Appropriate Drought Training Induces Optimal Drought Tolerance by Inducing Stepwise H2O2 Homeostasis in Soybean.

Soybean is considered one of the most drought-sensitive crops, and ROS homeostasis can regulate drought tolerance in these plants. Understanding the mechanism of H2O2 homeostasis and its regulatory effect on drought stress is important for improving drought tolerance in soybean. We used different concentrations of polyethylene glycol (PEG) solutions to simulate the progression from weak drought stress (0.2%, 0.5%, and 1% PEG) to strong drought stress (5% PEG). We investigated the responses of the soybean plant phenotype, ROS level, injury severity, antioxidant system, etc., to different weak drought stresses and subsequent strong drought stresses. The results show that drought-treated plants accumulated H2O2 for signaling and exhibited drought tolerance under the following stronger drought stress, among which the 0.5% PEG treatment had the greatest effect. Under the optimal treatment, there was qualitatively describable H2O2 homeostasis, characterized by a consistent increasing amplitude in H2O2 content compared with CK. The H2O2 signal formed under the optimum treatment induced the capacity of the antioxidant system to remove excess H2O2 to form a primary H2O2 homeostasis. The primary H2O2 homeostasis further induced senior H2O2 homeostasis under the following strong drought and maximized the improvement of drought tolerance. These findings might suggest that gradual drought training could result in stepwise H2O2 homeostasis to continuously improve drought tolerance.

Response and inversion of skewness parameters to meteorological factors based on RGB model of leaf color digital image.

In the natural environment, complex and changeable meteorological factors can influence changes in the internal physiology and phenotype of crops. It is important to learn how to convert complex meteorological factor stimuli into plant perception phenotypes when analyzing the biological data obtained under the natural field condition. We restored the true gradation distribution of leaf color, which is also known as the skewed distribution of color scale, and obtained 20 multi-dimensional color gradation skewness-distribution (CGSD) parameters based on the leaf color skewness parameter system. Furthermore, we analyzed the correlation between the five corresponding meteorological factors and canopy CGSD parameters of peppers growing in a greenhouse and cabbages growing in an open air environment, built response model and inversion mode of leaf color to meteorological factors. Based on the analysis, we find a new method for correlating complex environmental problems with multi-dimensional parameters. This study provides a new idea for building a correlation model that uses leaf color as a bridge between meteorological factors and plants internal physiological state.

α-naphthaleneacetic acid positively regulates soybean seed germination and seedling establishment by increasing antioxidant capacity, triacylglycerol mobilization and sucrose transport under drought stress.

Drought stress is an important constraint for the germination of soybean (Glycine max [L.] Merr.) seeds and seedling establishment. A pot experiment was conducted to determine the effects of priming soybean seeds with 5 µM α-naphthaleneacetic acid (NAA) and the mechanism responsible for the induced tolerance of drought stress (soil relative water content of 55%). NAA priming inhibited drought-induced oxidative damage in seeds, and further analysis indicated that it induced an early spike in hydrogen peroxide content by the upregulation of abscisic acid-dependent GmRbohC2, resulting in an enhancement of antioxidant capacity. Moreover, NAA priming also improved the hydrolysis of triacylglycerol (TAG) to sucrose in stressed cotyledons by causing a 2- to 5-fold increase in the transcript levels of GmSDP1, GmACX2, GmMFP2, GmICL, GmMLS, GmGLI1, GmPCK1, GmFBPase1, GmSPS1 and GmSPS2. Consistently, it upregulated the expression levels of GmSUT1, GmCWINV1 and GmMST2 under drought stress, thus enhancing the transport of sucrose from cotyledons to embryonic axes, providing carbon skeletons and energy for axis growth. The seed germination percentage increased by 208.1% at 21 h after sowing, and seedling establishment percentage increased by 47.8% at 14 days after sowing. Collectively, the positive effects of NAA priming on seed germination and seedling establishment can be attributed to enhanced antioxidant ability in seeds, TAG mobilization in cotyledons and sucrose transport from cotyledons to embryonic axes under drought stress.

Beneficial effects of triadimefon in overcoming drought stress in soybean at fluorescence stage.

Soybean (Glycine max [L.] Merr.) at fluorescence stage frequently experiences drought stress. Although triadimefon has been observed to improve drought tolerance of plants, reports on its role in drought resistance on leaf photosynthesis and assimilate transport are limited. This study examined the effects of triadimefon on leaf photosynthesis and assimilate transport at fluorescence stage of soybean experiencing drought stress. Results showed that triadimefon application relieved the inhibitory effects of drought stress on photosynthesis and increased RuBPCase activity. Drought increased soluble sugar contents, yet reduced starch content in the leaves by heightening the activities of sucrose phosphate synthase (SPS), fructose-1,6-bisphosphatase (FBP), invertase (INV), and amylolytic enzyme, impeding the translocation of carbon assimilates to roots and reducing plant biomass. Nevertheless, triadimefon elevated starch content and minimized sucrose degradation by augmenting sucrose synthase (SS) activity and restraining the activities of SPS, FBP, INV, and amylolytic enzyme compared with drought alone, regulating the carbohydrate balance of drought-stressed plants. Therefore, triadimefon application could reduce the photosynthesis inhibition and regulate the carbohydrate balance of drought-stressed soybean plants to lessen the impacts of drought on soybean biomass.

The Effect of Quadruple Therapy with Polaprezinc or Bismuth on Gut Microbiota after Helicobacter pylori Eradication: A Randomized Controlled Trial.

Background: Quadruple therapy with polaprezinc provided an alternative to Helicobacter pylori eradication; however, the effect on gut microbiota remains uncertain. This study aims to identify whether polaprezinc-containing quadruple therapy causes adverse microbiota effects among asymptomatic adults, compared with bismuth therapy. Methods: This was a randomized control trial. One hundred asymptomatic H. pylori-infected adults were randomly (1:1) assigned to two treatment groups (polaprezinc-containing therapy, PQT; or bismuth-containing therapy, BQT). Fecal samples were collected from subjects before and 4−8 weeks after therapy. Samples were sequenced for the V4 regions of the 16S rRNA gene. Results: The relative abundance of the three dominant bacterial phyla (Bacteroidota, Firmicutes, and Proteobacteria) accounted for more than 95% of each treatment group. The alpha diversity between eradications that succeeded and those that failed had no significant difference (p > 0.05). After successful eradication, the alpha diversity in the BQT group decreased in comparison with the baseline (p < 0.05). Subjects who were successfully eradicated by BQT showed considerably lower alpha diversity indices than those of the PQT at follow-up (p < 0.05). The abundance of Parasutterella in subjects who were successfully eradicated by PQT was four times greater than that of BQT (q < 0.05). Conclusion: A 14-day PQT may be superior to BQT in maintaining short-term gut microbiota homeostasis after H. pylori treatment. Our findings preliminarily provide evidence of the short-term impacts of the gut microbiota after PQT treatment of H. pylori infection.

The variants in PTPRB, TRAF3IP3, and DISC1 genes were associated with Graves' disease in the Chinese population.

Previously, a case series study was conducted on our part in which 5 patients with Graves' disease (GD) were collected from a 3-generation family to screen for susceptibility genes responsible for GD. The single nucleotide variants of Microtubule-associated protein 7 domain containing 2 c. 452C > T, p. Ala151Val, Solute carrier family 1 member 7 c. 1204C > T, p. Arg402Cys, tumor necrosis factor receptor-associated factor 3 interacting protein 3 (TRAF3IP3) c. 209A > T, p. Asn70Ile, protein tyrosine phosphatase receptor type B (PTPRB) c. 3472A > G, p. Ser1158Gly, Phosphoinositide-3-kinase regulatory subunit 3 c. 121C > T, p. Pro41Ser, disrupted in schizophrenia 1 (DISC1), c. 1591G > C p. Gly531Arg were associated with the familial GD. We then further confirmed these variants and investigated whether other mutations render susceptibility to GD. The case-control study collected patients with sporadic GD or no GD family history. A snapshot program was used for genotyping the selected SNPs in 235 GD patients (GD group 1) and 284 healthy patients (control group). Furthermore, another 184 GD patients were recruited (GD group 2) to sequence the specified exons of these genes. The sequenced data was compared with Chinese Millionome Database (CMDB). Several variants of PTPRB, phosphoinositide-3-kinase regulatory subunit 3, TRAF3IP3, and DISC1 were found in GD group 2 but not in CMDB. Moreover, the allele frequency of SNP rs2076150 (TRAF3IP3) and rs2492367 DISC1 in GD group 2 was significantly higher than that of in CMDB (all P < .05). When the control group or CMDB was set as a reference group, a significantly higher frequency in alter allele C of SNP rs186466118 PTPRB was observed in GD group 1 and GD group (constituted by GD group 1 and GD group 2). Equally importantly, there was a correlation between the allele C of SNP rs186466118 and the increased risk of GD susceptibility (all P < .05). PTPRB, TRAF3IP3, and DISC1 may be susceptibility genes for GD, and more variants of PTPRB, TRAF3IP3, and DISC1 were found in GD patients.

Cabergoline possesses a beneficial effect on blood-brain barrier (BBB) integrity against lipopolysaccharide (LPS).

Sepsis is a disease induced by severe systemic inflammation and contributes to multiple acute organic dysfunctions. It is reported that disrupted blood-brain barrier (BBB) integrity is involved in sepsis-associated encephalopathy (SAE), which can be alleviated by repairing the damaged tight junction structure. Cabergoline is a specific dopamine D2 receptor agonist developed to treat Parkinson's disease and hyperprolactinemia and is reported to exert promising anti-inflammatory properties. The present study aimed to explore the beneficial effect of Cabergoline for the treatment of sepsis. In the animal experiments, mice were separated into 4 groups: sham, LPS (5 mg/kg), Cabergoline (0.1 mg/kg/day), and Cabergoline+LPS. We found that the increased neurological deficits, disrupted BBB integrity, elevated production of inflammatory factors, and declined expression level of zonula occludens-1 (ZO-1) were observed in lipopolysaccharide (LPS)-treated mice, all of which were significantly reversed by the administration of Cabergoline. In the in vitro model, human brain microvascular endothelial cells (HBMECs) were challenged with 1 µg/mL LPS in the presence or absence of Cabergoline (10, 20 µM) for 24 hours. The elevated cell permeability Papp value of fluorescein disodium across the HBMECs monolayer and declined trans-endothelial electrical resistance (TEER) in the LPS-treated HBMECs were significantly alleviated by Cabergoline, accompanied by the upregulation of ZO-1. In addition, wnt1 and ß-catenin were found downregulated, which was reversed by Cabergoline. Importantly, the protective benefits of Cabergoline were all abolished by the overexpression of Dickkopf 3 (DKK3). Taken together, our data reveal that Cabergoline possessed a protective effect on BBB integrity against LPS.

High-Curie Temperature Multilayered Hybrid Double Perovskite Photoferroelectrics Induced by Aromatic Cation Alloying.

Due to the breakthrough development of layered hybrid perovskites, the multilayered hybrid double perovskites have emerged as outstanding semiconducting materials owing to their environmental friendliness and superior stability. Despite recent booming advances, the realization of above-room temperature ferroelectricity in this fascinating family remains a huge challenge. Herein, when the molecular design strategy of aromatic cation alloying is applied, an above-room temperature "green" bilayered hybrid double perovskite photoferroelectric, (C6H5CH2NH3)2CsAgBiBr7 (BCAB), is successfully developed with a notable saturation polarization of 10.5 µC·cm-2 and high-Curie temperature (Tc ∼ 483 K). Strikingly, such a Tc achieves a new record in multilayered hybrid perovskite ferroelectrics, which extends the ferroelectric working temperature to a high level. Further computational investigation reveals that the high-Tc originated from the high phase-transition energy barrier switched by the rotation of the aromatic cation in the confined environment of the inorganic layers. In addition, benefiting from the attractive polarization and remarkable photoelectric properties, a bulk photovoltaic effect (BPVE) with a prominent zero-bias photocurrent (2.5 µA·cm-2) is achieved. As far as we know, such a high-Tc multilayered hybrid double perovskite ferroelectric is unprecedented, which sheds light on the rational design of an environmental photoferroelectric for high performance photoelectric devices.

Giant room temperature electrocaloric effect in a layered hybrid perovskite ferroelectric: [(CH3)2CHCH2NH3]2PbCl4.

Electrocaloric effect driven by electric fields displays great potential in realizing highly efficient solid-state refrigeration. Nevertheless, most known electrocaloric materials exhibit relatively poor cooling performance near room temperature, which hinders their further applications. The emerging family of hybrid perovskite ferroelectrics, which exhibits superior structural diversity, large heat exchange and broad property tenability, offers an ideal platform. Herein, we report an exceptionally large electrocaloric effect near room temperature in a designed hybrid perovskite ferroelectric [(CH3)2CHCH2NH3]2PbCl4, which exhibits a sharp first-order phase transition at 302 K, superior spontaneous polarization (>4.8 µC/cm2) and relatively small coercive field (<15 kV/cm). Strikingly, a large isothermal entropy change ΔS of 25.64 J/kg/K and adiabatic temperature change ΔT of 11.06 K under a small electric field ΔE of 29.7 kV/cm at room temperature are achieved, with giant electrocaloric strengths of isothermal ΔS/ΔE of 0.86 J·cm/kg/K/kV and adiabatic ΔT/ΔE of 370 mK·cm/kV, which is larger than those of traditional ferroelectrics. This work presents a general approach to the design of hybrid perovskite ferroelectrics, as well as provides a family of candidate materials with potentially prominent electrocaloric performance for room temperature solid-state refrigeration.

Efficacy of quadruple regimen with polaprezinc for gastric Helicobacter pylori infection eradication: protocol for a single-centre, single-blind, non-inferiority, randomised clinical trial.

INTRODUCTION: Helicobacter pylori (H. pylori) is the most well-known risk factor for gastric cancer. At present, H. pylori shows varying levels of resistance to different treatments, leading to a lower rate of H. pylori eradication. The aim of this study is to evaluate the efficacy of polaprezinc-containing quadruple therapy (PQT) for the eradication of H. pylori infection and, thus, to provide more evidence to inform the clinical treatment of H. pylori infection in China. METHODS AND ANALYSIS: This is a single-centre, single-blind, non-inferiority, randomised controlled trial, enrolling 158 patients with H. pylori infection. Patients are randomised (1:1) to the two groups for a 14-day therapy. Treatment group: PQT (esomeprazole 20 mg, amoxicillin 1 g, clarithromycin 500 mg, polaprezinc 75 mg) two times per day; control group: bismuth-containing quadruple therapy (esomeprazole 20 mg, amoxicillin 1 g, clarithromycin 500 mg, bismuth potassium citrate 220 mg) two times per day. The primary outcome is the rate of H. pylori eradication. Secondary outcomes are the incidence of adverse events and the gastrointestinal microbiota distribution. The 16S ribosomal RNA (16S rRNA) next-generation sequencing (NGS) is used to evaluate the effect of two different therapies on the distribution of the gastrointestinal microbiota. ETHICS AND DISSEMINATION: This study was approved by the Ethics Committee of Sichuan Cancer Center & Hospital (No. SCCHEC-02-2019-015). Any amendment to the research protocol will be submitted for ethical approval. All participants must provide informed consent. On completion, the results of the study will be published in the appropriate peer-reviewed journal. TRIAL REGISTRATION NUMBER: ChiCTR1900025800; preresults.

Skewed distribution of leaf color RGB model and application of skewed parameters in leaf color description model.

BACKGROUND: Image processing techniques have been widely used in the analysis of leaf characteristics. Earlier techniques for processing digital RGB color images of plant leaves had several drawbacks, such as inadequate de-noising, and adopting normal-probability statistical estimation models which have few parameters and limited applicability. RESULTS: We confirmed the skewness distribution characteristics of the red, green, blue and grayscale channels of the images of tobacco leaves. Twenty skewed-distribution parameters were computed including the mean, median, mode, skewness, and kurtosis. We used the mean parameter to establish a stepwise regression model that is similar to earlier models. Other models based on the median and the skewness parameters led to accurate RGB-based description and prediction, as well as better fitting of the SPAD value. More parameters improved the accuracy of RGB model description and prediction, and extended its application range. Indeed, the skewed-distribution parameters can describe changes of the leaf color depth and homogeneity. CONCLUSIONS: The color histogram of the blade images follows a skewed distribution, whose parameters greatly enrich the RGB model and can describe changes in leaf color depth and homogeneity.

Proteomic analysis of tree peony (Paeonia ostii 'Feng Dan') seed germination affected by low temperature.

Seed germination is a critical process that is influenced by various factors. In the present study, the effect of low temperature (4 °C) on tree peony seed germination was investigated. Compared to seeds maintained at 25 °C, germination was inhibited when seeds were kept at 4 °C. Furthermore, low-temperature exposure of seeds resulted in a delay in water uptake, starch degradation, and soluble sugar consumption and a subsequent increase in soluble protein levels. Two-dimensional gel electrophoresis (2-DE) proteomic analysis identified 100 protein spots. Comparative analysis indicated that low-temperature exposure apparently mainly affected glycolysis and the tricarboxylic acid (TCA) cycle, while also significantly affecting proteometabolism-related factors. Moreover, low-temperature exposure led to the induction of abscisic acid, whereas the gibberellin pathway was not affected. Further comparison of the two temperature conditions showed that low-temperature exposure delays carbohydrate metabolism, adenosine triphosphate (ATP) production, respiration, and proteolysis and increases defense response factors. To further examine the obtained proteomic findings, four genes were evaluated by quantitative polymerase chain reaction (qPCR). The obtained transcriptional results for the GAPC gene coincided with the translational results, thus further suggesting that the delay in glycolysis may play a key role in low-temperature-induced inhibition of seed germination. However, the other three genes examined, which included FPP synthase, PCNT115, and endochitinase, showed non-correlative transcriptional and translational profiles. Our results suggest that the exposure of tree peony seeds to low temperature results in a delay in the degradation of starch and other metabolites, which in turn affects glycolysis and some other processes, thereby ultimately inhibiting seed germination.

Proteome Dynamics and Physiological Responses to Short-Term Salt Stress in Brassica napus Leaves.

Salt stress limits plant growth and crop productivity and is an increasing threat to agriculture worldwide. In this study, proteomic and physiological responses of Brassica napus leaves under salt stress were investigated. Seedlings under salt treatment showed growth inhibition and photosynthesis reduction. A comparative proteomic analysis of seedling leaves exposed to 200 mM NaCl for 24 h, 48 h and 72 h was conducted. Forty-four protein spots were differentially accumulated upon NaCl treatment and 42 of them were identified, including several novel salt-responsive proteins. To determine the functional roles of these proteins in salt adaptation, their dynamic changes in abundance were analyzed. The results suggested that the up-accumulated proteins, which were associated with protein metabolism, damage repair and defense response, might contribute to the alleviation of the deleterious effect of salt stress on chlorophyll biosynthesis, photosynthesis, energy synthesis and respiration in Brassica napus leaves. This study will lead to a better understanding of the molecular basis of salt stress adaptation in Brassica napus and provides a basis for genetic engineering of plants with improved salt tolerance in the future.

[Effects of exogenous α-naphthaleneacetic aid on the antioxidation system in soybean leaves subjected to long-term drought stress during flowering].

Two different drought tolerance soybean ( Glycine max) varieties (Nannong 99-6 and Kefeng 1) were used to study the effects of α-naphthaleneacetic acid (NAA) on the antioxidation system under long-term drought stress after flowering with pot experiment, which lasted for 110 days at Pailou Experiment Station, Nanjing Agricultural University, in 2012. The results showed that long-term stress decreased the shoot dry mass significantly, however, increased the level of reactive oxygen species (ROS) and malondialdehyde (MDA) content. It also obviously increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) , ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR) and glutathione peroxidase (GPX). The contents of ascorbic acid (AsA) and glutathione (GSH), and the ratios of AsA/DHA (dehydroascorbic acid) and GSH/GSSG (L-glutathione oxidized) were obviously enhanced. Kefeng 1 showed a higher antioxidation ability than Nannong 99-6, and could consequently maintain lower ROS and MDA levels. NAA distinctly enhanced the activities of APX, POD, CAT, MDHAR, GPX, and ratios of AsA/DHA and GSH/GSSG, while decreased the levels of ROS and MDA. The AsA content and dehydroascorbate reductase (DHAR) activity were significantly increased in Kefeng 1.

Adverse effects of preserved vegetables on squamous cell carcinoma of esophagus and precancer lesions in a high risk area.

INTRODUCTION: Squamous cell carcinoma of esophagus (ESCC) is one of the most common cancers in China. Preserved vegetables are processed foods, consumed in high amounts in the high risk areas for ESCC. This study aimed to investigate the relationships of preserved vegetable consumption with SCC and precancer lesions. METHODS: Cases from Yanting cancer hospital with pathological diagnosis of primary cancer, along with controls and individuals diagnosed with precancer lesions by endoscopy with iodine staining were interviewed. Trained staff collected data on dietary habits 1 year before the interview. An unconditional logistic regression model was used to estimate the risk odds ratios for preserved vegetable consumption with precancer lesions and cancer. RESULTS: Adjusting for potential confounders, intake of preserved vegetables (OR=2.92, 95%CI 1.32~6.47) and longer intake period (OR=5.78, 95%CI 2.26~14.80) were associated with higher risk of cancer. Compared with lowest intake frequency, the highest was associated with a 3.0-fold risk for precancer lesions and 3.59-fold risk for ESCC (both p<0.05). CONCLUSION: Consumption of preserved vegetables is a risk factor for esophageal lesions in high risk areas. The carcinogenicity of preserved vegetables needs investigation in further studies and public health strategies for reduction of consumption might be initiated in high risk areas.

Esophageal cancer mortality during 2004-2009 in Yanting County, China.

OBJECTIVE: Yanting County is a high risk area for esophageal cancer (EC) in China. The purpose of this study was to describe the mortality and mortality change of EC from 2004 to 2009 in Yanting County. METHODS: EC mortality data from 2004 to 2009 obtained from the Cancer Registry in Yanting were analyzed. Annual percentage changes (APC) were calculated to assess the trends in EC mortality. Age-standardized mortality was calculated based on world standard population of 2000. RESULTS: The average EC mortality was 54.7/105 in males and 31.6/105 in females over the 6 years. A decline in EC mortality with time was observed in both genders, with a rate of -8.70% per year (95% CI: -13.23%~-3.93%) in females and -4.11% per year (95%CI: -11.16%~3.50%) in males. CONCLUSION: EC mortality decreased over the six years in both genders, although it remained high in the Yanting area. There is still a need to carry out studies of risk factors for improved cancer prevention and further reduction in the disease burden.

[Influence of different duration of waterlogging on the growth and C and N metabolism of soybean at seedling and flowering stages].

A pot experiment with soybean cultivar Nannong 99-6 was conducted to study its growth and C and N metabolism at seedling and flowering stages under the stress of different duration of waterlogging. Waterlogging inhibited the soybean growth. The plant biomass, leaf area, leaf pigment content, and photosynthesis rate all decreased significantly, and the leaf malonyldialdehyde (MDA) content had a significant increase. The change ranges of the physiological indices increased with increasing waterlogging duration. After the release of the stress, the plants had some recovery. The recovery capability in treatment waterlogging for 10 d was stronger than that in the treatment waterlogging for 20 d. The leaf soluble C and N and key enzymes had different responses to waterlogging. Soluble sugar content and glutamine synthease and sucrose synthease activities all increased, but soluble protein content decreased. The influence of waterlogging on the plant biomass, leaf area, and MDA content was smaller at seedling stage than at flowering stage. It was suggested that the shorter the waterlogging duration was, the lesser damage the soybean plant suffered, and the stronger the recovery capability was. Soybean plant could recover itself after waterlogging for 10 d.

[Effects of simulated acid rain on oilseed rape (Brassica napus) physiological characteristics at flowering stage and yield].

A pot experiment was conducted to study the effects of different acidity simulated acid rain on the physiological characteristics at flowering stage and yield of oilseed rape (B. napus cv. Qinyou 9). Comparing with the control (pH 6.0), weak acidity (pH = 4.0-5.0) simulated acid rain stimulated the rape growth to some extent, but had less effects on the plant biomass, leaf chlorophyll content, photosynthetic characteristics, and yield. With the further increase of acid rain acidity, the plant biomass, leaf chlorophyll content, photosynthetic rate, antioxidative enzyme activities, and non-enzyme antioxidant contents all decreased gradually, while the leaf malonyldialdehyde (MDA) content and relative conductivity increased significantly. As the results, the pod number per plant, seed number per pod, seed weight, and actual yield decreased. However, different yield components showed different sensitivity to simulated acid rain. With the increasing acidity of simulated acid rain, the pod number per plant and the seed number per pod decreased significantly, while the seed weight was less affected.