Technical Performance Reduces during the Extra-Time Period of Professional Soccer Match-Play

Despite the importance of extra-time in determining progression in specific soccer tournament matches, few studies have profiled the demands of 120-minutes of soccer match-play. With a specific focus on the extra-time period, and using a within-match approach, we examined the influence of prolonged durations of professional soccer match-play on markers of technical (i.e., skilled) performance. In 18 matches involving professional European teams played between 2010 and 2014, this retrospective study quantified the technical actions observed during eight 15-minute epochs (E1: 00∶00–14∶59 min, E2: 15∶00–29∶59 min, E3: 30∶00–44∶59 min, E4: 45∶00–59∶59 min, E5: 60∶00–74∶59 min, E6: 75∶00–89∶59 min, E7: 90∶00–104∶59 min, E8: 105∶00–119∶59 min). Analysis of players who completed the demands of the full 120 min of match-play revealed that the cumulative number of successful passes observed during E8 (61±23) was lower than E1–4 (E1: 88±23, P = 0.001; E2: 77±21, P = 0.005; E3: 79±18, P = 0.001; E4: 80±21, P = 0.001) and E7 (73±20, P = 0.002). Similarly, the total number of passes made in E8 (71±25) was reduced when compared to E1 (102±22, P = 0.001), E3 (91±19, P = 0.002), E4 (93±22, P≤0.0005) and E7 (84±20, P = 0.001). The cumulative number of successful dribbles reduced in E8 (9±4) when compared to E1 (14±4, P = 0.001) and E3 (12±4, P≤0.0005) and the total time the ball was in play was less in E8 (504±61 s) compared to E1 (598±70 s, P≤0.0005). These results demonstrate that match-specific factors reduced particular indices of technical performance in the second half of extra-time. Interventions that seek to maintain skilled performance throughout extra-time warrant further investigation.     

Impact Loading and Locomotor-Respiratory Coordination Significantly Influence Breathing Dynamics in Running Humans

Locomotor-respiratory coupling (LRC), phase-locking between breathing and stepping rhythms, occurs in many vertebrates. When quadrupedal mammals gallop, 1∶1 stride per breath coupling is necessitated by pronounced mechanical interactions between locomotion and ventilation. Humans show more flexibility in breathing patterns during locomotion, using LRC ratios of 2∶1, 2.5∶1, 3∶1, or 4∶1 and sometimes no coupling. Previous studies provide conflicting evidence on the mechanical significance of LRC in running humans. Some studies suggest LRC improves breathing efficiency, but others suggest LRC is mechanically insignificant because ‘step-driven flows’ (ventilatory flows attributable to step-induced forces) contribute a negligible fraction of tidal volume. Yet, although step-driven flows are brief, they cause large fluctuations in ventilatory flow. Here we test the hypothesis that running humans use LRC to minimize antagonistic effects of step-driven flows on breathing. We measured locomotor-ventilatory dynamics in 14 subjects running at a self-selected speed (2.6±0.1 ms⁻¹) and compared breathing dynamics in their naturally ‘preferred’ and ‘avoided’ entrainment patterns. Step-driven flows occurred at 1-2X step frequency with peak magnitudes of 0.97±0.45 Ls⁻¹ (mean ±S.D). Step-driven flows varied depending on ventilatory state (high versus low lung volume), suggesting state-dependent changes in compliance and damping of thoraco-abdominal tissues. Subjects naturally preferred LRC patterns that minimized antagonistic interactions and aligned ventilatory transitions with assistive phases of the step. Ventilatory transitions initiated in ‘preferred’ phases within the step cycle occurred 2x faster than those in ‘avoided’ phases. We hypothesize that humans coordinate breathing and locomotion to minimize antagonistic loading of respiratory muscles, reduce work of breathing and minimize rate of fatigue. Future work could address the potential consequences of locomotor-ventilatory interactions for elite endurance athletes and individuals who are overweight or obese, populations in which respiratory muscle fatigue can be limiting.     

Response of tree growth to nitrogen addition in a Larix gmelinii primitive forest

 氮沉降对树木生长的影响是全球变化研究的一个核心问题。该文通过设置4种氮添加水平(对照(0)、低氮(20 kg N·hm^-2·a^-1)、中氮(50 kg N·hm^-2·a^-1)和高氮(100 kg N·hm^-2·a^-1)), 研究了模拟氮沉降对落叶松(Larix gmelinii)原始林树木胸径生长的影响。结果表明: 中氮和高氮添加对落叶松胸径相对生长率有显著影响, 而且这种影响随施氮年限的增加而增强。不同高度的树木对氮添加的响应有很大差异, 较低树木(树高<16.5 m)的生长对氮添加无显著响应, 而较高(树高>16.5 m)的树木在中氮和高氮处理下胸径生长有显著加速(胸径相对生长率增幅>79.5%), 但随着树木高度的进一步增加, 这种加速作用明显下降。研究结果显示氮添加会促进落叶松胸径生长, 这种促进作用主要发生在较高的落叶松个体中。     

Density and habitat use of one of the last jaguar populations of the Brazilian Atlantic Forest: Is there still hope?

The jaguar (Panthera onca) plays an important role in maintaining biodiversity and ecological processes. We evaluated the status of a jaguar population in one of the last stronghold habitats for its conservation in the Atlantic Forest, the Rio Doce State Park (RDSP). We used a random survey design from 2016/17 to estimate jaguar abundance and density as well as its occupancy and detection probabilities in the entire Park''s area. To monitor for temporal fluctuations in density and abundance, we used a systematic survey design in the southern portion of the Park where jaguars were more recorded when using the random approach. We then conducted two surveys in 2017/18 and 2020. Our 2016/17 random survey revealed that jaguar density (0.11 ± SE 0.28 individuals/100 km²) was the lowest obtained for the species across the Atlantic Forest. We noticed that jaguar density increased three times from 2017/18 (0.55 ± SE 0.45 individuals/100 km²) to 2020 (1.61 ± SE 0.6 individuals/100 km²). Jaguar occupancy and detection probability were 0.40 and 0.08, respectively. The low jaguar occupancy probability was positively associated with smaller distances from lakes and records of potential prey. The detection probability was positively associated with prey detection, the rainy season, and smaller distances from lakes. Our work contributes to a growing awareness of the potential conservation value of a protected area in a human‐dominated landscape as one of the last strongholds for jaguars across the Atlantic Forest.     

Dephytinisation with Intrinsic Wheat Phytase and Iron Fortification Significantly Increase Iron Absorption from Fonio (Digitaria exilis) Meals in West African Women

Low iron and high phytic acid content make fonio based meals a poor source of bioavailable iron. Phytic acid degradation in fonio porridge using whole grain cereals as phytase source and effect on iron bioavailability when added to iron fortified fonio meals were investigated. Grains, nuts and seeds collected in Mali markets were screened for phytic acid and phytase activity. We performed an iron absorption study in Beninese women (n = 16), using non-dephytinised fonio porridge (FFP) and dephytinised fonio porridge (FWFP; 75% fonio-25% wheat), each fortified with ⁵⁷Fe or ⁵⁸Fe labeled FeSO₄. Iron absorption was quantified by measuring the erythrocyte incorporation of stable iron isotopes. Phytic acid varied from 0.39 (bambara nut) to 4.26 g/100 g DM (pumpkin seed), with oilseeds values higher than grains and nuts. Phytase activity ranged from 0.17±1.61 (fonio) to 2.9±1.3 phytase unit (PU) per g (whole wheat). Phytic acid was almost completely degraded in FWFP after 60 min of incubation (pH≈5.0, 50°C). Phytate∶iron molar ratios decreased from 23.7∶1 in FFP to 2.7∶1 in FWFP. Iron fortification further reduced phytate∶iron molar ratio to 1.9∶1 in FFP and 0.3∶1 in FWFP, respectively. Geometric mean (95% CI) iron absorption significantly increased from 2.6% (0.8–7.8) in FFP to 8.3% (3.8–17.9) in FWFP (P<0.0001). Dephytinisation of fonio porridge with intrinsic wheat phytase increased fractional iron absorption 3.2 times, suggesting it could be a possible strategy to decrease PA in cereal-based porridges.     

鹫峰国家森林公园残次林物种多样性及生态位特征

残次林作为低效林的一种,林相及生态稳定性较差,造成景观差、生态功能低下。本文利用样地调查方法,对鹫峰森林公园主要残次林群落及不同立地条件下物种多样性进行分析,同时计算群落内优势种群生态位,旨在通过研究其生态系统的结构与功能,为提出林相改造技术提供科学依据。结果表明：（1）鹫峰国家森林公园残次林各群落多样性指数：油松林>刺槐林>五角枫、山桃混交林>荆条灌草林>侧柏林>栓皮栎林。（2）在不同的立地条件下,残次林的分布的影响较为明显,乔木层和草本层多样性指数呈现阴坡>半阴半阳坡>阳坡,厚土层>薄土层,而灌木层受坡向与土壤状况影响不明显。（3）鹫峰残次林的典型特点为林草匮乏,表现为乔木层破败、草本层覆盖度低种群分化。（4）生态位较宽的种群之间有可能具有较高重叠程度,但重叠程度不一定随宽度值增大而增大,分布频度是影响物种重叠程度的重要因素。该研究说明残次林与普通林物种多样性相比有很大的差别,且各林分残次程度不一,分化严重,物种单一和恶劣的立地条件是产生残次林的主要原因。     

Short-Term Effects of Particulate Matter on Stroke Attack: Meta-Regression and Meta-Analyses

Background and Purpose

Currently there are more and more studies on the association between short-term effects of exposure to particulate matter (PM) and the morbidity of stroke attack, but few have focused on stroke subtypes. The objective of this study is to assess the relationship between PM and stroke subtypes attack, which is uncertain now.

Methods

Meta-analyses, meta-regression and subgroup analyses were conducted to investigate the association between short-term effects of exposure to PM and the morbidity of different stroke subtypes from a number of epidemiologic studies (from 1997 to 2012).

Results

Nineteen articles were identified. Odds ratio (OR) of stroke attack associated with particular matter (“thoracic particles” [PM₁₀]<10 µm in aerodynamic diameter, “fine particles” [PM_2.5]<2.5 µm in aerodynamic diameter) increment of 10 µg/m³ was as effect size. PM₁₀ exposure was related to an increase in risk of stroke attack (OR per 10 µg/m³ = 1.004, 95%CI: 1.001∼1.008) and PM_2.5 exposure was not significantly associated with stroke attack (OR per 10 µg/m³ = 0.999, 95%CI: 0.994∼1.003). But when focused on stroke subtypes, PM_2.5 (OR per 10 µg/m³ = 1.025; 95%CI, 1.001∼1.049) and PM₁₀ (OR per 10 µg/m³ = 1.013; 95%CI, 1.001∼1.025) exposure were statistically significantly associated with an increased risk of ischemic stroke attack, while PM_2.5 (all the studies showed no significant association) and PM₁₀ (OR per 10 µg/m³ = 1.007; 95%CI, 0.992∼1.022) exposure were not associated with an increased risk of hemorrhagic stroke attack. Meta-regression found study design and area were two effective covariates.

Conclusion

PM_2.5 and PM₁₀ had different effects on different stroke subtypes. In the future, it''s worthwhile to study the effects of PM to ischemic stroke and hemorrhagic stroke, respectively.     

Venous Thromboembolism in an Industrial North American City: Temporal Distribution and Association with Particulate Matter Air Pollution

Background

Emerging evidence, mainly from Europe and Asia, indicates that venous thromboembolism (VTE) occurs most often in winter. Factors implicated in such seasonality are low temperature-mediated exacerbation of coagulation and high levels of particulate matter (PM) air pollution. However, in contrast to most European and Asian cities, particulate matter pollution peaks in the summer in many North American cities.

Objectives

We aimed to exploit this geographical difference and examine the temporal distribution of VTE in a cold-weather, North American city, Detroit, with a summer PM peak. Our goal was thereby to resolve the influence of temperature and PM levels on VTE.

Methods

Our retrospective, analytical semi-ecological study used chart review to confirm 1,907 acute, ambulatory VTE cases, divided them by location (Detroit versus suburban), and plotted monthly VTE frequency distributions. We used Environmental Protection Agency data to determine the temporal distribution of PM pollution components in Detroit. Suburban PM air pollution is presumed negligible and therefore not monitored.

Results

Acute VTE cases in Detroit (1,490) exhibited a summer peak (June 24^th) and differed from both a uniform distribution (P<0.01) and also that of 1,123 no-VTE cases (P<0.02). Levels of 10 µm diameter PM and coarse particle (2.5 to 10 µm) PM also exhibited summer peaks versus a winter peak for 2.5 µm diameter PM. Contrary to their urban counterparts, suburban cases of acute VTE (417) showed no monthly variation.

Conclusions

The summer peak of acute VTE in Detroit indicates that low temperature is not a major factor in VTE pathogenesis. In contrast, concordance of the 10 µm diameter PM, coarse particle, and the Detroit VTE monthly distributions, combined with no monthly suburban VTE variation, is consistent with a role for PM pollution. Furthermore, divergence of the VTE and 2.5 µm PM distributions suggests that particle size may play a role.     

无锡惠山三种城市游憩林内细颗粒物(PM2.5)浓度变化特征

在2011年秋、冬季和2012年春、夏季的游憩时段内(5:00-19:00),对无锡惠山香樟林、湿地松林和栓皮栎林3种游憩林内PM_2.5质量浓度进行实时监测,并同步观测气象因子,分析了游憩林内PM_2.5浓度的时间变化规律及其影响因素.结果表明: 惠山3种游憩林内PM_2.5浓度年均值低于道路,湿地松林和香樟林内PM_2.5浓度年均值低于栓皮栎林;3种游憩林和道路的PM_2.5年均浓度低于无锡市背景值.游憩林内PM_2.5浓度的季节变化规律为夏季最低,秋季次之,春季最高;PM_2.5浓度在春、夏、冬季最低的是湿地松林,秋季最低的是香樟林,栓皮栎林在各季节的PM_2.5浓度都较高.PM_2.5浓度在四季的日变化近似于“单峰单谷”型,7:00-9:00和15:00-19:00各出现最大值和最小值.4个季节的湿度和温度与PM_2.5浓度均极显著相关,光照仅在冬季显著影响PM_2.5浓度,较小风速对PM_2.5浓度的影响不大.     

Interaction between HLA-DRB1-DQB1 Haplotypes in Sardinian Multiple Sclerosis Population

We performed a case-control study in 2,555 multiple sclerosis (MS) Sardinian patients and 1,365 healthy ethnically matched controls, analyzing the interactions between HLA-DRB1-DQB1 haplotypes and defining a rank of genotypes conferring a variable degree of risk to the disease. Four haplotypes were found to confer susceptibility (*13∶03-*03∶01 OR = 3.3, Pc 5.1×10⁻⁵, *04∶05-*03∶01 OR = 2.1, Pc 9.7×10⁻⁸, *15∶01-*06∶02 OR = 2.0, Pc = 9.1×10⁻³, *03∶01-*02∶01 OR = 1.7 Pc = 7.9×10⁻²²) and protection (*11, OR = 0.8, Pc = 2.7×10⁻², *16∶01-*05∶02 OR = 0.6, Pc = 4.8×10⁻¹⁶, *14∶01-4-*05∶031 = OR = 0.5, Pc = 9.8×10⁻⁴ and *15∶02-*06∶01 OR = 0.4, Pc = 5.1×10⁻⁴). The relative predispositional effect method confirms all the positively associated haplotypes and showed that also *08 and *04 haplotypes confers susceptibility, while the *11 was excluded as protective haplotype. Genotypic ORs highlighted two typologies of interaction between haplotypes: i) a neutral interaction, in which the global risk is coherent with the sum of the single haplotype risks; ii) a negative interaction, in which the genotypic OR observed is lower than the sum of the OR of the two haplotypes. The phylogenic tree of the MS-associated DRB1 alleles found in Sardinian patients revealed a cluster represented by *14∶01, *04∶05, *13∶03, *08∶01 and *03∶01 alleles. Sequence alignment analysis showed that amino acids near pocket P4 and pocket P9 differentiated protective from predisposing alleles under investigation. Furthermore, molecular dynamics simulation performed on alleles revealed that position 70 is crucial in binding of MBP 85–99 peptide. All together, these data suggest that propensity to MS observed in Sardinian population carried by the various HLA-DRB1-DQB1 molecules can be due to functional peculiarity in the antigen presentation mechanisms.     

Evaluating the Impact of Flexible Alcohol Trading Hours on Violence: An Interrupted Time Series Analysis

Background

On November 24^th 2005, the Government of England and Wales removed regulatory restrictions on the times at which licensed premises could sell alcohol. This study tests availability theory by treating the implementation of Licensing Act (2003) as a natural experiment in alcohol policy.

Methods

An interrupted time series design was employed to estimate the Act’s immediate and delayed impact on violence in the City of Manchester (Population 464,200). We collected police recorded rates of violence, robbery, and total crime between the 1st of February 2004 and the 31st of December 2007. Events were aggregated by week, yielding a total of 204 observations (95 pre-, and 109 post-intervention). Secondary analysis examined changes in daily patterns of violence. Pre- and post-intervention events were separated into four three-hour segments 18∶00–20∶59, 21∶00–23.59, 00∶00–02∶59, 03∶00–05∶59.

Results

Analysis found no evidence that the Licensing Act (2003) affected the overall volume of violence. However, analyses of night-time violence found a gradual and permanent shift of weekend violence into later parts of the night. The results estimated an initial increase of 27.5% between 03∶00 to 06∶00 (ω = 0.2433, 95% CI = 0.06, 0.42), which increased to 36% by the end of the study period (δ = −0.897, 95% CI = −1.02, −0.77).

Conclusions

This study found no evidence that a national policy increasing the physical availability of alcohol affected the overall volume of violence. There was, however, evidence suggesting that the policy may be associated with changes to patterns of violence in the early morning (3 a.m. to 6 a.m.).     

Rabbit as a Novel Animal Model for Hepatitis E Virus Infection and Vaccine Evaluation

Background

The identification of hepatitis E virus (HEV) from rabbits motivated us to assess the possibility of using rabbits as a non-human primate animal model for HEV infection and vaccine evaluation.

Methodology/Principal Findings

First, 75 rabbits were inoculated with seven strains of genotypes 1, 3, 4, and rabbit HEV, to determine the appropriate strain, administrative route and viral dosage. Second, 15 rabbits were randomly divided into three groups and vaccinated with 0 µg (placebo), 10 µg and 20 µg of HEV candidate vaccine, HEV p179, respectively. After three doses of the vaccination, the rabbits were challenged with 3.3×10⁵ genome equivalents of genotype 4 HEV strain H4-NJ703. The strain of genotype 1 HEV was not found to be infectious for rabbits. However, approximately 80% of the animals were infected by two rabbit HEV strains. All rabbits inoculated with a genotype 3 strain were seroconverted but did not show viremia or fecal viral shedding. Although two genotype 4 strains, H4-NJ153 and H4-NJ112, only resulted in part of rabbits infected, another strain of genotype 4, H4-NJ703, had an infection rate of 100% (five out of five) when administrated intravenously. However, only two out of fifteen rabbits showed virus excretion and seroconversion when inoculated orally with H4-NJ703 of three different dosages. In the vaccine evaluation study, rabbits vaccinated with 20 µg of the HEV p179 produced anti-HEV with titers of 1∶10⁴–1∶10⁵ and were completely protected from infection. Rabbits vaccinated with 10 µg produced anti-HEV with titers of 1∶10³–1∶10⁴ and were protected from hepatitis, but two out of the five rabbits showed virus shedding.

Conclusions/Significance

Rabbits may be served as an alternative to the non-human primate models for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected.     

Patterns of Biomass and Carbon Distribution across a Chronosequence of Chinese Pine (Pinus tabulaeformis) Forests

Patterns of biomass and carbon (C) storage distribution across Chinese pine (Pinus tabulaeformis) natural secondary forests are poorly documented. The objectives of this study were to examine the biomass and C pools of the major ecosystem components in a replicated age sequence of P. tabulaeformis secondary forest stands in Northern China. Within each stand, biomass of above- and belowground tree, understory (shrub and herb), and forest floor were determined from plot-level investigation and destructive sampling. Allometric equations using the diameter at breast height (DBH) were developed to quantify plant biomass. C stocks in the tree and understory biomass, forest floor, and mineral soil (0–100 cm) were estimated by analyzing the C concentration of each component. The results showed that the tree biomass of P. tabulaeformis stands was ranged from 123.8 Mg·ha^–1 for the young stand to 344.8 Mg·ha^–1 for the mature stand. The understory biomass ranged from 1.8 Mg·ha^–1 in the middle-aged stand to 3.5 Mg·ha^–1 in the young stand. Forest floor biomass increased steady with stand age, ranging from 14.9 to 23.0 Mg·ha^–1. The highest mean C concentration across the chronosequence was found in tree branch while the lowest mean C concentration was found in forest floor. The observed C stock of the aboveground tree, shrub, forest floor, and mineral soil increased with increasing stand age, whereas the herb C stock showed a decreasing trend with a sigmoid pattern. The C stock of forest ecosystem in young, middle-aged, immature, and mature stands were 178.1, 236.3, 297.7, and 359.8 Mg C ha^–1, respectively, greater than those under similar aged P. tabulaeformis forests in China. These results are likely to be integrated into further forest management plans and generalized in other contexts to evaluate C stocks at the regional scale.     

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ cotransport in plant cells such that extracellular K⁺ stimulated OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. This study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.Intracellular Na⁺ and K⁺ homeostasis play vital roles in growth and development of higher plants (Clarkson and Hanson, 1980). Low cytosolic Na⁺ and high K⁺/Na⁺ ratios aid in maintaining an osmotic and biochemical equilibrium in plant cells. Na⁺ and K⁺ influx and efflux across membranes require the function of transmembrane Na⁺ and K⁺ transporters/channels. Several Na⁺-permeable transporters have been characterized in plants (Zhu, 2001; Horie and Schroeder, 2004; Apse and Blumwald, 2007). Na⁺/H⁺ antiporters mediate sequestration of Na⁺ into vacuoles under salt stress conditions in plants (Blumwald and Poole, 1985, 1987; Sze et al., 1999). Na⁺ (cation)/H⁺ antiporters are encoded by six AtNHX genes in Arabidopsis (Arabidopsis thaliana; Apse et al., 1999; Gaxiola et al., 1999; Yokoi et al., 2002; Aharon et al., 2003). A distinct Na⁺/H⁺ antiporter, Salt Overly Sensitive1, mediates Na⁺/H⁺ exchange at the plasma membrane and mediates cellular Na⁺ extrusion (Shi et al., 2000, 2002; Zhu, 2001; Ward et al., 2003). Electrophysiological analyses reveal that voltage-independent channels, also named nonselective cation channels, mediate Na⁺ influx into roots under high external Na⁺ concentrations (Amtmann et al., 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000); however, the underlying genes remain unknown.Potassium is the most abundant cation in plants and an essential nutrient for plant growth. The Arabidopsis genome includes 13 genes encoding KUP/HAK/KT transporters (Quintero and Blatt, 1997; Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998), and 17 genes have been identified encoding this family of transporters in rice (Oryza sativa ‘Nipponbare’; Bañuelos et al., 2002). Several KUP/HAK/KT transporters have been characterized as mediating K⁺ uptake across the plasma membrane of plant cells (Rigas et al., 2001; Bañuelos et al., 2002; Gierth et al., 2005).Ionic balance, especially the Na⁺/K⁺ ratio, is a key factor of salt tolerance in plants (Niu et al., 1995; Maathuis and Amtmann, 1999; Shabala, 2000; Mäser et al., 2002a; Tester and Davenport, 2003; Horie et al., 2006; Apse and Blumwald, 2007; Chen et al., 2007; Gierth and Mäser, 2007). Salinity stress is a major problem for agricultural productivity of crops worldwide (Greenway and Munns, 1980; Zhu, 2001). The Arabidopsis AtHKT1;1 transporter plays a key role in salt tolerance of plants by mediating Na⁺ exclusion from leaves (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005; Rus et al., 2006; Davenport et al., 2007; Horie et al., 2009). athkt1;1 mutations cause leaf chlorosis and elevated Na⁺ accumulation in leaves under salt stress conditions in Arabidopsis (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005). AtHKT1;1 and its homolog in rice, OsHKT1;5 (SKC1), mediate leaf Na⁺ exclusion by removing Na⁺ from the xylem sap to protect plants from salinity stress (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006, 2009; Davenport et al., 2007).The land plant HKT gene family is divided into two classes based on their nucleic acid sequences and protein structures (Mäser et al., 2002b; Platten et al., 2006). Class 1 HKT transporters have a Ser residue at a selectivity filter position in the first pore loop, which is replaced by a Gly in all but one known class 2 HKT transporter (Horie et al., 2001; Mäser et al., 2002b; Garciadeblás et al., 2003). While the Arabidopsis genome includes only one HKT gene, AtHKT1;1 (Uozumi et al., 2000), seven full-length OsHKT genes were found in the japonica rice cv Nipponbare genome (Garciadeblás et al., 2003). Members of class 1 HKT transporters, AtHKT1;1 and SKC1/OsHKT1;5, have a relatively higher Na⁺-to-K⁺ selectivity in Xenopus laevis oocytes and yeast than class 2 HKT transporters (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Ren et al., 2005). The first identified plant HKT transporter, TaHKT2;1 from wheat (Triticum aestivum), is a class 2 HKT transporter (Schachtman and Schroeder, 1994). TaHKT2;1 was found to mediate Na⁺-K⁺ cotransport and Na⁺ influx at high Na⁺ concentrations in heterologous expression systems (Rubio et al., 1995, 1999; Gassmann et al., 1996; Mäser et al., 2002b). Thus, class 1 HKT transporters have been characterized as Na⁺-preferring transporters with a smaller K⁺ permeability (Fairbairn et al., 2000; Uozumi et al., 2000; Su et al., 2003; Jabnoune et al., 2009), whereas class 2 HKT transporters function as Na⁺-K⁺ cotransporters or channels (Gassmann et al., 1996; Corratgé et al., 2007). In addition, at millimolar Na⁺ concentrations, class 2 HKT transporters were found to mediate Na⁺ influx, without adding external K⁺ in Xenopus oocytes and yeast (Rubio et al., 1995, 1999; Gassmann et al., 1996; Horie et al., 2001). However, the differential cation transport selectivities of the two types of HKT transporters have not yet been analyzed and compared in plant cells.A study of the barley (Hordeum vulgare) and wheat class 2 transporters has suggested that the transport properties of HvHKT2;1 and TaHKT2;1 expressed in yeast are variable, depending on the constructs from which the transporter is expressed, and have led to questioning of the K⁺ transport activity of HKT transporters characterized in Xenopus oocytes and yeast (Haro et al., 2005). It was further proposed that the 5′ translation initiation of HKT proteins in yeast at nonconventional (non-ATG) sites affects the transporter selectivities of HKT transporters (Haro et al., 2005), although direct evidence for this has not yet been presented. However, recent research has shown a K⁺ permeability of OsHKT2;1 but not of OsHKT1;1 and OsHKT1;3 in Xenopus oocytes. These three OsHKT transporters show overlapping and also distinctive expression patterns in rice (Jabnoune et al., 2009).The report of Haro et al. (2005) has opened a central question addressed in this study: are the Na⁺/K⁺ transport selectivities of plant HKT transporters characterized in heterologous systems of physiological relevance in plant cells, or do they exhibit strong differences in the cation transport selectivities in these nonplant versus plant systems? To address this question, we analyzed the Na⁺/K⁺ transport selectivities of the OsHKT2;1 and OsHKT2;2 transporters expressed in cultured tobacco (Nicotiana tabacum ‘Bright-Yellow 2’ [BY2]) cells. OsHKT2;1 and OsHKT2;2 are two highly homologous HKT transporters from indica rice cv Pokkali, sharing 91% amino acid and 93% cDNA sequence identity (Horie et al., 2001). OsHKT2;1 mediates mainly Na⁺ uptake, which correlates with the presence of a Ser residue in the first pore loop of OsHKT2;1 (Horie et al., 2001, 2007; Mäser et al., 2002b; Garciadeblás et al., 2003). In contrast, OsHKT2;2 mediates Na⁺-K⁺ cotransport in Xenopus oocytes and yeast (Horie et al., 2001). Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediates Na⁺ influx in the absence of added K⁺ (Horie et al., 2001). Recent research on oshkt2;1 loss-of-function mutant alleles has revealed that OsHKT2;1 from japonica rice mediates a large Na⁺ influx component into K⁺-starved roots, thus compensating for lack of K⁺ availability (Horie et al., 2007). But the detailed Na⁺/K⁺ selectivities of Gly-containing, predicted K⁺-transporting class 2 HKT transporters have not yet been analyzed in plant cells.Here, we have generated stable OsHKT2;1- and OsHKT2;2-expressing tobacco BY2 cell lines and characterized the cell lines by ion content measurements and tracer influx studies to directly analyze unidirectional fluxes (Epstein et al., 1963). These analyses showed that OsHKT2;1 exhibits Na⁺ uptake activity in plant BY2 cells in the absence of added K⁺, but little K⁺ (Rb⁺), influx activity. In contrast, OsHKT2;2 was found to function as a Na⁺-K⁺ cotransporter/channel in plant BY2 cells, showing K⁺-stimulated Na⁺ influx and Na⁺-stimulated K⁺ (Rb⁺) influx. The differential K⁺ selectivities of the two OsHKT2 transporters were consistently reproduced by voltage clamp experiments using Xenopus oocytes here, as reported previously (Horie et al., 2001). OsHKT2;2 was also found to mediate K⁺-independent Na⁺ influx at millimolar external Na⁺ concentrations. These findings demonstrate that the cation selectivities of OsHKT2;1 and OsHKT2;2 in plant cells are consistent with past findings obtained from heterologous expression analyses under similar ionic conditions (Horie et al., 2001; Garciadeblás et al., 2003; Tholema et al., 2005). Furthermore, the shift in OsHKT2;2 Na⁺-K⁺ selectivity depending on ionic editions is consistent with the model that HKT transporters/channels are multi-ion pores (Gassmann et al., 1996; Corratgé et al., 2007). Classical studies of ion channels have shown that ion channels, in which multiple ions can occupy the pore at the same time, can change their relative selectivities depending on the ionic conditions (Hille, 2001). Moreover, the presence of external K⁺ and Ca²⁺ was found here to down-regulate OsHKT2;1-mediated Na⁺ influx both in tobacco BY2 cells and in rice roots. The inhibitory effect of external K⁺ on OsHKT2;1-mediated Na⁺ influx into intact rice roots, however, showed a distinct difference in comparison with that of BY2 cells, which indicates a possible posttranslational regulation of OsHKT2;1 in K⁺-starved rice roots.     

Effects of Simulated Nitrogen Deposition on Soil Respiration in a Populus euphratica Community in the Ebinur Lake Area,a Desert Ecosystem of Northwestern China

One of the primary limiting factors for biological activities in desert ecosystems is nitrogen (N). This study therefore examined the effects of N and investigated the responses of an arid ecosystem to global change. We selected the typical desert plant Populus euphratica in a desert ecosystem in the Ebinur Lake area to evaluate the effects of N deposition on desert soil respiration. Three levels of N deposition (0, 37.5 and 112.5 kg·N·ha^-1·yr^-1) were randomly artificially provided to simulate natural N deposition. Changes in the soil respiration rates were measured from July to September in both 2010 and 2013, after N deposition in April 2010. The different levels of N deposition affected the total soil N, soil organic matter, soil C/N ratio, microorganism number, and microbial community structure and function. However, variable effects were observed over time in relation to changes in the magnitude of N deposition. Simulated high N deposition significantly reduced the soil respiration rate by approximately 23.6±2.5% (P<0.05), whereas low N deposition significantly increased the soil respiration rate by approximately 66.7±2.7% (P<0.05). These differences were clearer in the final growth stage (September). The different levels of N deposition had little effect on soil moisture, whereas N deposition significantly increased the soil temperature in the 0–5 cm layer (P<0.05). These results suggest that in the desert ecosystem of the Ebinur Lake area, N deposition indirectly changes the soil respiration rate by altering soil properties.     

Filtration Parameters Influencing Circulating Tumor Cell Enrichment from Whole Blood

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·10⁴∶10²∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm² track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.     

Analysis of the Setup Uncertainty and Margin of the Daily ExacTrac 6D Image Guide System for Patients with Brain Tumors

This study evaluated the setup uncertainties for brain sites when using BrainLAB’s ExacTrac X-ray 6D system for daily pretreatment to determine the optimal planning target volume (PTV) margin. Between August 2012 and April 2015, 28 patients with brain tumors were treated by daily image-guided radiotherapy using the BrainLAB ExacTrac 6D image guidance system of the Novalis-Tx linear accelerator. DUON^TM (Orfit Industries, Wijnegem, Belgium) masks were used to fix the head. The radiotherapy was fractionated into 27–33 treatments. In total, 844 image verifications were performed for 28 patients and used for the analysis. The setup corrections along with the systematic and random errors were analyzed for six degrees of freedom in the translational (lateral, longitudinal, and vertical) and rotational (pitch, roll, and yaw) dimensions. Optimal PTV margins were calculated based on van Herk et al.’s [margin recipe = 2.5∑ + 0.7σ - 3 mm] and Stroom et al.’s [margin recipe = 2∑ + 0.7σ] formulas. The systematic errors (∑) were 0.72, 1.57, and 0.97 mm in the lateral, longitudinal, and vertical translational dimensions, respectively, and 0.72°, 0.87°, and 0.83° in the pitch, roll, and yaw rotational dimensions, respectively. The random errors (σ) were 0.31, 0.46, and 0.54 mm in the lateral, longitudinal, and vertical rotational dimensions, respectively, and 0.28°, 0.24°, and 0.31° in the pitch, roll, and yaw rotational dimensions, respectively. According to van Herk et al.’s and Stroom et al.’s recipes, the recommended lateral PTV margins were 0.97 and 1.66 mm, respectively; the longitudinal margins were 1.26 and 3.47 mm, respectively; and the vertical margins were 0.21 and 2.31 mm, respectively. Therefore, daily setup verifications using the BrainLAB ExacTrac 6D image guide system are very useful for evaluating the setup uncertainties and determining the setup margin.     

NaCl-Induced Alternations of Cellular and Tissue Ion Fluxes in Roots of Salt-Resistant and Salt-Sensitive Poplar Species

Using the scanning ion-selective electrode technique, fluxes of H⁺, Na⁺, and Cl⁻ were investigated in roots and derived protoplasts of salt-tolerant Populus euphratica and salt-sensitive Populus popularis 35-44 (P. popularis). Compared to P. popularis, P. euphratica roots exhibited a higher capacity to extrude Na⁺ after a short-term exposure to 50 mm NaCl (24 h) and a long term in a saline environment of 100 mm NaCl (15 d). Root protoplasts, isolated from the long-term-stressed P. euphratica roots, had an enhanced Na⁺ efflux and a correspondingly increased H⁺ influx, especially at an acidic pH of 5.5. However, the NaCl-induced Na⁺/H⁺ exchange in root tissues and cells was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H⁺-ATPase inhibitor). These results indicate that the Na⁺ extrusion in stressed P. euphratica roots is the result of an active Na⁺/H⁺ antiport across the plasma membrane. In comparison, the Na⁺/H⁺ antiport system in salt-stressed P. popularis roots was insufficient to exclude Na⁺ at both the tissue and cellular levels. Moreover, salt-treated P. euphratica roots retained a higher capacity for Cl⁻ exclusion than P. popularis, especially during a long term in high salinity. The pattern of NaCl-induced fluxes of H⁺, Na⁺, and Cl⁻ differs from that caused by isomotic mannitol in P. euphratica roots, suggesting that NaCl-induced alternations of root ion fluxes are mainly the result of ion-specific effects.Soil salinity causes increasingly agricultural and environmental problems on a worldwide scale, especially in arid areas. When plant roots are subjected to saline environments with high NaCl content, external Na⁺ and Cl⁻ establish a large electrochemical gradient favoring the passive entry of salt ions through a variety of cation and anion channels and/or transporters in the plasma membrane (PM; Blumwald et al., 2000; Hasegawa et al., 2000; White and Broadley, 2001; Roberts, 2006; Demidchik and Maathuis, 2007). The entry and accumulation of toxic ions lead to disruption of ion homeostasis and finally cause secondary stress, e.g. oxidative bursts (Zhu, 2001, 2003). Accordingly, the maintenance of low salt concentration in the cytosol is of great importance for salt adaptation of plants (Greenway and Munns, 1980; Munns and Tester, 2008).Active Na⁺ extrusion to the apoplast or external environment is essential for sustaining Na⁺ homeostasis in the cytosol (Blumwald et al., 2000; Tester and Davenport, 2003; Zhu, 2003; Apse and Blumwald, 2007). PM Na⁺/H⁺ antiporters have been widely considered to play a crucial role in active Na⁺ extrusion under saline conditions (Shi et al., 2000, 2002; Qiu et al., 2002; Martínez-Atienza et al., 2007). NaCl-induced activity of PM Na⁺/H⁺ antiporter has been reported in crop species, tomato (Solanum lycopersicum; Wilson and Shannon, 1995), Arabidopsis (Arabidopsis thaliana; Qiu et al., 2002, 2003), and rice (Oryza sativa; Martínez-Atienza et al., 2007). Furthermore, overexpression of the Na⁺/H⁺ antiporter gene AtSOS1 decreases the accumulation of Na⁺ in transgenic Arabidopsis under NaCl stress (Shi et al., 2003). These PM Na⁺/H⁺ antiporters depend on electrochemical H⁺ gradients, which are generated by PM H⁺-ATPase (Blumwald et al., 2000; Zhu, 2003). Using an ion-selective microelectrode, Shabala and a colleague suggested the involvement of PM H⁺-ATPase in the Na⁺/H⁺ antiport according to H⁺ kinetics on salt shock (Shabala, 2000; Shabala and Newman, 2000). Therefore, the NaCl-induced H⁺ pumping is fundamental to Na⁺/H⁺ exchange and salinity tolerance (Ayala et al., 1996; Vitart et al., 2001; Chen et al., 2007; Gévaudant et al., 2007). However, the active Na⁺/H⁺ antiport across PM and the contribution to salt exclusion have been rarely investigated in tree species.Munns and Tester (2008) claimed that Cl⁻ toxicity is more important than Na⁺ toxicity in some woody species, e.g. citrus. Similarly, we have noticed that the inability to restrict Cl⁻ uptake contributes to the NaCl-induced salt damage in salt-sensitive poplar (Populus spp.) species, in addition to toxicity of excess Na⁺ (Chen et al., 2001, 2002, 2003). The differences in Cl⁻ tolerance exhibited by plants are usually related to the ability to restrict Cl⁻ transport to the aerial part (Greenway and Munns, 1980; White and Broadley, 2001). Excluding Cl⁻ from the xylem seems to be an effective mechanism for lotus to cope with the interactive effect of salt and water logging (Teakle et al., 2007). An influx of Cl⁻, immediately after addition of NaCl, was observed in bean (Vicia faba) mesophyll tissue (Shabala, 2000). The Cl⁻ flux response to salt shock is helpful to reveal the rapid adjustments of plants to salinity. However, Cl⁻ fluxes in salt-adapted roots, which are necessary to clarify plant adaptations to long durations of salinity, have not been examined.Populus euphratica has been widely considered as a model plant to elucidate physiological and molecular mechanisms of salt tolerance in woody species (Chen et al., 2001, 2002, 2003; Gu et al., 2004; Ottow et al., 2005a, 2005b; Junghans et al., 2006; Wang et al., 2007, 2008; Wu et al., 2007; Zhang et al., 2007). Comparative studies have shown that salt-stressed P. euphratica seedlings accumulate less Na⁺ and Cl⁻ in root and shoot tissues than salt-sensitive poplar species (Chen et al., 2001, 2002). It is suggested that the greater capacity to exclude NaCl in P. euphratica is likely the result of salt uptake and transport restriction in roots (Chen et al., 2002, 2003). However, this needs further investigations, e.g. by electrophysiology, to clarify.In this study, we used a noninvasive ion flux technique to measure the tissue and cellular fluxes of H⁺, Na⁺, and Cl⁻ in roots of the salt-tolerant P. euphratica and salt-sensitive P. popularis 35-44. The aim was to compare the NaCl-induced alternations of ion fluxes in poplar species differing in salt tolerance. Furthermore, we examined the effects of pH, salt shock, and PM transport inhibitors on Na⁺ and H⁺ fluxes in root-derived protoplasts of the salt-tolerant species, P. euphratica, which exhibited an evident Na⁺ exclusion under saline conditions.     

The Road to Gold: Training and Peaking Characteristics in the Year Prior to a Gold Medal Endurance Performance

Purpose

To describe training variations across the annual cycle in Olympic and World Champion endurance athletes, and determine whether these athletes used tapering strategies in line with recommendations in the literature.

Methods

Eleven elite XC skiers and biathletes (4 male; 28±1 yr, 85±5 mL. min⁻¹. kg⁻¹ , 7 female, 25±4 yr, 73±3 mL. min⁻¹. kg⁻¹ ) reported one year of day-to-day training leading up to the most successful competition of their career. Training data were divided into periodization and peaking phases and distributed into training forms, intensity zones and endurance activity forms.

Results

Athletes trained ∼800 h/500 sessions.year⁻¹, including ∼500 h. year⁻¹ of sport-specific training. Ninety-four percent of all training was executed as aerobic endurance training. Of this, ∼90% was low intensity training (LIT, below the first lactate threshold) and 10% high intensity training (HIT, above the first lactate threshold) by time. Categorically, 23% of training sessions were characterized as HIT with primary portions executed at or above the first lactate turn point. Training volume and specificity distribution conformed to a traditional periodization model, but absolute volume of HIT remained stable across phases. However, HIT training patterns tended to become more polarized in the competition phase. Training volume, frequency and intensity remained unchanged from pre-peaking to peaking period, but there was a 32±15% (P<.01) volume reduction from the preparation period to peaking phase.

Conclusions

The annual training data for these Olympic and World champion XC skiers and biathletes conforms to previously reported training patterns of elite endurance athletes. During the competition phase, training became more sport-specific, with 92% performed as XC skiing. However, they did not follow suggested tapering practice derived from short-term experimental studies. Only three out of 11 athletes took a rest day during the final 5 days prior to their most successful competition.     

Ice Nucleation Activity in Lichens

A newly discovered form of biological ice nucleus associated with lichens is described. Ice nucleation spectra of a variety of lichens from the southwestern United States were measured by the drop-freezing method. Several epilithic lichen samples of the genera Rhizoplaca, Xanthoparmelia, and Xanthoria had nuclei active at temperatures as warm as −2.3°C and had densities of 2.3 × 10⁶ to more than 1 × 10⁸ nuclei g⁻¹ at −5°C (2 to 4 orders of magnitude higher than any plants infected with ice nucleation-active bacteria). Most lichens tested had nucleation activity above −8°C. Lichen substrates (rocks, plants, and soil) showed negligible activity above −8°C. Ice nucleation-active bacteria were not isolated from the lichens, and activity was not destroyed by heat (70°C) or sonication, indicating that lichen-associated ice nuclei are nonbacterial in origin and differ chemically from previously described biological ice nuclei. An axenic culture of the lichen fungus Rhizoplaca chrysoleuca showed detectable ice nucleation activity at −1.9°C and an ice nucleation density of 4.5 × 10⁶ nuclei g⁻¹ at −5°C. It is hypothesized that these lichens, which are both frost tolerant and dependent on atmospheric moisture, derive benefit in the form of increased moisture deposition as a result of ice nucleation.