data should also be addressed. In addition, most studies mainly focus on the general trends without the consideration of the abrupt change in the temporal processes. Overall trend estimations may leave out detailed information and thus make these revealed changing patterns deviate from the real situation. Recently, contrary to the famous “evaporation paradox”, an increase in ETpan in China along with continuously rising temperature during the recent two decades was identified by Liu et al.23 with more recent observations. This phenomenon was further regionally verified in the hyper-arid region of Northwest of China by Li et al.24. Moreover, Wang et al.25 suggested that there was actually a zigzag increasing-decreasing-increasing pattern of regional average ET0 series with two joint points in 1973 and 1993 in the Tibetan Plateau. It is therefore worthwhile to re-examine the ET0 patterns in larger area with different climatic and geomorphologic conditions based on the updated data. However, nationwide investigation on the changing patterns of ET0 and its related trigger mechanism is not available.
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The goal of this study is to examine whether the “evaporation paradox” is still true by investigating the response of ET0 to changing climatic environment based on recently updated nationwide observation in China. Specifically, we begin by identifying the homogenously sensitive regions of ET0 using a rotated empirical orthogonal function clustering method. Then we assess the spatial patterns of ET0 trends and analyze the abrupt change in average series for individual homogenous regions. Meanwhile, special efforts are further made in characterizing the underlying aerodynamic and radiative driving mechanism for the ET0 changes. Finally, the driving mechanisms under natural large atmosphere system are explored by identifying the correlation between changes in ET0 and Pacific Decadal Oscillation.
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To intensively characterize the spatial anomalies of ET0 change nationwide, it is necessary to make a reasonable partition to the whole country. Traditionally, the whole China is always divided into eight climatic regions according to the latitude and longitude, and the climate regions are roughly coincide with the socioeconomic division1323. However, there may be some uncertainty in sub-region selection based on visual inspection of the geographical distribution or administrative practices due to its vulnerability to subjective discretion9. This is especially true for the study of ET0 changing patterns due to the fact that ET0 involve in multifarious factors including geomorphologic features, topography factors and climate conditions. Therefore, the rotated empirical orthogonal function (REOF) method, a widely used clustering algorithm to objectively capture realistic spatial information, is employed in the current study to identify the homogenous regions regarding ET0 in the whole China. The first 11 EOFs together explain 80.8% of the variance and the first 8 EOFs together can explain 73.7% of the variance. The fact that more than 70% of the total variability is captured in the first 8 EOFs indicates that the complexity of spatial patterns of ET0 all over the country can mostly be explained by a small number of spatial structures. The percentages of variance of the new set of REOF modes generated by rotating the first eight loading vectors of the initial EOFs (Table S1) and correspondent isolines of the loading factor values (Figure S1) suggest that Mainland China can be categorized into eight homogenous regions (Fig. 1) by REOF analysis based on the annual ET0 series from 602 stations for 1961–2011. Eight leading modes deduced sub-regions can roughly be described respectively as following: East Center China (EC), Southeast China (SE), Southwest China (SW), Tibet Plateau (TP), North Center China (NC), Northwest China (NW), North China Plain (NP), and Northeast China (NE). In term of the regional average series of these eight homogenous regions, investigation on temporal patterns and their attributions across the whole China are presented in following sections.
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We conduct trends and change point analysis for the area-averaged ET0 in the resulted eight sub-regions by the means of the segmented regression model (shown in Fig. 2 and Table 1). The overall impression from the temporal patterns in ET0 series is that shift trends can be evidently found in all eight sub-regions, although the turning years and the number of change points are regionally different. The decreasing-increasing (DI) patterns (with one joint point) are detected in EC, SE and SW regions with joint points happening at about 1992, 1992 and 1995, respectively. The zigzag increasing-decreasing-increasing (IDI) patterns (with two joint points) are depicted in TP (1973 and 1993) and NW (1977 and 1995) regions. With three joint points, approximating to periodic variation, more complex zigzag increasing-decreasing-increasing-decreasing (IDID) patterns are identified in NC (1973, 1991 and 2000) and NE (1978, 1993 and 2003) regions. It should be noted that although only two points of 1992 and 2002 are found in NP region, the overall changing pattern of decreasing-increasing-decreasing for ET0 are similar to NC and NE regions, especially for the recent two decades. Interestingly, irrespective of complex shift trends reflected by different number of joint points and turning modes, multidecadal changing features in ET0 are characterized by an evident spatial clustering partition. This clustering partition can roughly indicates that DI patterns mainly occur in southern region in China with a humid climate, whereas IDI mostly emerge in western Plateau regions with a cold and drought climate and IDID mainly appear in north and northeast regions with a cold climate. We also investigate the trends for the 602 stations during the segmented periods according to the corresponding regional change points. Taking EC region for example, as shown in Fig. 3, annual ET0 at all stations decrease during 1961–1991 by 0–8 mm yr−1 (over 23% stations by 4–8 mm yr−1), of which over 80% stations are statistically significant at 95% confidence level (p < 0.05). As for as the second segmented period, from 1992–2011, more than 65% stations (over 30% stations by 4–12 mm yr−1) are dominated by increasing trends in ET0 despite that only 35% stations are statistically significant at 95% confidence level (p < 0.05). Such extremely distinct spatial patterns in ET0 during the adjacent segmented period are also identified in other homogeneous regions (shown in Figure S2–S8), giving us confidence in detecting multidecadal changes in ET0 with shift trends in China.
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The “evaporation paradox” is stated based on measured pan evaporation3, therefore, the correlation analyses of ET0 with ETpan (data collected from183 stations with the same records of ET0) is conducted before the contribution assessment of climatic factors. The close relationship between annual ETpan and ET0 with correlation coefficient (R) of more than 0.9 suggests that ETpan is a good indicator of ET0 across the whole China (Figure S9). We identify the driving factors of changing ET0 by quantifying the contribution of climatic factors to ET0 change using partial derivatives (see Equation 10). For the eight homogeneous regions, the maximum relative error ρ(δ) representing the ratio of error δ to observed trend is found in NC region during the fourth segmented period of 2000–2011 with the value of −14.78%. The minimum ρ(δ) is only 1.70%, which occurs in NP region during the first segmented period of 1961–1991 (Table S2). The calculated ET0 trends for all regions during all the segmented periods match very well with those detected from the computed ET0 with P-M method (always regarded as observed ones) with quite satisfactory R2 value of 0.99. The high accordance of calculated ET0 trends with estimated ones reflected by high R2 and low error indicates the differential equation method is applicable to quantify the contributions of climatic variables to changes in ET0. The contributions of climatic factors to the trends in ET0 in categorized eight sub-regions during different periods are shown in Fig. 4. Taking NC regions with most complex shift trends in ET0 as an example, during 1961–1972, the decreasing air temperature (T) causes a decrease of ET0 at the rate of 0.42 mm yr−1. Meanwhile, the increase of wind speed (U) and net radiation (Rn) as well as the decease of relative humidity (RH) lead to the increase of ET0 at the rate of 2.53 mm yr−1, 0.85 mm yr−1 and 1.18 mm yr−1, respectively. Consequently, U is the dominant factor for the change in ET0 in NC region during 1961–1972. Likewise, U, RH and Rn are responsible for the decrease of ET0 during 1973–1990, the increase of ET0 during 1991–1999 and the decrease of ET0 during 2000–2011 in NC region, respectively. Overall, although the change in ET0 of the whole country is characterized by complicated spatial and temporal variability in the dominating contribution factors, some interesting phenomena can be summarized as below. Before the early 1990s, the decreases of ET0 in EC, SE and SW regions, mostly located in South China, are mainly attributed to the decrease of Rn. While for the rest regions including TP, NC, NW, NP and NE, mainly distributed in western and northern part of China, the changes of ET0 before the early 1990s (all consist of increasing period before 1970s and subsequent decreasing period except NP region) mainly resulted from the changes of U, except the NE region during 1961–1977 with the dominating factor RH. From early 1990s to 2011, the ET0 in EC, SE, SW, TP and NW regions present monotonous increase. While for the NC, NP and NE regions, changes in ET0 are separated into two stages, i.e., increases from early 1990s to around 2000 and decreases after 2000. Although Rn, T and U play respectively the most import role in the decrease of ET0 at NC, NP and NE regions after around 2000, decreasing trends in RH is the most crucial factor for the increasing trends in ET0 detected after the early 1990s in all the eight sub-regions.
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In order to investigate the decadal characteristic of ET0 in China and seek for the possible driving mechanisms, the regional mean anomalies of annual ET0, precipitation (P) and the climatic water availability (CWD, defined as P minus ET0) as well as its relationship to the Pacific Decadal Oscillation index (PDO) are analyzed. Although as a whole, the regional ET0 record shows a significant decrease trend of 0.298 mm yr−2 (p < 0.1) during the past 50-years period, the evolving process of continental mean ET0 can be clearly divided into three stages. From 1961 to 1979, ET0 present a slight but not significant decline (0.008 mm yr−2) with a high mean value. A prolonged significant decline starts in the year of 1980 and ends in the year of 1995, being contrary to the previously reported upward actual evapotranspiration in this period262728. This may partly be explained by the complementary relationship29 between ET0 and actual evapotranspiration. After 1996, a pronounced recovery growth rate in ET0 can be clearly revealed. An overall oscillation in regional mean ET0 is found in China, which was not revealed by the previous studies, suggesting that the variation of ET0 from 1961 to 2011 should be a phenomenon of earth’s atmospheric circulation. PDO is considered as the important earth’s climatic driving power for the continental hydro-meteorology abnormality such as shift of dry/wet in Asia, especially in China30313233. The PDO driven climate oscillations impart clear influences to the regional ET0 in China as a whole, indicated by significant negative correlation between annual PDO index and annual mean ET0 (r = −0.384; p < 0.001). Years with high ET0 mostly coincide with PDO cold phase although ET0 responses to PDO may differ in different sub-regions in China. On the other hand, years with low ET0 correspond to PDO warm phase conditions (Fig. 5). The regional mean P in China present large temporal variability with a negative but not significant trend of 0.257 mm yr−2 (p = 0.564) during 1961–2011, while the CWD present a significantly no trend although the slope is slightly positive with the value of 0.041 mm yr−2 (p = 0.938), suggesting that there is a weak tendency to reduce the deficit between atmospheric moisture requirement and available water supply for evapotranspiration. Despite of the strong relationship between regional ET0 and PDO activities, the CWD is not significantly correlated with PDO (r = 0.164, p > 0.1) due to the weak relationship between P and PDO (r = 0.045, p > 0.1).
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The “evaporation paradox”, reported in many regions of the world including China15, has drawn a considerable attention to explore the reason of the decreases in ETpan and/or ET0 with increases in air temperature. However, with more recent observations or longer record, an increase in ETpan and/or ET0 since 1980s or 1990s has been found in China or some regions of China2324343536. Specially, a zigzag increasing-decreasing-increasing pattern with two joint points in 1973 and 1993 has been identified in our previous study on the changes in ET0 across the Tibetan Plateau25. However, the results from most previous studies on ET0 change and driving mechanism are only based on subjective sub-regions selection. Meanwhile, the natural large atmosphere system triggering mechanisms for ET0 change have not been investigated in previous studies. Consequently, a nationwide investigation in China is naturally performed in this study to detect the real multidecadal changing patterns of ET0 and the correlation with PDO with extended data from 1961 to 2011. The temporal patterns of ET0 in eight homogenous regions identified by REOF evidently reveal that there is no monotonous decreasing trend in ET0 along with the continuously increasing temperature for the entire period (from 1961 to 2011), suggesting “paradox” phenomenon just exist in a certain stage of the multidecadal changes in ET0 in China. With one turning point, the finding that decreasing trends of ET0 reverse into increasing trends in early 1990s across EC, SE and SW regions, is in agreement with region studies on ETpan/ET0 by Liu and Zhang (2012)37 and Li et al.24. When other sub-regions are considered, coarse periodic behaviors of ET0 change with two/or three turning point seems to be reasonable as compared with total decreasing-increasing patterns of evaporation in entire China reported by Cong et al.38 and Liu et al.23. The combined effects of climatic variables to ET0 changes are revealed in all the sub-regions with the sensitive analysis, and are effective with a good agreement between the observed and calculated ET0 trends (see Table S2). Although relative humidity is the most sensitive variable (followed by net radiation, wind speed and air temperature) for all the period as well as all the sub-regions except SW and TP region, there are different dominating factors for ET0 change because the contributions of climate factors depend on the combined effect between sensitivity and trends of a variable itself9. Decline of net radiation only plays the important role for the ET0 decrease before early 1990s rather than the entire term in EC, SE and SW regions located in south China, highlighting the potential implications of “global dimming” in China over a roughly 1960–1990 period and a recovery (coined with “global brightening”) thereafter3940, which is consistent with previous studies in China1320233841, the United States3, the northeast of India42, and Greece43. There is consensus among many researchers that cloud coverage and aerosol, which are not completely independent variables with interaction in various ways44, are considered as the most likely candidates for the explanation of global dimming and brightening3945464748. Particularly strong evidence for aerosol effects on surface solar radiation with increasing air pollution in China were noted in various studies49505152. As far as the other regions with larger inter-annual variability in ET0 are concerned, variation of wind speed generally plays more decisive role in the change of ET0 from 1961 to early 1990s, in line with the evidences reported in many places and summarized by McVicar et al.11. Although the dynamic mechanism of recent slow-down in near-surface global winds is complicated, it can generally be attributed to the increases of terrestrial surface roughness5354 or large scale atmospheric circulations55565758. Focusing on the periods after 1990s, our preliminary analyses have shown that dominating factors for the increases of ET0 transited from net radiation and wind speed to relative humidity for all the sub-regions. This coincides with the recent regional study in the arid regions of northwest china by Li et al.24 and national wide study in China by Cong et al.38. A global evidence of reduction in surface atmospheric humidity revealed in this study in national scale was presented by Simmons et al.59. They inferred the recent reduction in relative humidity over land may be due to limited moisture supply from the oceans. For the purpose of simplification, the systemic errors of contribution analysis reflected by the differences between observed ET0 and complied trends may be due to the interactions among climatic factors (which were not considered here but may exist in practice) and ignorance of other factors such as aerosol and dust.
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From global perspective, there is an indication for a shift that occurred in the late 1970s with minor change occurred around 1990 and 2000 for PDO6061. Having the strong correlation with PDO, the decadal variations of ET0 in China with two turning points during 1961–2011 are likely dominated by the episodic dynamics of climatic system, indicating that the recent ET0 trends in China reflect PDO conditions and are not the consequence of a persistent reorganization of the hydro-meteorological cycle. The similar conclusions have also been obtained from the global analysis for continental evaporation2728. Consequently, the widely reported “evaporation paradox”, a counterintuitive behavior, representing that evapotranspiration decrease despite the increase of air temperature, seem to be more reasonably explained as part of a climate oscillation, at least in China33. The emerging picture of enhanced of ET0 in southern China highlights the possible threat posed by acceleration of the terrestrial hydrological cycle to water resources management and food security in an enhanced-greenhouse-affected climate.
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| 99.94 |
Daily measurements of air temperatures (minimum, maximum and average) at 2 m height, relative humidity, wind speed at 10 m height, and sunshine duration obtained from the National Climatic Centre (NCC) for 602 ground-based stations in China during the period 1961–2011, provided by the National Meteorological Information Centre of China (NMIC) of the China Meteorological Administration (CMA), are used for estimating ET0 by Penman-Montieth method. Wherein, wind speed was adjusted to 2 m height by virtue of wind profile relationship proposed by Allen et al.62. Net radiation at 51 stations was available. Moreover, precipitation data from these meteorological stations were also collected.
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| 75.94 |
From the reference surface defined as “a hypothetical reference grass with an assumed crop height of 0.12 m, a fixed surface resistance of 70s m−1 and an albedo of 0.23”, the daily reference evapotranspiration (ET0) is estimated by the Penman-Montieth (P-M) combination method, which is recommended by the FAO as a standard to calculate ET0 wherever the required input data are available and is proven to have good performance for various climatic conditions worldwide9. The P-M equation can be expressed as (Allen et al.)62:
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| 99.3 |
where ET0 is the reference evapotranspiration (mmd−1), Rn is the net radiation at the crop surface (MJ m−2 d−1), G is the soil heat flux density (MJ m−2 d−1), T is the mean daily air temperature (°C), u2 is the daily average wind speed at 2 m above ground level (ms−1), es is the saturation vapor pressure (kPa), ea is the actual vapor pressure (kPa), es − ea is the saturation vapor pressure deficit (kPa), Δ is the slope of the saturated vapor pressure in relation to air temperature (kPa °C −1) and γ is the psychrometric constant (kPa °C −1).
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| 99.9 |
where Rs is the solar or shortwave radiation (MJ m−2 d−1), n is the actual duration of sunshine (h), N is the maximum possible duration of sunshine or daylight hours (h), ( is thus the relative sunshine duration), Ra is the extraterrestrial radiation (MJ m−2 d−1), as and bs are the Angstrom coefficients. We calibrate the Angstrom coefficient using the observation of n and Rs from 51 stations widespread the whole China. Meanwhile, Ra and N are computed based on date and latitude according to the equations provided by the FAO P-M method. Overall, the high R2 values (see Table S3) between observed and estimated Rs indicate that the Angstrom model is suitable for daily global radiation estimation in China. For stations with no observation of solar radiation but sunshine duration, as and bS are estimated by Kringing interpolation method.
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The soil heat flux, G, is the energy that is utilized in heating the soil. G is positive when the soil is warming and negative when the soil is cooling. The soil heat flux is small compared to Rn and may often be ignored in daily evapotranspiration estimating.
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| 99.9 |
A simple linear regression method was used to calculate the slope of linear least squares regression line fit to the inter-annual variation of ET0 and other climatic variables inputted in the P-M equation. The significance of changes of these variables across China was mapped and assessed with the help of the two-tailed significance tests. The segmented regression with constraints method developed by Shao and Campbell63, which can detect both shift trends and step changes simultaneously without knowing the number of trend segments and change points and their location over time64, is employed to investigate the possible turning points of the regional average ET0 series across China. The method was summarily described as follow636465:
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| 100.0 |
with Jl, 0 = r−1, where (t−Jl, k)+ = u+ is defined as u+ = u if u ≥ 0. A modified Akaike’s information criterion (AICc), derived by Hurvich and Tasi66 with c standing for the second order correction67, is employed to select an optimal numbers of break and join points and their locations, and the optimal model can be obtained by minimizing AICc.
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| 99.25 |
To characterize in detail the spatial variability of ET0 at nationwide scale and identify the homogenous regions, we applied REOF to analyze the most dominant spatial patterns. The aim of the EOF method is to find a relatively small number of independent variables conveying as much of the original information as possible without redundancy by decomposing a multivariate data set into uncorrelated linear combination of separate function of the original variables. However, the physical interpretability of the obtained patterns is sometimes a matter of controversy because of orthogonality in both space and time68. This limitation has brought about the development of the rotated empirical orthogonal function (REOF)69, which can cluster within each mode a small number of high valued variables and a large number of near-zero value variables through a rotation to simple structure. In a comparison study by Kim and Wu70, REOF was found to be better in dividing climatic patterns. In this paper, with maximizing the variance of the squared correlation between each rotated principal component (RPCs) and each variable, the varimax REOF method was chosen to give the simplest pattern description while explaining the maximum amount of variance.
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| 100.0 |
Assuming that y and xi are the time series variables, and should be the long-term trend for y and xi, respectively. The term of , the product of long-term change in xi and the partial derivative, can then be considered as the contribution of change in xi to the long-term variation of y. Thus, following P-M equation for ET0 estimation, contributions of changes in key climatic factors to ET0 variation can be approximately as55:
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| 76.9 |
For such a multi-variable model of P-M equation, it is difficult to compare the sensitivity by partial derivatives due to the different dimensions and ranges of values for related variables. Hence, the partial derivative is transformed into a non-dimensional form in term of McCuen71 and Beven72 to express the sensitivity of the climatic variables mathematically:
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other
| 99.5 |
where S(xi) is the sensitivity coefficient of ET0 related to xi, the i th variable. Being first employed by McCuen71, the sensitivity coefficient has been widely used in evaluating the climatic response of evapotranspiration, especially during recent two decades7374757677.
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| 99.9 |
Sodium iodide symporter is a transmembrane glycoprotein (codified by the SLC5A5 gene) expressed almost exclusively in the basolateral plasma membrane of thyroid follicular cells. It plays a central role in thyroid metabolism, mediating the active transport of iodine from the bloodstream into the follicular cells, the first step for thyroid hormones’ synthesis. NIS plays an essential role in the treatment of differentiated thyroid carcinomas (DTC), which usually maintain NIS expression, allowing the recognition and the treatment of recurrences and metastases with radioactive iodine (RAI) (1). Nonetheless, a significant subgroup of DTC patients with advanced disease lose NIS expression and become refractory to 131I; some of these patients die within 3–5 years (2). Furthermore, a study performed by Yildririm-Poyraz and coworkers (3) demonstrated that NIS expression in nontumoral thyroid tissues associates with higher rates of delayed structural response. NIS expression has been widely studied in normal thyroid and tumor tissues, on one hand to verify if its downregulation could be the molecular cause for the decrease of RAI uptake and on the other hand to understand the impairing mechanisms of NIS expression and function. However, no clear answer emerged from the results obtained in the previous studies. Despite the central role of NIS in diagnosis, treatment and follow-up of thyroid cancer patients, reliable methods for ascertaining NIS expression and functionality in clinical samples are not available.
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review
| 97.4 |
In the majority of the studies, SLC5A5 mRNA levels are lower in thyroid carcinomas than in adenomas (4) and normal adjacent thyroid (5, 6, 7); furthermore, SLC5A5 expression presents some limitations in predicting NIS expression and functionality: whereas a negative or low mRNA level may lead to reduced protein expression, a positive or high mRNA expression does not always correspond to higher protein levels or higher functionality (7, 8).
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| 98.9 |
These observations suggest that in thyroid carcinomas, besides transcription regulation, NIS expression appears to be modulated by post-transcriptional events. Therefore, studies of NIS expression by immunohistochemistry (IHC) (1, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25), may be, theoretically, more informative since they ‘grab’ NIS a step forward in its biological processing and allow the evaluation of the localization of NIS in the basolateral plasma membrane of follicular cells (the functional transporter).
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study
| 99.94 |
According to the published data, NIS expression (evaluated by IHC) varies in different thyroid tissues. In normal thyroid, it is low and very heterogeneous; only a few follicular cells within some follicles express NIS in the basolateral plasma membrane (10, 14, 17, 21, 26), suggesting that, NIS expression is tightly regulated in thyroid gland. In carcinomas, when NIS is present, it is usually expressed in a higher number of cells than in normal tissue and the expression is mainly intracytoplasmic, poorly targeted to the basolateral plasma membrane (1, 11, 12, 13, 14, 17, 21, 22, 23). The increased intracytoplasmic NIS staining in thyroid tumors compared to normal tissue has been pointed out as a reason for the decreased RAI uptake in tumors, reflecting a mislocalization of NIS from the basolateral membrane, which would impair its activity (17). This assumption has been questioned, because the real significance of intracytoplasmic NIS detected by immunostaining remains unclarified (21).
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study
| 99.94 |
The molecular mechanisms responsible for the downregulation and/or not targeting to the basolateral membrane of NIS in thyroid tumors remain poorly understood, but some studies demonstrated that both mRNA and protein are differentially expressed according to the genetic background of the tumor. In fact, papillary thyroid carcinomas (PTCs) harboring the BRAFV600E mutation present lower SLC5A5 mRNA and NIS protein expression as well as less targeting to the basolateral membrane compared to PTCs BRAFWT (19, 24, 27). Nonetheless, a recent study reported different association between NIS membrane expression and BRAFV600E mutation in a series of 96 cPTCs, demonstrating that the ones harboring BRAFV600E mutation expressed more often NIS in the cell membrane of tumor cells compared to BRAFWT cPTCs (28). Less is known about the impact of mutations in other genes (i.e. RAS and TERTp) on SLC5A5 and NIS expression/targeting to the basolateral membrane.
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study
| 99.94 |
NIS being the central molecule for DTC treatment, it is logical to study if its expression in the primary tumor would be helpful in predicting therapy response as well as tumor behavior and prognosis. Some studies tried to understand if NIS immunohistochemical expression in thyroid primary tumors would be helpful in predicting 131I uptake in recurrences and distant metastases. Although positive NIS immunostaining in primary tumors seemed to be predictive of positive recurrences and metastases on 131I scans, other studies did not distinguish whether NIS was expressed in the cell basolateral membrane, and negative NIS staining did not predict 131I scan-negative metastases (13, 15, 18). Even when only NIS membrane staining was considered, a negative NIS staining in the primary tumor was still not predictive of a negative 131scan of subsequent recurrences (29). To the best of our knowledge, there is only one study that addressed possible associations between NIS expression, evaluated by immunohistochemistry (IHC), and clinicopathological features and prognosis in a large series of thyroid primary tumors (1), reporting a significantly lower NIS expression in older patients (≥45 years) and also that NIS expression in the primary tumor was not useful as a prognostic marker.
|
study
| 99.25 |
So, in our opinion, more retrospective studies in larger series of primary tumors are still necessary, to understand the role of NIS expression in therapy response, tumor behavior and prognosis, and also if other factors besides BRAFV600E mutation can contribute to NIS downregulation and/or misdirecting to the basolateral membrane. Furthermore, it is also important to understand the advantages and limitations of the analysis of SLC5A5 and NIS expression and evaluate what is the better/more informative method to study NIS expression.
|
study
| 75.75 |
Having this in mind, we addressed SLC5A5 expression by qPCR and NIS expression by IHC analysis, in a large series of primary thyroid carcinomas and looked for possible associations with some clinicopathological and molecular features, as well as to the response to RAI therapy and outcome. In order to validate our results of SLC5A5 mRNA expression associations’ with clinicopathological and molecular features and also to get new evidences, we used the data available about SLC5A5 in TCGA Research Network that completed an integrated genomic analysis of 496 PTCs using NGS and other pan-genomic technologies, together with detailed pathologic and clinical data (30).
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study
| 100.0 |
Our series was composed by 255 thyroid samples from 229 patients. Cases were collected from the files of the Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP, Porto, Portugal), corresponding to patients with thyroid tumors (n = 229) operated and followed in two university hospitals. Samples from normal thyroid (n = 25) and Graves’ disease (n = 1) were obtained from the contralateral lobe of the surgical specimens. Carcinomas series was composed by 193 PTCs (123 cases of classical PTC (cPTC), 47 cases of follicular variant of PTC (fvPTC) and 23 cases of other PTC variants), 23 follicular thyroid carcinomas (FTC) and 13 poorly differentiated thyroid carcinomas (PDTC). In 166 of the cases, there was only formalin-fixed paraffin-embedded (FFPE) representative tissue; in 45 cases, there were FFPE samples and correspondent frozen tissue (the tumors were divided at the time of surgery) and in 18 cases, there was only frozen tissue available. Frozen material was collected at the time of surgery and conserved at −80°C. The histology of all tumor samples was reviewed by three pathologists (CE, ER, MSS) according to the criteria of the World Health Organization (31). Clinicopathological and molecular data of the 229 patients with carcinoma are summarized in Supplementary Table 1 (see section on supplementary data given at the end of this article). In 141 cases, follow-up data were available. The number of 131I treatments varied from 1 to 5 treatments (mean 1.9), and the cumulative total dose of RAI was between 30 and 1146 mCi (mean 251 mCi). This work was approved by the Ethic Committee for Health (CES) of the Hospital Center of São João (CHSJ)/Faculty of Medicine of the University of Porto (FMUP) (CES 137 284–13) and by the Ethic Committee of the Faculty of Medicine of the University of Coimbra (n° 1309). All the procedures described in this study were in accordance with national ethical standards (Law n° 12/2005) and Helsinki declaration. Patients signed an informed consent form.
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study
| 99.94 |
Patients were treated and followed in accordance with the international protocols available at the time. Data regarding the number of radioiodine treatments and cumulative activity were retrieved from hospital records. Patients were considered as being disease-free at the end of follow-up if they had undetectable stimulated thyroglobulin (in the absence of thyroglobulin antibodies), and no evidence of the disease on radiographic or radionuclide imaging. Follow-up time (in years) varied from 0.3 up to 38.9 years, with a mean value of 8.0 ± 6.6 years. For statistical analysis, we defined the category ‘additional treatments’, in which we included other treatment modalities in addition to radioiodine, including extra surgery, external beam irradiation and treatment with tyrosine kinase inhibitors.
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study
| 99.94 |
There were 378 tumor cases for which there was information for the main driver somatic mutations (TERTp, BRAF and RAS), gender and SLC5A5 expression. Of these, we eliminated 4 cases, for which the SLC5A5 expression was above the 99 percentile, being outliers. A total of 353 of the cases had information about tumor size, 362 had information for extrathyroidal extension, 282 had information for lymph node metastases (at the time of diagnosis) and all 374 had information about new tumor event (lymph node metastases or local recurrence (grouped in locoregional recurrence) and distant metastases). The SLC5A5 expression was inferred from RNA-seq data and quantification reflects reads per kilobase per million mapped reads (RPKM).
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study
| 100.0 |
DNA extraction from FFPE tissues was performed from 10 μm sections after careful microdissection. DNA extraction was performed using Ultraprep tissue DNA kit (AHN Biotechnologie, Nordhausen, Germany) following the manufacturer’s instructions. The genetic characterization of part of the tumors regarding BRAF, NRAS and TERT promoter mutations (TERTp) had been reported previously; mutations were screened as previously described (32, 33, 34).
|
study
| 99.94 |
Total RNA was extracted from tumors and from contralateral normal adjacent thyroid, from which frozen samples were available (n = 84), using a TRIzol commercial kit (Thermo Scientific/GIBCO) according to the manufacturer’s protocol. RNA was quantified by spectrophotometry, and its quality was checked by analysis of 260/280 nm and 260/230 nm ratios. For cDNA preparation, 1 μg of total RNA was reverse-transcribed using the RevertAid first-strand cDNA synthesis kit (Thermo Scientific/Fermentas).
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study
| 99.94 |
Immunohistochemistry was performed in normal thyroid and in 211 carcinomas. Briefly, deparaffinized and rehydrated sections were subjected to heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 3% of hydrogen peroxide and nonspecific binding with Large Volume Ultra V Block reagent (Thermo Scientific/Lab Vision). Sections were then incubated overnight at 4°C with anti-NIS antibody (1:400) clone FP5A (Thermo Scientific/Lab Vision) and in 24 carcinomas with anti-NIS pAb 795 IgG (20 µg/mL) (kindly supplied by Dr Ruby) (36). Additionally, Tyramide Signal Amplification (TSA) Biotin System (Perkin-Elmer) was used for signal amplification in 44 carcinomas, according to manufacturer’s instructions. The detection was performed with a labeled, streptavidin–biotin immunoperoxidase detection system (Thermo Scientific/Lab Vision) followed by 3,3′-diaminobenzidine (Dako) and counterstained with hematoxylin. Graves’ disease sample was used as a positive control and the negative control consisted in omission of the primary antibody.
|
study
| 100.0 |
Slides were evaluated by two observers and were analyzed according to the percentage of tumor-stained cells, the intensity and the cellular localization of the staining. In order to compare our results to the literature, we considered cases with >5% of stained tumor cells (regardless of the cellular localization) as positive. Nevertheless, all our statistical analyses were performed considering two groups; cases that presented membrane staining in tumor cells and all the other cases. Photographs were acquired using Nikon DS-L1 camera in 100× and 400× magnifications.
|
study
| 99.94 |
Statistical analysis was performed using 21.0 SPSS Statistical Package (SPSS, 2003). Fisher’s exact test and independent-samples t-test were performed to correlate NIS and SLC5A5 mRNA expression with clinicopathological and molecular features. When parametric tests were not applicable, we used alternative tests, specifically Mann–Whitney (independent samples). Wilcoxon (related samples) was used to compare SLC5A5 expression between tumor samples and their adjacent normal counterparts. Kruskal–Wallis test was used to correlate SLC5A5 expression (retrieved from TCGA and database) with clinicopathological and molecular features. Values of P ˂ 0.05 were considered statistically significant.
|
study
| 100.0 |
SLC5A5 expression was significantly lower in carcinomas than that in normal adjacent counterparts (Fig. 1). No significant difference was observed between the three different carcinoma histotypes (PTC, FTC and PDTC). Considering the analysis in DTC, SLC5A5 expression was significantly lower in males and in cases with vascular invasion (P = 0.003 and P = 0.03, respectively) (Table 1). SLC5A5 expression in normal thyroid from males was not significantly different from that of females (data not shown). In addition, there was a tendency to lower SLC5A5 levels in cases with extrathyroidal extension (P = 0.06) and in PTCs harboring BRAFV600E mutation (P = 0.07). When the statistical analysis was performed only in the PTC group, all the significant associations described in the DTC group were maintained. Figure 1SLC5A5 mRNA expression in thyroid carcinomas and paired normal adjacent counterparts. Table 1Associations between SLC5A5 mRNA expression with clinicopathological and molecular features in DTCs.SLC5A5 expressionP valueGenderF (n = 47)1.2 ± 2.2M (n = 12)0.2 ± 0.20.003Age<45 years (n = 30)1.0 ± 1.5≥45 years (n = 29)1.1 ± 2.40.8Tumor capsulePresent (n = 27)1.1 ± 1.6Absent (n = 30)0.7 ± 1.60.4Tumor capsule invasionYes (n = 17)0.9 ± 1.6No (n = 11)1.4 ± 1.40.4Extrathyroidal extensionYes (n = 17)0.5 ± 1.1No (n = 37)1.4 ± 2.40.06Lymphocytic infiltrationPresent (n = 19)0.9 ± 1.9Absent (n = 37)1.2 ± 2.20.7Vascular invasionPresent (n = 28)0.4 ± 0.8Absent (n = 29)1.5 ± 2.60.03Lymph node metastasesPresent (n = 13)0.5 ± 0.8Absent (n = 18)0.4 ± 0.70.8BRAF*WT n = (27)1.6 ± 2.7V600E (n = 20)0.5 ± 1.00.07NRASWT (n = 54)1.0 ± 2.0Mut (n = 6)1.3 ± 1.70.7*PTC only.
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study
| 100.0 |
The SLC5A5 expression was around 200 times higher in normal tissue than in tumor tissue in both genders, but no differences in tumor and in adjacent tissue between genders were found (Fig. 2A and B). SLC5A5 expression was significantly higher in smaller tumors ≤2 cm (median = 5.85) compared to those with >2 cm (median = 2.51) (P = 0.028; Fig. 2C). There was no statistical difference in SLC5A5 expression in primary tumors with (median = 3.0) or without (median = 5.4) lymph node metastases at the time of diagnosis (P = 0.253) (Fig. 2D). Figure 2SLC5A5 expression in primary PTCs (RPKM), data retrieved from TCGA database. Comparative analysis of SLC5A5 expression. (A) Between genders in tumor (TT) and normal tissue (NT); (B) between genders only in tumor tissue (TT); (C) in tumors with ≤2 cm and >2 cm; (D) in cases with or without lymph node metastases at the time of diagnosis; (E) in cases without, with minimal (T3) and with moderate/advanced extrathyroidal extension (T4a+b); (F) in cases with and without recurrence and (G) between cases with different genetic backgrounds (WT, RAS mutation, TERTp mutation, BRAF mutation, BRAF + TERTp mutation). The boxes represent the interquartile range; the whiskers are the 5% and 95% quartiles; the small open boxes are the mean values and the lines are the median values. Significant values for the Kruskal–Wallis test are indicated.
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study
| 100.0 |
SLC5A5 expression in primary PTCs (RPKM), data retrieved from TCGA database. Comparative analysis of SLC5A5 expression. (A) Between genders in tumor (TT) and normal tissue (NT); (B) between genders only in tumor tissue (TT); (C) in tumors with ≤2 cm and >2 cm; (D) in cases with or without lymph node metastases at the time of diagnosis; (E) in cases without, with minimal (T3) and with moderate/advanced extrathyroidal extension (T4a+b); (F) in cases with and without recurrence and (G) between cases with different genetic backgrounds (WT, RAS mutation, TERTp mutation, BRAF mutation, BRAF + TERTp mutation). The boxes represent the interquartile range; the whiskers are the 5% and 95% quartiles; the small open boxes are the mean values and the lines are the median values. Significant values for the Kruskal–Wallis test are indicated.
|
study
| 100.0 |
The SLC5A5 expression was reduced with the level of the extrathyroidal extension (median values: 5.4 for ‘none’; 2.8 for ‘minimal (T3)’ and 0.9 for ‘moderate/advanced (T4a + b)’), reaching statistical significance for comparisons between ‘none’ vs the ‘moderate/advanced (T4a + b)’ class and ‘minimal (T3)’ vs ‘moderate/advanced (T4a + b)’ (P = 0.018 and P = 0.039, respectively Fig. 2E). We also observed a statistical significant decrease (from a median of 3.8 to 0.8; P = 0.002) of the SLC5A5 expression in cases with new tumor events (Fig. 2F), lumping together 12 cases of distant metastasis (6 lung; 1 lung + bone; 1 lymph node only; 1 lung + femur + neck + pleura + liver; 1 bone; 2 unknown) and 14 locoregional recurrences (10 lymph node only; 2 left thyroid; 1 lymph node + soft tissue; 1 unknown). Finally, SLC5A5 expression was significantly higher in the absence (median = 21.77) of the evaluated mutations: RAS (P = 0.034), TERTp (P = 0.0072) and BRAFV600E (P = 3.1 × 10−8). The PTCs that harbored only TERTp, only BRAF or simultaneous TERTp and BRAF mutations displayed significantly lower expression of SLC5A5 than the WT tumors. The group with RAS mutations displayed the second highest expression value (median = 7.50), reaching statistical significance when compared with the groups including BRAF mutation only and BRAF + TERTp mutations (median = 2.27 in BRAF (P = 0.042); median = 1.89 in TERT+BRAF (P = 0.027)) (Fig. 2G).
|
study
| 100.0 |
In normal thyroid tissues, NIS immunohistochemical expression was mainly localized in the basolateral plasma membrane of follicular cells. NIS positivity was detected in a few foci of isolated follicles throughout the tissue and within the positive follicles most of the cells were positive. Positivity was more frequently detected in small follicles composed by cuboidal and columnar cells and rarely detected in large follicles limited by flattened cells (Fig. 3A). In Graves’ disease, NIS was widely expressed and present in the basolateral plasma membrane of the great majority of follicular cells (Fig. 3B). In carcinomas, NIS staining was observed in 71.6% of the cases (74.8% of cPTCs, 69.8% of fvPTCs, 80.9% of other PTC variants, 55% of FTC and 67% of PDTC). Its location was predominantly in the cytoplasm (124/211) (Fig. 3C) and nucleus (15/211) and only 12/211 of the cases presented NIS in the basolateral plasma membrane of tumor cells (Fig. 3D). Figure 3NIS immunoexpression in different thyroid tissues. (A) Normal thyroid; (B) Graves’ disease; (C) cytoplasmatic staining in an oncocytic PTC; (D) membrane staining in a fvPTC; (E and F) NIS immunoexpression in a fvPTC without and with TSA amplification signal, respectively; (G and H) NIS immunoexpression in a FTC without and with TSA amplification signal, respectively; (I) negative staining in a cPTC and strong membrane staining in the surrounding Graves’ disease. In F and H, notice the loss of cytoplasmatic staining after the use of TSA amplification system. Bar 100 μm.
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study
| 100.0 |
NIS immunoexpression in different thyroid tissues. (A) Normal thyroid; (B) Graves’ disease; (C) cytoplasmatic staining in an oncocytic PTC; (D) membrane staining in a fvPTC; (E and F) NIS immunoexpression in a fvPTC without and with TSA amplification signal, respectively; (G and H) NIS immunoexpression in a FTC without and with TSA amplification signal, respectively; (I) negative staining in a cPTC and strong membrane staining in the surrounding Graves’ disease. In F and H, notice the loss of cytoplasmatic staining after the use of TSA amplification system. Bar 100 μm.
|
study
| 99.9 |
Since we observed a low percentage of carcinomas with NIS staining in the basolateral membrane, we hypothesized that our IHC approach was not being sensitive enough to detect small amounts of NIS. To clarify this issue, we used two strategies: a TSA signal amplification method and the use of another NIS antibody characterized by a different specificity compared to the commercial antibody (36).
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study
| 100.0 |
The TSA signal amplification method was applied in a subset of 44 carcinomas with different staining patterns (16 with cytoplasmic staining in the tumor and membrane staining in adjacent thyroid; 3 with membrane staining in the tumor; 5 negative both in the in tumor and the adjacent thyroid and, finally, 20 with only cytoplasmic staining in tumor and adjacent thyroid). When we compared the slides performed with and without TSA signal amplification, we verified that only the membrane staining remained and appeared more intense with the amplification method. In these cases, the staining involved almost always the same foci of cells that already presented membrane staining (Fig. 3E, F, G and H) i.e. it did not stain additional cells. The intra-cytoplasmic staining vanished both in cancer and in normal tissues. Furthermore, we performed IHC using a homemade antibody for human NIS, pAb 795 against a peptide corresponding to the C-terminal sequence of hNIS pAb 795 (36) in 24 carcinomas (12 cPTC, 4 fvPTC, 2 micro PTC, 2 tall cell PTC, 2 FTC and 2 PDTC). The results were similar to those obtained with clone FP5A (Thermo Scientific/Lab Vision).
|
study
| 100.0 |
Since some doubts remained about the specificity of the cytoplasmic staining, and because NIS is only active when present in the basolateral membrane of the cells, we performed statistical analysis dividing our series in two groups: with and without membrane staining.
|
study
| 99.94 |
We did not find any significant association between NIS expression in the membrane and age, tumor size, tumor capsule, multifocality, lymphocytic infiltration, vascular invasion, lymph node metastases, tumor margins, distant metastases, staging, BRAF, NRAS and TERTp status, additional treatments, disease-free status at one year, disease-free status at the end of follow-up or disease-specific survival in the DTC group. When we analyzed NIS expression between WT PTCs and those harboring any of the studied mutations, we verified that NIS-positive expression was significantly more frequent in WT PTCs (P = 0.01) (Table 2). The number of RAI therapies, as well as the cumulative dose of RAI, did not differ significantly between patients with or without NIS expression in the basolateral membrane of primary tumor’s cells. Table 2Associations between NIS expression and clinicopathological and molecular features in PTCs.NIS immunoexpressionP valueNegativePositiveGenetic background n = 118 WT45 (41.3%)8 (88.9%) Mutated#64 (58.7%)1 (11.1%)0.011#BRAF, NRAS or TERTp mutations.
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study
| 100.0 |
The thorough analysis of the few cases with membrane staining (n = 12) revealed that all but one carcinoma were wild type for the studied mutations (NRAS, BRAF or TERTp). These cases presented variable outcomes i.e. presence of distant metastases, number of RAI therapies, cumulative dose of RAI, the need of additional treatments, disease-free status and death (disease caused), that are apparently unrelated with the presence of NIS membrane expression (Table 3). Table 3Clinicopathological and molecular data of cases presenting NIS membrane staining.DiagnosisBRAFNRASTERTpLymph node metastasesDistant metastasesNumber of 131I therapiesCumulative dose (mCi)Aditional treatmentsOne year DFS*DFS*,#DeathsCase 1cPTCWTWTWTNoBone3457.5NoNoNoNoCase 2cPTCWTWTWTNoNo163NoYesYesNoCase 3cPTCWTWTWTYesNo34592 surgeriesNoNoNoCase 4fvPTCWTWTWTYesNo137NoYesYesNoCase 5fvPTCWTWTWTNoNo2382NoNoNoNoCase 6PDTCWTWTWTNoLung + bone57982 surgeriesYesNoNoCase 7cPTCWTWT124G>AYesNo4527U/INoNoNoCase 8sclPTCWTWTWTYesNo3400U/INoNoNoCase 9FTCWTWTWTNoNo1102U/IU/IYesNoCase 10cPTCWTWTWTYesU/IU/IU/IU/IU/IU/IU/ICase 11fvPTCWTWTWTU/IU/IU/IU/IU/IU/IU/IU/ICase 12cPTCWTWTWTYesU/IU/IU/IU/IU/IU/IU/I*DFS disease-free survival; #at the end of follow-up.sclPTC, sclerosing variant of PTC; U/I, unavailable information.
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study
| 100.0 |
In this work, we tried to clarify the impact of NIS expression (mRNA and protein) on thyroid tumors’ aggressiveness and therapy success and, as a result of the above, the putative prognostic significance of SLC5A5 mRNA and NIS protein expression. Moreover, we also addressed the impact of the genetic background of the tumor on SLC5A5 and NIS expression as well as its targeting to the basolateral cell membrane.
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study
| 100.0 |
We found that SLC5A5 expression was always lower in tumors than in normal adjacent counterparts as reported by other groups (6, 7, 37). We observed a significantly lower SLC5A5 expression in male gender patients, and in cases with vascular invasion, as well as a tendency to lower SLC5A5 expression in cases with extrathyroidal extension, but no differences were found in cases with and without lymph node metastases (Table 1). When we compare the results from our series to those from TCGA data, we confirmed that tumors express significantly less SLC5A5 compared to normal adjacent tissue, that SLC5A5 was not differently expressed in the presence or absence of lymph node metastases (at the time of diagnosis) and a significant lower SLC5A5 expression was found in tumors with extrathyroidal extension (moderate/advanced) compared to those without extrathyroidal extension (Fig. 2D). However, the differential expression of SLC5A5 between genders was not confirmed (Fig. 2B). Unfortunately, in TCGA database, there was no information about vascular invasion, so we could not validate this result in this large series.
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study
| 100.0 |
The significantly lower SLC5A5 expression in cases presenting vascular invasion and extrathyroidal extension suggests that a decreased SLC5A5 expression may be associated to an aggressive tumor behavior and thus may help to characterize patients at risk for poor therapy response. Further analysis of TCGA data demonstrated that SLC5A5 expression is significantly lower in cases that had locoregional recurrences and/or distant metastases (Fig. 2E). Given the high prognostic impact of recurrences and distant metastases (38), these results suggest that a lower expression of SLC5A5 in thyroid primary tumor seems to be associated with features of higher aggressiveness of the primary tumor and also with a worse prognosis and with poor response to therapy. Two groups reported that SLC5A5 was significantly less expressed in DTCs larger than 2 cm and PTCs larger than 1 cm (in comparison to ≤2 cm and <1 cm, respectively) (1, 39), TCGA results corroborated the literature by showing that larger PTCs (>2 cm) expressed significantly less SLC5A5 compared to those with ≤2 cm. Since larger tumors are associated with higher recurrence rates and worse prognosis (40, 41), the significantly lower SLC5A5 expression in tumors larger than 2 cm may be considered as an additional fact linking lower SLC5A5 mRNA expression with higher tumor aggressiveness. Nevertheless, we must interpret this information carefully: larger tumors may present higher levels of necrosis/fibrosis and also additional non-carcinomatous tissue as part of the nodule, which may contribute to a lower SLC5A5 expression. In our series, we did not include microcarcinomas, so the group of tumors with ≤2 cm was very small, precluding any meaningful analysis (data not shown).
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study
| 100.0 |
Previous studies reported a lower SLC5A5 expression in cases harboring BRAFV600E, and there is experimental evidence showing that BRAFV600E can impair SLC5A5 expression (1, 19, 27, 39), nevertheless the impact of other relevant mutations found in thyroid tumors on SLC5A5 expression remained unknown. In our series, SLC5A5 expression was lower but did not reach statistical significance in the BRAFV600E PTC compared to that of BRAF wild-type group. The lack of significance in our series may be due to differences in size and composition of the series, since the above mentioned studies addressing SLC5A5 expression and BRAF V600E (1, 19, 27, 39) used larger series of PTC.
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study
| 100.0 |
When we compared SLC5A5 expression (retrieved from TCGA database) between PTCs harboring different mutations (BRAFV600E, TERTp and RAS) and WT PTCs, we observed that independently of the mutation, SLC5A5 expression was always significantly lower compared to WT PTCs. Moreover, we also observed that RAS mutation was the one with lower impact on SLC5A5 expression. PTCs with RAS mutation displayed significantly higher levels of SLC5A5 compared to BRAFV600E and BRAF + TERTp-mutated PTCs. In fact, it has been previously reported that a distinct profile of expression of genes involved in thyroid hormone biosynthesis (being SLC5A5 one of these genes) between BRAFV600E and RAS-driven PTCs, with RAS-like PTCs having relatively high thyroid differentiation score (30).
|
study
| 100.0 |
Our results on the immunohistochemical NIS expression in normal thyroid and Graves’ disease (an autoimmune condition known to express high levels of NIS) (42) were in accordance to data previously reported (10, 12, 14, 17, 21), i.e., focal membrane expression of NIS in normal thyroid gland and a strong and widespread membrane NIS expression in Graves’ disease. The great difference observed in NIS expression between normal thyroid and in Graves’ disease may be considered as an example of how TSH is able to regulate NIS expression and the targeting to the membrane. Regarding carcinomas, we observed that the majority (71.6%) displayed NIS immunostaining, which is in accordance to the literature (13, 14, 17, 18, 19, 20, 21, 22, 23) (Table 4), but only a minority presented NIS in the basolateral membrane of tumor cells (5.7%). If one compares the percentage of cases with NIS plasma membrane staining, there are large differences between studies (Table 4). Such differences may be due to the variable size of the series and to differences in the antibodies used to perform the IHC (almost every study uses its own antibody, Table 4). In fact, one study compared NIS immunostaining using two different antibodies in a large series of thyroid carcinomas and observed some differences in the percentage of positive cells (29). To be sure that we were not missing any signal, we performed the IHC for NIS with TSA signal amplification in a subset of carcinomas with different immunostaining patterns and observed a complete vanish of intracytoplasmic staining and an amplification of the membrane staining. These results, like those from Peyrottes and coworkers (21) rise some questions about the real significance of NIS intracytoplasmic staining, so we decided to perform our analysis considering positive only the cases with membrane staining. Table 4Bibliographic revision and present results of NIS protein evaluation by IHC in thyroid carcinomas.ReferenceNo. of carcinomasAnti-NIS antibodies used in the studyNegative cases (%)Positive cases (%)*Cases with membrane staining (%)**(26)4 DTCsProduced by authors(10)14 DTCsProduced by authorsDescriptive studies(11)12 PTCsProduced by authors(12)9 DTCsProduced by authors(13)60 DTCsClone FP-1326.773.3N/A(14)57 (53 DTCs; 2 ATC; 2 MTC)Produced by authors29.870.215.8(15)67 DTCsDonated by Dr SM Jhiang of Ohio State University, USA67.232.8N/A(17)90 (87 DTCs; 3 ATC)Produced by authors22.577.5Some(24) 67 PTCsPohlenz et al. (2000)N/ASome(18)17 PTCsClone Ab-1010058.8(19)40 PTCsBrahms Diagnostica GmbH, Berlin, Germany010052.5(20)29 (25 DTCs; 4 ATC)Clone FP5A37.562.5N/A(21)47 (42 DTCs; 5MTC)Clones 39S, Ab-1 and FP5A49510(25)50 PTCsTazebay et al. (2000)N/A8(22)32 DTCsZhongshan Goldbridge Biotechnology, Beijin China010018.8(1)#265 DTCsClone FP5A881212(29)#380 (283 DTCs; 97 ATCs)Donated by N. Morgenthaler (Brahms Diagnostics, Henningsdorf, Germany)87.412.612.6(23)370 PTCsClone SPM18632.767.30.8(present study)211 (199 DTCs; 12 PDTCs)Clone FP5A28.471.65.7*Percentage of positive cases (independently of the cellular location); **percentage of cases with NIS membrane staining with or without simultaneous cytoplasmic staining; #these specific studies only considered positive cases with membrane staining.ATC, anaplastic thyroid carcinoma; MTC, medullary thyroid carcinoma; N/A, not addressed; PDTC, poorly differentiated thyroid carcinoma.
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study
| 99.94 |
The presence of NIS in the basolateral membrane of thyroid primary carcinomas did not associate with clinicopathological features, response to therapy or prognosis. If we look to the treatment of thyroid carcinoma (surgery followed by RAI ablation), only the remnants, metastases and eventually the recurrences are subjected to RAI. Prior to RAI ablation patients are subjected to TSH stimulation, either by withdrawal of thyroid hormones or by the administration of recombinant TSH (2). Since TSH has a major role in NIS expression and targeting to the membrane (43), we can hypothesize that levels of membrane NIS in stimulated recurrences and metastases may be different from those in non-stimulated primary tumors because they may reflect two different biological conditions. It seems pertinent that further studies of NIS expression should be performed in recurrences and/or remaining thyroid tissues rather than in the primary tumors. In fact, one study addressed NIS expression in nontumoral thyroid tissue and reported a significant association between lower NIS expression and delayed structural response (3). Even though the nontumoral tissue used in this study was obtained at the time of surgery (normal TSH levels), this might corroborate our hypothesis. Altogether, this information may help to explain why NIS expression in the primary tumor does not predict RAI therapy success and/or prognosis.
|
study
| 99.94 |
Another interesting finding of our study was the observation that the cases with NIS membrane staining were predominantly wild type for the analyzed mutations (NRAS, BRAF and TERTp) (Table 3). Although this membrane expression was not associated with any outcome (clinicopathological features or prognosis), it is tempting to advance that the genetic background of tumors influence NIS targeting to the membrane. There are in vitro evidence that BRAFV600E mutation affects NIS targeting to the membrane (24), but the impact of the other mutations (NRAS and TERTp) remains unknown.
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study
| 100.0 |
In summary, the absence of BRAF and NRAS mutations in every carcinoma displaying NIS membrane staining at immunohistochemistry supports the assumption that the genetic background of tumors may be of major importance to SLC5A5 expression as well as to NIS targeting to the basolateral membrane. On the other hand, NIS immunohistochemical expression did not predict tumor behavior, therapy response or outcome. Moreover, SLC5A5 mRNA expression was significantly lower in mutated PTCs and a lower SLC5A5 mRNA expression was associated with tumor aggressiveness and worse prognosis. Thus, the study of SLC5A5 mRNA expression is much more informative compared to NIS expression evaluated by IHC.
|
study
| 100.0 |
This study was supported by FCT (‘Portuguese Foundation for Science and Technology’) through PhD grants to Catarina Tavares (SFRH/BD/87887/2012), Ana Pestana (SFRH/BD/110617/2015), Rui Batista (SFRH/BD/111321/2015) and by a CNPq PhD grant (‘National Counsel of Technological and Scientific Development’, Brazil), Science without Borders, Process n# 237322/2012-9 for Luciana Ferreira. Miguel Melo received a grant from Genzyme for the research project ‘Molecular biomarkers of prognosis and response to therapy in differentiated thyroid carcinomas’. Further funding was obtained from FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274) and by the project ‘Advancing cancer research: from basic knowledgement to application’; NORTE-01-0145-FEDER-000029; ‘Projetos Estruturados de I&D&I’, funded by Norte 2020-Programa Operacional Regional do Norte. This work was also financed by Sociedade Portuguesa de Endocrinologia Diabetes e Metabolismo through a grant ‘Prof. E Limbert Sociedade Portuguesa de Endocrinologia Diabetes e Metabolismo/Sanofi-Genzyme in thyroid pathology’.
|
other
| 99.9 |
The ability to think ahead and anticipate future events consistently distinguishes expert performers from their less-expert counterparts (Triolet et al., 2013). In domains such as the military, aviation, and invasion sports, the importance of anticipation is magnified given the dynamic nature and strict time constraints under which performers must make decisions before executing complex motor skills. The ability to utilize perceptual–cognitive processes to inform decision-making and motor actions has been proposed to be a key factor that distinguishes expert performers from those less-expert across domains (Williams et al., 2011).
|
other
| 97.8 |
At a conceptual level, following extended domain-specific practice, experts develop highly specialized and refined knowledge structures which enable them to disregard non-relevant information and attend to only the most critical cues within the display (cf., Ericsson and Kintsch, 1995). These differences are believed to underpin the expert’s ability to identify advance cues in the environment (Savelsbergh et al., 2002), as well as the localized relative motion information between these cues (Diaz et al., 2012), to assess the likelihood of situational probabilities (Farrow and Reid, 2012), and to perceive patterns in displays comprising multiple discrete features (North et al., 2011). Experts encode information more efficiently and effectively, resulting in quicker and more accurate decisions and superior motor execution when compared with novice or less-expert individuals who have accrued less practice.
|
study
| 99.75 |
Perception and knowledge of patterns is typically assessed using recall and recognition paradigms. In the former, participants must recall the positions of display features after a brief exposure, whereas in the latter familiarity judgments are made as to whether or not stimuli have been previously viewed. The typical finding is that experts show a memory advantage for structured stimuli (representing typical formations that one would expect to see), but that this advantage is lost when attempting to recall or recognize unstructured stimuli (where information is randomly organized). These results were originally reported in the domain of chess (De Groot, 1965; Chase and Simon, 1973; Goldin, 1978, 1979), but subsequently the findings have been replicated across multiple domains including diagnostic imaging (Nodine and Kundel, 1987), business (McKelvie and Wiklund, 2004), and in numerous sports such as basketball (Allard et al., 1980), field hockey (Starkes, 1987), Australian Rules Football (Berry et al., 2004), and soccer (Williams and Davids, 1995). It is believed that the ability to quickly recall or recognize previously encountered situations “buys time” and facilitates more accurate anticipation judgments. In dynamic sports like soccer it is likely that participants will never truly encounter the exact same situation more than once; however, it is proposed that the critical features of patterns will remain consistent with some room for variability (see Gobet and Simon, 1996). Expert performers are proposed to perceive and encode these key features and relations in displays when recognizing patterns (North et al., 2017). In other words, judging the current situation against those instances previously encountered allows the observer to assess the most likely courses of action and anticipate effectively in a timely manner.
|
review
| 99.7 |
Practice history data from expert performers reveal that vast amounts of deliberate practice are required over numerous years to attain high levels of perceptual–cognitive–motor expertise (see Ericsson et al., 1993; Williams et al., 2012b). Consequently, researchers have started to consider whether training interventions may be developed that facilitate the more rapid acquisition of perceptual–cognitive skills. The majority of researchers have focused on training advance cue utilization using relatively closed skills such as goalkeepers saving penalty kicks in soccer (Savelsbergh et al., 2010) or players attempting to return serve in tennis (Farrow and Abernethy, 2002). These interventions seek to direct attention toward the most critical cues (as determined from process measures such as gaze behavior and verbal reports) and the effectiveness of this training is assessed by comparing performance post-intervention to an earlier pre-test. In general, these training programs have reported positive findings across sports (e.g., Scott et al., 1998; Williams et al., 2003; Murgia et al., 2014).
|
review
| 99.75 |
Although researchers have highlighted the potential benefit of training perceptual–cognitive skills, at least in micro-situations (i.e., one vs. one), there have been relatively few attempts to train perception of patterns in macro-situations (i.e., full-sided games). The later observation is surprising given the fairly substantive literature base focusing on identifying the processes and mechanisms underpinning this skill (Smeeton et al., 2004; North et al., 2009, 2011). Moreover, the ability to recall and recognize patterns is considered one of the key attributes of expert performers (Abernethy et al., 2005).
|
review
| 59.47 |
In one rare exception, Gorman and Farrow (2009) attempted to train the perception of patterns in basketball. However, these authors failed to report any advantage for their experimental groups over a control group and there was no positive transfer to on-court performance. A potential limitation to their approach was the mode of presentation since the viewing perspective employed during the intervention was different to that experienced in the on-court transfer task, potentially explaining the lack of on-court improvement. Another potential limitation was the use of highly skilled participants only. The majority of researchers who have reported the benefits of perceptual–cognitive training programs have used novice or intermediate performers. The benefits of such training programs may be restricted to, or are optimized in, more novice or intermediate populations.
|
review
| 99.6 |
A challenge when devising perceptual–cognitive training interventions is how to direct attention toward the critical cues. Typically, explicit verbal instructions have been used to focus the learner’s attention on the desired display features. A series of “if-then” statements are employed to highlight how these cues related to the eventual event outcome (e.g., see Smeeton et al., 2005). Although these explicit instructional methods have produced positive training effects, it has been argued that the use of such methods can be detrimental in the long term and especially when performing under anxiety (Abernethy et al., 2012). Learning under explicit instruction is thought to result in the development of declarative knowledge, making performers prone to reinvest in this consciously controlled information when under pressure. In contrast, implicit modes of instruction seek to facilitate learning without accruing declarative knowledge, with published reports suggesting that performance is more robust when subsequently performing under pressure given the relative absence of declarative knowledge in which to reinvest (for a review, see Masters, 1992; Jackson and Farrow, 2005; Masters and Maxwell, 2008; Hill et al., 2010). In this paper, our focus was to compare different modes of implicit instruction that guided attention to relevant cues without explicitly stating how these were to be used.
|
review
| 99.9 |
It appears that methods which guide the learner’s attention, as opposed to being told explicitly, and permit performers to self-discover and learn independently are the most effective strategies to train perceptual–cognitive skills as they show both short- and long-term advantages. However, there are various means by which the attention of learners can be directed. Although Smeeton et al. (2005) used simple verbal instructions to guide attention, technology allows video footage to be edited so that additional information can be overlaid on top of the footage to direct attention to the pertinent cues. Hagemann et al. (2006) and Abernethy et al. (2012) have used transparent colored masks to highlight critical cues when anticipating shots in badminton and handball, respectively. Moreover, prompts such as arrows may be overlaid on the screen to direct attention (see Ryu et al., 2013). However, the empirical evidence supporting the effectiveness of such methods is equivocal and there is no consensus as to whether one of these strategies is better than the others or if they offer any advantages at all over simply directing attention using verbal instructions (as per Smeeton et al., 2005).
|
review
| 99.9 |
The most effective method of conveying information remains unclear and conflicting results mean there is a need to further investigate the value of perceptual-training programs. In addition, there remains a paucity of research examining whether pattern recognition skill can be trained. Also, while experts may be differentiated from less-expert counterparts on their ability to recognize patterns, it has been argued such a task is only an indirect measure of expertise and not a skill that is explicitly employed in performance contexts. A debate exists as to whether recognition simply represents a by-product of exposure to the domain and does not directly contribute to the expertise they demonstrate in the performance environment (see Ericsson and Lehmann, 1996; North et al., 2009, 2011). In light of this debate, we have participants complete an anticipation test before and after the pattern recognition training intervention to assess if any benefits of training pattern recognition transferred to what may be considered a more representative measure of expertise (cf., Mann et al., 2007).
|
study
| 99.8 |
In sum, we investigate whether it is possible to train the perceptual–cognitive skill of pattern recognition between display features (i.e., players) using soccer as the vehicle. In light of the absence of any significant effects in the study by Gorman and Farrow (2009), which used elite basketball players, we examined whether this skill was amenable to training using a more novice population group. Also, we compared the relative effectiveness of four different instructional methods. Participants were assigned to either a verbal cueing, visual cueing, video only with no cueing, or a control condition. Finally, given recent findings which have suggested recognition skill may not be as closely related to anticipation as previously thought (North et al., 2009, 2011), we examined whether the benefits of training pattern recognition transfers to improvements in anticipation accuracy. Since previously researchers have shown a variety of instructional approaches to be effective in training perceptual–cognitive skill in micro-contexts, we hypothesized that all three experimental conditions would improve recognition performance from pre- to post-test in comparison to a control group. Also, we expected the verbal cueing and visual cueing groups to improve more than the video only group given that their attention was being directed to those features identified as most important in successful recognition judgments (see North et al., 2009, 2011; Williams et al., 2012a). Since only a few researchers have directly tested different instructional methods, producing contradictory results, we had no a priori hypothesis as to whether visual or verbal cueing would be more effective in training recognition. As knowledge and awareness of patterns of play has consistently been identified as a characteristic of expert performers (Abernethy et al., 2005; Williams et al., 2006), and even published reports suggesting the skill may not be central to anticipation performance (North et al., 2009, 2011) report positive correlations between the two, our final hypothesis was that successfully training the ability to recognize patterns of play would result in improvements in anticipation accuracy.
|
study
| 99.94 |
Altogether, 64 amateur soccer players volunteered to participate. The performance of participants was rank-ordered based on their pre-test recognition accuracy scores, following which participants were then assigned to one of four equally matched groups of N = 16: control (M age = 19.5 years, SD = 2.07); visual attention guided (M age = 20.2 years, SD = 3.24); verbal instruction guided (M age = 19.8 years, SD = 3.67); and video only (M age = 20.9 years, SD = 2.47). Participants were considered as amateurs if they had only played soccer at recreational or school level. Participants reported having played soccer at this level for an average of 9.24 years (SD = 2.55). All reported normal or corrected to normal visual function and none reported color blindness. The research was conducted according to the ethical guidelines and approval of the second author’s institution. Participants provided written informed consent and were free to withdraw at any stage.
|
study
| 100.0 |
We used three different test films. An anticipation test film, a perceptual training test film, and a recognition test film. All test films used video footage which was recorded using a fixed, tripod-mounted video camera (Canon XM-2, Tokyo, Japan) in a raised position (approximate height 9 m) set back behind the goal (approximate distance 15 m). The camera position ensured that all players were visible at all times and that information was not excluded from wide areas. Although the raised viewing perspective is different to that which players would typically experience during game situations, construct-validity has previously been established for the approach. When using the same viewing perspective, expert–novice differences have been reported using recognition (North et al., 2011), recall (Abernethy et al., 2005), situational probability (Ward and Williams, 2003), and anticipation (North et al., 2016) paradigms. All test films comprised of a number of separate clips, each showing a developing pattern of play which culminated in a penetrative attacking pass to a teammate. All the action sequences used showed patterns of play developing in the direction of the camera (i.e., coming toward the participant) and were all “structured” in nature. Clips were classified as being structured on the basis of three expert coaches independently rating a battery of clips as being either low or high in structure using a Likert-type scale (0 = very low in structure, 10 = very high in structure). The clips rated most highly for structure were those judged to be most representative of tactics, strategies, and plans that would typically be observed in attacking play. Only clips with a mean rating above 7 were used.
|
study
| 100.0 |
The action sequences used for the recognition test films were sampled from three English Premier League reserve team matches. The recognition test was comprised of a viewing phase and a recognition phase. Each individual clip in both viewing and recognition phases was 7 s in duration. The initial 2 s showed a static image of the first frame in the sequence, during which participants were cued to the location of the ball by a red circle. The clip then played normally for 5 s, showing a developing pattern of play before it occluded to black. There was then a 3 s inter-trial interval after which the next 7 s clip played in the same fashion. Both viewing and recognition test films contained 40 clips, however, for the recognition test film 20 of these were also present in the initial viewing test film and 20 were novel.
|
study
| 99.7 |
At the start of each clip in the anticipation phase, participants were shown a freeze-frame of the clip’s opening frame for 2 s. During this time, a red circle was shown on the screen to cue participants as to the ball’s location. The red circle then disappeared and the clip played normally, showing 6 s of action in which possession started in the defensive half (that furthest away from the participant) and ended in the attacking half (that nearest the participant). Each clip stopped when the player in possession was about to make a penetrative pass to a teammate in an attacking position. The final frame when the clip ended was paused and presented to participants for 2-s, during which time possible passing options were highlighted using red, blue, black, and yellow squares. The clip then occluded to a black screen and the next clip commenced after a 5 s inter-trial interval. In total, there were 24 clips in the anticipation test film with each being presented for a total of 10 s. An example of the first and final frames of a clip used in the anticipation test film is presented in Figures 1, 2.
|
study
| 100.0 |
An example of a freeze-frame shown at the end of each clip in the anticipation paradigm with the passing options highlighted by yellow, black, blue, and red circles. Note: White square shown here is for illustrative purposes to highlight ball location and was not used in the actual test film.
|
other
| 99.9 |
The match footage used in the perceptual training films was taken from a sample of two Football Association under 18 years Youth Cup matches. In total, there were 120 clips spread over four perceptual training sessions. Participants were assigned to one of four different perceptual training groups. The precise nature of footage in the perceptual training film was dependent on which group participants were assigned to following the pre-test.
|
other
| 98.6 |
Film clips were presented using a DVD player (Panasonic, DMR-E50, Osaka, Japan) and projector (Sharp, XG-NV2E, Manchester, United Kingdom) to project images onto a 9′ × 12′ screen (Cinefold, Spiceland, IN, United States) at a rate of 25 frames/s with XGA resolution. Verbal instructions were recorded onto a dictaphone and transferred onto test films using video editing software (Adobe Premiere, Adobe Systems Incorporated, San Jose, CA, United States). The same video editing software was used to create the test films and insert freeze-frames in sequences. To highlight the ball and players of interest, the Microsoft Paint Program (Microsoft Corporation 2010, Redmond, WA, United States) was used.
|
other
| 99.9 |
Participants completed pre- and post-tests to assess anticipation and recognition performance which were separated by 2 weeks. During the intervening 2 weeks, participants completed the perceptual training program spread over four separate sessions, with approximately 2.5 days between each perceptual training session.
|
study
| 99.8 |
Participants initially completed the recognition test. This involved participants being presented with the viewing test film which comprised of 40 individual clips. Participants were informed that the ball’s starting location would be highlighted by a red circle, after which the clip would play normally and show a developing attacking sequence that culminated in a player being about to make a forward attacking pass, but that the clip would occlude before this pass was played. Participants were instructed to watch the clip as if they were playing in the match as a central defensive player, but that no specific response was required. After the viewing film had been presented there was a 10 min comfort break. Participants were then presented with the recognition film which comprised of 40 clips. The participants were told that some of the clips in the recognition film had been included in the viewing phase and that others were novel; their task was to make a recognition decision for each clip as to whether they had seen it in the viewing film or not. Participants were instructed to watch each clip for its full duration before making a recognition response (yes or no) by writing down their answer using pen and paper. When each clip was occluded in the recognition phase, participants were presented with a “Respond Now” image on the screen and were instructed to respond quickly and accurately. The recognition test took approximately 20 min to complete.
|
study
| 100.0 |
After completing the recognition test, participants were provided with another 10 min break during which they completed a short questionnaire that requested demographic information as well as information about their practice history and involvement in soccer. Participants then completed an anticipation test. Participants were informed they would be presented with a further 24 clips showing developing patterns of play, which culminated in a player about to make an attacking pass and that they should watch the clips as if they were a central defensive player. The participants were told that a red circle would highlight the ball’s position at the start of the clip before playing and then pausing on the final frame of the sequence. Participants were told that four different passing options would be highlighted using colored circles and that their task was to select the player they thought was most likely to receive the ball by writing down the respective colored circle on a pen and paper response sheet. The final frame was paused for 2 s, after which the message “Respond Now” was presented on the screen. Participants were instructed to respond quickly and accurately. The anticipation test took approximately 10 min to complete.
|
study
| 99.94 |
Participants were allocated to one of four equally matched perceptual-training groups based on their pre-test recognition scores. There were four training sessions, each comprising of 30 clips with an inter-trial interval of 5 s, with each training session taking approximately 20 min to complete. Participants were not required to make any responses during the training sessions, but they were informed to watch the clips and pay attention to any instructions or guidance provided within these session.
|
study
| 99.94 |
When the first frame was presented and “frozen” for 2 s to cue participants to the location of the ball, participants in this group were provided with verbal instructions about where they should direct their attention during the clip. The instruction provided was based on findings reported by Williams et al. (2006) and North et al. (2009), with participants being told to focus their attention on the positions and movements of central attacking players without explicitly stating the purpose of the movements that were to be made or exactly what information this might convey. Generic verbal instructions were provided at the onset of the sequence to focus on these specific players with subsequent verbal instructions individually tailored for each clip. For example, a clip in which two strikers would move in order to create space for another teammate would play as normal after the initial 2 s freeze-frame, before a second freeze-frame would be inserted at an appropriate point in order for verbal instructions to be provided to highlight the specific movements and runs of interest. Once the verbal instructions had been provided the clip resumed and played as normal. Each clip contained 6 s of dynamic activity although the total presentation time varied due to each clip being individually tailored with freeze-frames and additional verbal instructions inserted as appropriate.
|
study
| 99.94 |
The clips used, and their order of presentation, were the same as in the verbal instruction training group. During the initial 2 s freeze-frame in which ball location was identified using a black circle, participants in this group were cued as to the most important players and where they should direct their attention using red circles (to highlight the players) and red arrows (to highlight their movements). The visual cues to guide attention were all presented during the freeze-frame only so as to avoid potentially obstructing information once clips played normally. To ensure consistency, the same players were highlighted as in the verbal instruction group and each clip was played for the same length of time with the same number of freeze-frames inserted for the same length of time. An example of a freeze-frame from the visual guidance group is shown in Figure 3.
|
study
| 100.0 |
In this group, participants were presented with the same clips, in the same order, as in the verbal instruction and video guidance groups. As with these groups, participants were initially presented with a 2 s freeze-frame during which the position of the ball was highlighted using a red circle. Participants in this group though received no further information and after the initial 2 s freeze-frame the clip played normally for 6 s before the screen was occluded.
|
other
| 69.7 |
Participants completed recognition and anticipation post-tests 2 days after the final perceptual training session. The post-test recognition and anticipation tests were conducted following the same procedures used for the pre-tests. To prevent familiarity bias and expectancy effects, the order of presentation for clips was changed from the pre-test and the clips that were repeated in the recognition test film were different to those in the pre-test. Also, the order of clips was changed for the anticipation paradigm.
|
study
| 100.0 |
The data were analyzed based on signal detection theory. This analysis method is used to measure the effectiveness of participants in distinguishing meaningful signals that may be present in displays from non-meaningful noise. Signal detection theory provides two dependent measures which were used to analyze recognition performance; a parametric measure of sensitivity (d′) and criterion (c) which is a measure of response bias (Green and Swets, 1966). The measure of sensitivity (d′) assesses discriminability: how well two conditions can be distinguished from one another (signal present or absent). The larger the d′ value the more sensitive a person is in discriminating between signal present and signal absent stimuli, while a value of 0 indicates chance (i.e., guessing) performance. Criterion (c) measures bias and refers to the extent to which one response (i.e., responding yes or no) is more probable than the other. If the c value is negative it indicates a bias toward “yes” responses (resulting in more “hits,” but also more “false alarms”), whereas if c is a positive value then it indicates the participants favor a bias to “no” responses, with fewer hits and fewer false alarms (MacMillan, 2002).
|
study
| 100.0 |
Anticipation performance was measured by dividing the total number of correct judgments by the total number of trials (n = 24) and then multiplying by 100 to create a percentage accuracy score. The data for d′, c, and anticipation accuracy were analyzed using separate two-way mixed-design ANOVAs in which the between participant factor was Group (verbal instruction vs. visual guidance vs. video only vs. control) and the within participants factor was Time of Test (pre-test vs. post-test).
|
study
| 100.0 |
Prior to conducting the analyses, all data were tested for normality using the Shapiro–Wilks test. Partial eta squared (ηp2) values are provided as a measure of effect size for all main effects and interactions and, where appropriate, Cohen’s d measures are reported for comparisons between two means. For repeated measures, violations of sphericity were corrected by adjusting the degrees of freedom using the Greenhouse–Geisser correction when the sphericity estimate was less than 0.75 and the Huynh–Feldt correction when greater than 0.75 (Girden, 1992). The alpha level for significance was set at p < 0.05.
|
study
| 100.0 |
An analysis of d′ revealed a significant main effect of Time of Test on recognition sensitivity, F(1,60) = 16.53, p < 0.05, ηp2 = 0.216. Participants were more sensitive in their recognition decisions at post-test (M = 0.55, SD = 0.5) than pre-test (M = 0.37, SD = 0.52), d = 0.34. However, the effect of Group, F(3,60) = 0.49, p > 0.05, ηp2 = 0.02, and the Group × Time of Test interaction, F(3,60) = 1.53, p > 0.05, ηp2 = 0.07, was not significant. The mean recognition sensitivity scores for each experimental group at pre- and post-tests are shown in Figure 4.
|
study
| 100.0 |
For c, ANOVA showed that the effects of Time of Test, F(1,60) = 0.39, p > 0.05, ηp2 = 0.01, Group, F(3,60) = 0.31, p > 0.05, ηp2 = 0.02, and the Group × Time of Test interaction, F(3,60) = 1.41, p > 0.05, ηp2 = 0.07, were all non-significant. These data demonstrate that neither the experimental group or time of test made participants more biased to responding : “yes” or “no” when making recognition decisions.
|
study
| 100.0 |
In this paper, we had three aims. First, we examined whether participants were able to improve the perceptual–cognitive skill of recognizing patterns between features following a perceptual–cognitive training intervention. Also, we tested the relative effectiveness of different instructional approaches to train this skill. Finally, given debate as to the importance of recognition in anticipation, we examined whether our training interventions, which focused on enhancing pattern recognition would transfer to improvements in anticipation accuracy.
|
study
| 100.0 |
We hypothesized that recognition performance would improve from pre- to post-test (cf., Farrow and Abernethy, 2002; Williams et al., 2003; Smeeton et al., 2005). This hypothesis was supported. Participants became more sensitive in distinguishing previously seen from novel patterns on the post- relative to the pre-test. However, contrary to our second hypothesis, there was no main effect of group and no Group × Time of Test interaction, indicating that the mode of instruction did not affect how well participants learned to recognize patterns. This pattern of findings closely mirrors that reported by Gorman and Farrow (2009) who showed significant improvements from pre- to post-test but reported that the improvements observed in the training groups did not differ when compared with the placebo and control groups. It is often suggested that the absence of significant effects is due to the small sample size and relatively short intervention period. However, in the current study, we present one of the most extensive perceptual training interventions conducted in the literature. The intervention period (2 weeks) was longer than, or comparable to, other studies which have trained perceptual skills and reported significant effects (e.g., 3 days, Abernethy et al., 2012; 7 days, Hagemann et al., 2006; 7 days, Ryu et al., 2013; 3 weeks, Serpell et al., 2011; and 45 min, Williams et al., 2002). Over the course of the intervention period, participants were exposed to 120 training trials which is a higher number than employed in other similar perceptual training studies (e.g., 64 trials, Ryu et al., 2013; 40 trials, Serpell et al., 2011; 8 trials; Williams et al., 2002; and 30 trials, Smeeton et al., 2005).
|
study
| 99.9 |
Our intervention failed to replicate the benefits evident using perceptual–cognitive training programs designed to improve the ability to use advance postural cues. However, perceiving patterns between independent display features (i.e., players) and perceiving advance postural cues (and potentially relations between these interrelated features) are two distinct perceptual–cognitive skills. Perceiving global patterns between features may represent a higher order and more strategic skill, whereas perceiving postural cues could represent a lower-order process. As a consequence, the higher-order, more strategic skills may require an extended training program with more prolonged exposure to stimuli and game patterns in order to see the same extent of performance improvements that are observed when training the ability to perceive more localized cues. In both Gorman and Farrow’s (2009) study and the current paper, there are trends in the data (albeit non-significant) for training intervention groups to improve in spite of relatively short-term interventions. Although the duration of our intervention was comparable (or longer) to that reported in other published reports where attempts have been made to train the pick-up of advance postural cues, researchers should seek to undertake more longitudinal interventions to investigate whether the trends observed lead to significant differences over time.
|
study
| 99.94 |
The information we highlighted using our training interventions was driven by research which had identified the central attacking players, and specifically the relative motion between these features, as being the critical information to convey structure and meaning in order to perceive patterns in dynamic, interactive displays (see Williams et al., 2006, 2012a; North and Williams, 2008; North et al., 2009, 2011). However, in seeking to ensure participants were attending to these critical features we provided a lot of detail (either through verbal instruction or visual highlighting) that was tailored to each individual sequence. It is possible we were overly prescriptive with the information provided and unintentionally we may have promoted an explicit style of learning. More pronounced benefits of the intervention may have been seen had participants only had their attention oriented to these features at the outset and then subsequently been allowed to discover the movement patterns and relations for themselves. Such an intervention would have likely promoted a more implicit style of learning, which is considered preferable and more advantageous than learning explicitly (Magill, 1998; Smeeton et al., 2005). Orienting attention toward the most critical features at the outset before subsequently allowing the sequence to play would have the advantage of allowing relative motion information to emerge more clearly. The level of detail we sought to present necessitated that “freeze-frames” be inserted in video sequences, which in itself is likely to have disturbed or distorted the relative motion information emerging. Williams et al. (2012a) have demonstrated that although the relationships between display features are important, specifically it is the relative motion information emerging through dynamic interactions between these features that are critical. The use of “freeze-frames” to highlight features may have served to prevent participants from extracting this critical source of information, in turn impairing their ability to perceive patterns within the displays. An intervention which simply directed attention before allowing sequences to play uninterrupted would both encourage a more implicit style of learning and enhance the potential for the critical relative motion information to emerge.
|
study
| 99.94 |
The highly prescriptive approach used to orientate attention may have resulted in participants adopting a narrow focus of attention (cf., Nideffer, 1976). While a narrow focus of attention can be advantageous in situations where the visual information is largely invariant (Nougier et al., 1991), in dynamic contexts, such as soccer, where visual information is highly variable in nature, a broader focus of attention is considered preferable (Ripoll, 1988). Although the relative motions between central attacking players have been demonstrated as critical information sources, it is likely that participants need an awareness of how these more localized relations fit within the broader and more global pattern.
|
other
| 99.7 |
Our final aim was to examine whether any improvements in pattern recognition would transfer to improvements in anticipation. We did not observe any change in anticipation accuracy from pre- to post-test. Gorman and Farrow (2009) similarly did not report any main effects or interactions. It may be that perception of patterns does not contribute to anticipation, but rather that it is merely a by-product of task experience (cf., Ericsson and Lehmann, 1996), or at the very least its contribution to anticipation is less than has been previously argued. A number of distinct perceptual–cognitive skills contribute to anticipation and decision-making (see Vaeyens et al., 2007; Williams and Ward, 2007; Roca et al., 2013). The relative importance of these perceptual–cognitive skills varies as a function of the task constraints under which one is performing. In soccer, Roca et al. (2013) and North et al. (2016) have demonstrated that when the ball is far away from the performer, they seek to perceive patterns between features to inform their decision-making. In contrast, as the ball moves closer to the performer, attention switches to utilizing postural cues, with perception of patterns between players becoming less important. The stimuli used in the anticipation paradigm in the present study all showed action sequences where the final pass was about to be made in relatively close proximity to the participant. Therefore, the task constraints used in this study may have dictated that participants seek to process postural cues rather than perceive patterns. The nature of the clips (i.e., a raised viewing perspective) is likely to have made it difficult to extract fine postural cues, meaning that while participants may have looked to use this source of information, their ability to do so will have been impaired and so this may explain why anticipation accuracy did not improve. Alternatively, the skill of perceiving patterns between features may not have been required to inform anticipation judgments in this study and may be one explanation for the lack of transfer to anticipation accuracy following the perceptual training intervention. It may also be that the methods employed to examine transfer lack sufficient sensitivity to capture any benefits that may emerge. In future, researchers may wish to consider supplementing the anticipation paradigm used here with some more direct field-based measures of performance using match analysis data or the ratings of expert coaches on in situ assessment of anticipation and decision-making using behavioral assessment scales (e.g., see French and Thomas, 1987; Oslin et al., 1998).
|
study
| 99.94 |
When assessing transfer effects, an important factor to consider is viewing perspective. While the raised viewing perspective used in this study has distinguished skilled and less-skilled performers in recall (Abernethy et al., 2005), recognition (Williams et al., 2006), situational probability (Ward and Williams, 2003), and anticipation tasks (North et al., 2016) it nevertheless provides a very different perspective to that which players would encounter on the field. The encoding specificity principle (Tulving and Thompson, 1973) gives reason to be skeptical that any training benefits using such third person perspectives might transfer to field environments given the clear differences in perceptual information during encoding and retrieval processes across these two contexts. A potentially fruitful area for researcher and practitioners is the use of immersive technologies (such as virtual and augmented reality), which can more faithfully represent the perceptual variables experienced in performance environments.
|
study
| 99.7 |
A final potential issue is that all of the conditions employed a perception-only mode of response (i.e., pen and paper). Some researchers argue for the need to ensure perception and action are tightly coupled when studying perceptual–cognitive–motor skills (see Mann et al., 2010) to ensure both ventral and dorsal streams are engaged and that tasks more faithfully represent those undertaken in performance environments. However, there are numerous previous examples whereby perceptual–cognitive skills have been improved using uncoupled training methods (e.g., see Farrow and Abernethy, 2002; Williams et al., 2002) and there is evidence that the motor system remains engaged during perception-only tests (e.g., see Urgesi et al., 2006). Furthermore, in applied contexts, some elite sporting organizations are opposed to athletes engaging in overt physical practice beyond formally scheduled training and competition situations due to increasing concern over overuse injuries through excessive physical exertion (see Pimenta et al., 2017; van Mechelen et al., 2017). Also, perception-only interventions have practical utility since they can be employed when players are injured or in transit to and from training and matches.
|
review
| 99.8 |
Wu attempted to train the perceptual–cognitive skill of recognizing patterns between display features. Although our analyses revealed non-significant effects, we have employed novel and innovative methods and presented a foundation for follow-up research. We have raised a number of important points that should be valuable and informative for scientists and practitioners when designing interventions to improve perception of patterns in future. A body of research now exists which identifies critical information sources for pattern perception (see North and Williams, 2008; North et al., 2009, 2011; Williams et al., 2012a). When seeking to enhance pattern perception by improving awareness of these critical information sources, we suggest that in future, researchers need to employ more longitudinal interventions. Finally, any interventions should not disturb relative motion information and any attempts to examine transfer should ensure the task constraints encourage the perception of patterns between features over and above any other perceptual–cognitive skills that performers may have available.
|
study
| 99.06 |
This study was carried out in accordance with the recommendations of Liverpool John Moores University ethics committee (ethics approval number: 09/SPS/010) with written informed consent from all participants. All participants gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Liverpool John Moores University ethics committee (ethics approval number: 09/SPS/010).
|
other
| 99.94 |
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