Different responses of Japanese encephalitis to weather variables among eight climate subtypes in Gansu, China, 2005-2019.

This study evaluated epidemic temporal aspects of Japanese encephalitis (JE) and investigated the weather threshold of JE response across eight climate subtypes between 2005 and 2019 in Gansu Province, China. Epidemiological data were collected from the China Information System for Disease Control and Prevention (CISDCP). Three epidemic temporal indices [frequency index (α), duration index (ß), and intensity index (γ)] were adopted for the comparison of epidemic features among different climate subtypes. In addition, the local indicators of spatial association (LISA) technique was used to detect the hot-spot areas. The category and regression tree (CART) model was used to detect the response threshold of weather variables in hot-spot areas across climate subtypes. Among eight climate subtypes in Gansu, in most hot-spot areas (i.e., high-high clusters), α, ß, and γ were detected in the climate subtypes of subtropical winter dry (Cwa), temperate oceanic continental (Cwb), and continental winter dry (Dwa and Dwb). According to the CART analysis, a minimum monthly temperature is required for Japanese encephalitis virus (JEV) transmission, with different threshold values among the climatic subtypes. In temperate climate zones (Cwa and Cwb), this threshold is 19 °C at a 1-month lag. It is lower in continental winter dry climate zones: 18 °C in Dwa (snow climate, dry winter, and hot summer) and 16 °C in Dwb (snow climate, dry winter, and warm summer). Additionally, some areas of the areas with temperate arid (BWk and BSk) had the first JE cases. Further studies to detect whether the climate change influence the JEV's distribution in Gansu Province are needed.

Selenium nanoparticles improve nickel-induced testosterone synthesis disturbance by down-regulating miR-708-5p/p38 MAPK pathway in Leydig cells.

The present study was designed to investigate the role of miR-708-5p/p38 mitogen-activated protein kinase (MAPK) pathway during the mechanism of selenium nanoparticles (Nano-Se) against nickel (Ni)-induced testosterone synthesis disorder in rat Leydig cells. We conducted all procedures based on in vitro culture of rat primary Leydig cells. After treating Leydig cells with Nano-Se and NiSO4 alone or in combination for 24 h, we determined the cell viability, reactive oxygen species (ROS) levels, testosterone production, and the protein expression of key enzymes involved in testosterone biosynthesis: steroidogenic acute regulatory (StAR) and cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1). The results indicated that Nano-Se antagonized cytotoxicity and eliminated ROS generation induced by NiSO4 , suppressed p38 MAPK protein phosphorylation and reduced miR-708-5p expression. Importantly, we found that Nano-Se upregulated the expression of testosterone synthase and increased testosterone production in Leydig cells. Furthermore, we investigated the effects of p38 MAPK and miR-708-5p using their specific inhibitor during Nano-Se against Ni-induced testosterone synthesis disorder. The results showed that Ni-inhibited testosterone secretion was alleviated by Nano-Se co-treatment with p38 MAPK specific inhibitor SB203580 and miR-708-5p inhibitor, respectively. In conclusion, these findings suggested Nano-Se could inhibit miR-708-5p/p38 MAPK pathway, and up-regulate the key enzymes protein expression for testosterone synthesis, thereby antagonizing Ni-induced disorder of testosterone synthesis in Leydig cells.

Nano-selenium attenuates mitochondrial-associated apoptosis via the PI3K/AKT pathway in nickel-induced hepatotoxicity in vivo and in vitro.

The aim of this study was to investigate the protective effects of Nano-Se against nickel (Ni)-induced hepatotoxicity and the potential mechanism. Hence, we constructed in vivo and in vitro models of Ni-induced hepatotoxicity. Sprague-Dawley (SD) rats were exposed to nickel sulfate (NiSO4 , 5.0 mg/kg, i.p.) with or without Nano-Se (0.5, 1, and 2 mg/kg, oral gavage) co-administration for 14 days, and HepG2 cells were exposed to NiSO4 (1500 µM) with or without Nano-Se (20 µM) for 24 h. Nano-Se obviously prevented Ni-induced hepatotoxicity indicated by ameliorating pathological change and decreasing Ni accumulation in rat livers. Ni induced a significant increase in hepatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSH-Px), and malondialdehyde (MDA) level, decreased the glutathione (GSH) content while compared to those in the control group. Nano-Se administration improved the hepatic antioxidant capacity through increase hepatic GSH contents and GSH-Px activity, decrease the activities of SOD, CAT, and MDA level. Nano-Se improved the cell viability, decreased active oxygen (ROS) generation and ameliorated morphological changes of nuclear structures in Ni-treated HepG2 cells. In addition, Nano-Se inhibited the Ni-induced increases of cytochrome c, caspase-9, cleaved caspase-3, increased PI3K and AKT phosphorylation both in vivo and in vitro. Besides, the PI3K inhibitor Y294002 could inhibit the protective effects of Nano-Se on apoptosis. Thus, Nano-Se significantly activates PI3K/AKT signaling to ameliorate apoptosis in Ni-induced hepatotoxicity.

[Differences in non-suicidal self-injury behaviors between only-child and non-only-child adolescents with mood disorders: a cross-sectional study]. / ç‹¬ç”Ÿä¸Žéžç‹¬ç”Ÿé’å°‘å¹´æƒ ç»ªéšœç¢æ‚£è€ éžè‡ªæ€æ€§è‡ªä¼¤è¡Œä¸ºå·®å¼‚çš„æ¨ªæ–­é¢ç ”ç©¶.

OBJECTIVES: To investigate the differences in non-suicidal self-injury (NSSI) behaviors between only-child and non-only-child adolescents with mood disorders. METHODS: A three-stage sampling method was used to perform a cross-sectional survey of 529 adolescents, aged 12-18 years, who had mood disorders and NSSI behaviors. These adolescents were sampled from the outpatient service of 20 mental hospitals in 9 provinces of China from August to November 2020. A self-made questionnaire was used to collect general demographic data. The Functional Assessment of Self-Mutilation, Beck Scale for Suicide Ideation, Kessler Psychological Distress Scale, Stress Mindset Measure-General, Multidimensional Scale of Perceived Social Support, Multidimensional Students' Life Satisfaction Scales, and Rosenberg Self-Esteem Scale were used to collect the information on self-injury behaviors and psychological factors in these adolescents. RESULTS: A total of 529 adolescents with mood disorders and NSSI behaviors were surveyed, among whom 375 were only-child adolescents and 154 were non-only-child adolescents. Compared with the non-only-child group, the only-child group had a significantly higher total score of Functional Assessment of Self-Mutilation (P<0.05) .The type and frequency of self-injury in the only-child group were significantly higher than those in the non-only-child group (P<0.05). Psychological analysis showed that compared with the non-only-child group, the only-child group had a significantly lower score of self-esteem (P<0.05) and significantly higher scores of psychological distress and depressive symptoms (P<0.05). The multiple linear regression analysis showed that the score of suicidal ideation was positively correlated with the frequency of NSSI behaviors in both only-child and non-only-child adolescents with mood disorders (P<0.05); in the only-child adolescents, the level of self-esteem was negatively correlated with the frequency of NSSI behaviors (P<0.05), and the score of stress perception was positively correlated with the frequency of NSSI behaviors (P<0.05); in the non-only-child adolescents, the score of anxious emotion was positively correlated with the frequency of NSSI behaviors (P<0.05). CONCLUSIONS: Among the adolescents with mood disorders and NSSI behaviors, the only-child adolescents tend to have a higher frequency of self-injury and poorer mental health, and therefore, the only-child adolescents with mood disorders and NSSI behaviors need more attention.

Overexpression of HbMBF1a, encoding multiprotein bridging factor 1 from the halophyte Hordeum brevisubulatum, confers salinity tolerance and ABA insensitivity to transgenic Arabidopsis thaliana.

KEY MESSAGE: HbMBF1a was isolated and characterized in H. brevisubulatum, and overexpressed HbMBF1a could enhance the salt tolerance and ABA insensitivity in Arabidopsis thaliana. The transcript levels of stress-responsive genes were significantly increased in the transgenic lines under salt and ABA conditions. Salinity is an abiotic stress that considerably affects plant growth, yield, and distribution. Hordeum brevisubulatum is a halophyte that evolved to become highly tolerant to salinity. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator and an important regulator of stress tolerance. In this study, we isolated and characterized HbMBF1a based on the transcriptome data of H. brevisubulatum grown under saline conditions. We overexpressed HbMBF1a in Arabidopsis thaliana and compared the phenotypes of the transgenic lines and the wild-type in response to stresses. The results indicated that HbMBF1a expression was induced by salt and ABA treatments during the middle and late stages. The overexpression of HbMBF1a in A. thaliana resulted in enhanced salt tolerance and ABA insensitivity. More specifically, the enhanced salt tolerance manifested as the increased seed germination and seedling growth and development. Similarly, under ABA treatments, the cotyledon greening rate and seedling root length were higher in the HbMBF1a-overexpressing lines, suggesting the transgenic plants were better adapted to high exogenous ABA levels. Furthermore, the transcript levels of stress-responsive genes were significantly increased in the transgenic lines under salt and ABA conditions. Thus, HbMBF1a is a positive regulator of salt and ABA responses, and the corresponding gene may be useful for producing transgenic plants that are salt tolerant and/or ABA insensitive, with few adverse effects. This study involved a comprehensive analysis of HbMBF1a. The results may provide the basis and insight for the application of MBF1 family genes for developing stress-tolerant crops.

Emerging crosstalk between two signaling pathways coordinates K+ and Na+ homeostasis in the halophyte Hordeum brevisubulatum.

K+/Na+ homeostasis is the primary core response for plant to tolerate salinity. Halophytes have evolved novel regulatory mechanisms to maintain a suitable K+/Na+ ratio during long-term adaptation. The wild halophyte Hordeum brevisubulatum can adopt efficient strategies to achieve synergistic levels of K+ and Na+ under high salt stress. However, little is known about its molecular mechanism. Our previous study indicated that HbCIPK2 contributed to prevention of Na+ accumulation and K+ reduction. Here, we further identified the HbCIPK2-interacting proteins including upstream Ca2+ sensors, HbCBL1, HbCBL4, and HbCBL10, and downstream phosphorylated targets, the voltage-gated K+ channel HbVGKC1 and SOS1-like transporter HbSOS1L. HbCBL1 combined with HbCIPK2 could activate HbVGKC1 to absorb K+, while the HbCBL4/10-HbCIPK2 complex modulated HbSOS1L to exclude Na+. This discovery suggested that crosstalk between the sodium response and the potassium uptake signaling pathways indeed exists for HbCIPK2 as the signal hub, and paved the way for understanding the novel mechanism of K+/Na+ homeostasis which has evolved in the halophytic grass.

Halophytic Hordeum brevisubulatum HbHAK1 Facilitates Potassium Retention and Contributes to Salt Tolerance.

Potassium retention under saline conditions has emerged as an important determinant for salt tolerance in plants. Halophytic Hordeum brevisubulatum evolves better strategies to retain K+ to improve high-salt tolerance. Hence, uncovering K+-efficient uptake under salt stress is vital for understanding K+ homeostasis. HAK/KUP/KT transporters play important roles in promoting K+ uptake during multiple stresses. Here, we obtained nine salt-induced HAK/KUP/KT members in H. brevisubulatum with different expression patterns compared with H. vulgare through transcriptomic analysis. One member HbHAK1 showed high-affinity K+ transporter activity in athak5 to cope with low-K+ or salt stresses. The expression of HbHAK1 in yeast Cy162 strains exhibited strong activities in K+ uptake under extremely low external K+ conditions and reducing Na+ toxicity to maintain the survival of yeast cells under high-salt-stress. Comparing with the sequence of barley HvHAK1, we found that C170 and R342 in a conserved domain played pivotal roles in K+ selectivity under extremely low-K+ conditions (10 µM) and that A13 was responsible for the salt tolerance. Our findings revealed the mechanism of HbHAK1 for K+ accumulation and the significant natural adaptive sites for HAK1 activity, highlighting the potential value for crops to promote K+-uptake under stresses.

Unconscious versus conscious thought in creative science problem finding: Unconscious thought showed no advantage!

Unconscious thought theory (UTT) suggests that creativity benefits more from unconscious thought than conscious thought. However, previous studies have only focused on creative problem solving. This study aims to explore the effect of unconscious thought and conscious thought in creative science problem finding (CSPF). The ability of CSPF was measured by fluency, flexibility and originality. Participants accomplished the CSPF task after 3 min of distraction, during which unconscious thought was supposed to take place, or after 3 min of conscious thought. Results showed that unconscious thought had no advantage over conscious thought on CSPF. For the CSPF task with open instructions, conscious thought was comparable to unconscious thought in fluency, flexibility and originality. What's more, for the CSPF task with closed instructions, unconscious thought was even overtaken by conscious thought in fluency, flexibility and originality. These findings extend the unconscious thought theory and provide practical guidance on how to propose a creative science problem.

Foxtail millet SiHAK1 excites extreme high-affinity K+ uptake to maintain K+ homeostasis under low K+ or salt stress.

KEY MESSAGE: This is the first evidence that SiHAK1 acts as a K+ transporter and is modulated by internal and external K+, which expands our understanding of the significant physiological roles of large HAK/KUP/KT transporters in crops. Crop genomes have shown the richness of K+ transporters in HAK/KUP/KT (High Affinity K+/K+ Uptake Proteins/K+ Transporter) family, and much progress have been achieved toward understanding the diverse roles of K+ uptake and translocation, and abiotic stresses resistance in this family. The HAK/KUP/KT family has increasingly been recognized to be at a pivotal status in the mediation of K+ translocation and long-term transport; however, our understanding of the molecular mechanisms remains limited. Foxtail millet is an ideal plant for studying long-distance potassium (K) transport because of its small diploid genome and better adaptability to arid lands. Here, we identified 29 putative HAK/KUP/KT proteins from the Setaria italica genome database. These genes were distributed in seven chromosomes of foxtail millet and divided into five clusters. SiHAK1 exhibited widespread expression in various tissues and significant up-regulation in the shoots under low K condition. SiHAK1 was localized in the cell membrane and low K elicited SiHAK1-meidated high-affinity K+ uptake activity in Cy162 yeast cells and Arabidopsis athak5 mutants. The transport activity of SiHAK1 was coordinately modulated by external K+ supply and internal K+ content in the cell under low K and high salt environment. Our findings reveal the K uptake mechanisms of SiHAK1 and indicated that it may be involved in the mediation of K homeostasis in S. italica under K+-deficiency and salt stress.

X1-homologous genes family as central components in biotic and abiotic stresses response in maize (Zea mays L.).

X1-homologous genes (XHS) encode plant specific proteins containing three basic domains (XH, XS, zf-XS). In spite of their physiological importance, systematic analyses of ZmXHS genes have not yet been explored. In this study, we isolated and characterized ten ZmXHS genes in a whole-of-genome analysis of the maize genome. A total of ten members of this family were identified in maize genome. The ten ZmXHS genes were distributed on seven maize chromosomes. Multiple alignment and motif display results revealed that most ZmXHS proteins share all the three conserved domains. Putative cis-elements involved in abiotic stress responsive, phytohormone, pollen-specific and quantitative, seed development and germination, light and circadian rhythms regulation, Ca(2+)-responsive, root hair cell-specific, and CO(2)-responsive transcriptional activation were observed in the promoters of ZmXHS genes. Yeast hybrid assay revealed that the XH domain of ZmXHS5 was necessary for interaction with itself and ZmXHS2. Microarray data showed that the ZmXHS genes had tissue-specific expression patterns in the maize developmental steps and biotic stresses response. Quantitative real-time PCR analysis results indicated that, except ZmXHS9, the other nine ZmXHS genes were induced in the seedling leaves by at least one of the four abiotic stresses applied.

Isolation, structural analysis, and expression characteristics of the maize (Zea mays L.) hexokinase gene family.

Hexokinases (HXKs, EC 2.7.1.1) play important roles in metabolism, glucose (Glc) signaling, and phosphorylation of Glc and fructose and are ubiquitous in all organisms. Despite their physiological importance, the maize HXK (ZmHXK) genes have not been analyzed systematically. We isolated and characterized nine members of the ZmHXK gene family which were distributed on 3 of the 10 maize chromosomes. A multiple sequence alignment and motif analysis revealed that the maize ZmHXK proteins share three conserved domains. Phylogenetic analysis revealed that the ZmHXK family can be divided into four subfamilies. We identified putative cis-elements in the ZmHXK promoter sequences potentially involved in phytohormone and abiotic stress responses, sugar repression, light and circadian rhythm regulation, Ca(2+) responses, seed development and germination, and CO2-responsive transcriptional activation. To study the functions of maize HXK isoforms, we characterized the expression of the ZmHXK5 and ZmHXK6 genes, which are evolutionarily related to the OsHXK5 and OsHXK6 genes from rice. Analysis of tissue-specific expression patterns using quantitative real time-PCR showed that ZmHXK5 was highly expressed in tassels, while ZmHXK6 was expressed in both tassels and leaves. ZmHXK5 and ZmHXK6 expression levels were upregulated by phytohormones and by abiotic stress.

Genome-wide identification and characterization of FAD family genes in barley.

Fatty acid desaturases (FADs) play pivotal roles in determining plant stress tolerance. Barley is the most salt-tolerant cereal crop. In this study, we performed genome-wide identification and characterization analysis of the FAD gene family in barley (Hordeum vulgare). A total of 24 HvFADs were identified and divided into four subfamilies based on their amino acid sequence similarity. HvFADs unevenly distributed on six of seven barley chromosomes, and three clusters of HvFADs mainly occurred on the chromosome 2, 3 and 6. Segmental duplication events were found to be a main cause for the HvFAD gene family expansion. The same HvFAD subfamily showed the relatively consistent exon-intron composition and conserved motifs of HvFADs. Cis-element analysis in HvFAD promoters indicated that the expression of HvFADs may be subject to complex regulation, especially stress-responsive elements that may involve in saline-alkaline stress response. Combined transcriptomic data with quantitative experiments, at least five HvFADs highly expressed in roots under salt or alkali treatment, suggesting they may participate in saline or alkaline tolerance in barley. This study provides novel and valuable insights for underlying salt/alkali-tolerant mechanisms in barley.

Structural evolution of pea-derived albumins during pH and heat treatment studied with light and X-ray scattering.

Pea albumins are found in the side stream during the isolation of pea proteins. They are soluble at acidic pH and have functional properties which differ from their globulin counterparts. In this study, we have investigated the aggregation and structural changes occurring to pea albumins under different environmental conditions, using a combination of size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and small-angle X-ray scattering (SAXS). Albumins were extracted from a dry fractionated pea protein concentrate by precipitating the globulin fraction at acidic pH. The albumins were then studied at different pH (3, 4, 4.5, 7, 7.5, and 8) values. The effect of heating at 90 °C for 1, 3, and 5 min on their structural changes was investigated using SAXS. In addition, size exclusion of the albumins showed 4 distinct populations, depending on pH and heating conditions, with two large aggregates peaks (∼250 kDa): a dimer peak (∼24 kDa) containing predominantly pea albumin 2 (PA2), and a monomer peak of a molar mass of about 12 kDa (PA1). X-ray scattering intensities as a function of q were modeled as polydisperse spheres, and their aggregation was followed as a function of heating time. Albumins was most stable at pH 3, showing no aggregation during heat treatment. While albumins at pH 7.5 and 8 showed aggregation after heating, solutions at pH 4, 4.5, and 7 already contained aggregates even before heating. This work provides new knowledge on the overall structural development of albumins under different environmental conditions, improving our ability to employ these as future ingredients in foods.

Effects of Heating Rates on Microstructural Evolution of Hot Extruded 7075 Aluminum Alloy in the Semi-Solid State and Thixotropic Deformation Behavior.

The non-dendritic microstructure plays a crucial role in determining the rheological properties of semi-solid alloys, which are of the utmost importance for the successful industrial application of the thixoforging process. To further understand the impact of the reheating process on the evolution of microstructure and thixotropic deformation behavior in the semi-solid state, a hot extruded and T6 treated 7075 aluminum alloy was reheated to the selected temperature ranges using varying heating rates. Subsequently, thixo-compression tests were performed. The study found that during reheating and isothermal holding, the elongated microstructure of the as-supplied alloy can transform into equiaxed or spherical grains. The presence of recrystallized grains was found to be closely linked to the penetration of the liquid phase into the recrystallized grain boundaries. Furthermore, it was observed that higher heating rates resulted in smaller grain sizes. The thixotropic flow behavior of the alloy with various microstructures was analyzed using the true stress-strain curves obtained by thixo-compression experiments, which exhibited three stages: a rapid increase in true stress to a peak value, followed by a decrease in true stress and a steady stress until the end of compression. The stress fluctuated with strain during the formation of the slurry at a strain rate of 10 s-1, indicating the significant role of strain rate in material flow during semisolid formation.

The effects of different types of high-amylose maize starches on viscosity and digestion of acidified milk gels.

High-amylose maize starch (HAMS) can provide dietary fiber to foods. In this study, we investigated the effects of three HAMSs (Gelose 50, Hylon VII, and NAFU50) on the functionality of casein (CA) and/or whey protein (WP) networks in acidified milk gels using normal maize starch (NMS) as a control thickener. When compared with NMS, HAMSs performed better in increasing the resistant starch content (RS), storage modulus, loss modulus, and complex viscosity of all the milk gels. The results are attributed to the retention of the starch granular integrity of HAMSs during the preparation of the milk gels, as observed by microscopy. HylonVII exhibited the highest RS and viscosity in all milk gel systems, especially in WP gels (71.8 % RS and >3000 Pa.s at 1 Hz viscosity). Our data provide support for the potential of using HAMS to develop healthier yogurt products using functional thickeners from natural sources.

A Comparative Study on Microstructure, Segregation, and Mechanical Properties of Al-Si-Mg Alloy Parts Processed by GISS-HPDC and SEED-HPDC.

There are multiple routes to prepare semi-solid slurries with a globular microstructure for semi-solid forming. The variations in the microstructure of semi-solid slurries prepared using different routes may lead to significant differences in the flow behavior and mechanical properties of rheo-diecasting parts. Therefore, it is crucial to have a comprehensive understanding of the microstructure evolution associated with different slurry preparation routes and their resulting effects. In this study, the gas-induced semi-solid process (GISS) and the swirl enthalpy equilibrium device (SEED) routes were employed to prepare semi-solid Al-Si-Mg slurries for their simplicity and productivity in potential industrial applications. The prepared slurries were then injected into the shoot sleeves of a high-pressure die casting (HPDC) machine to produce tensile test bars. Subsequently, the bars underwent T6 treatment to enhance their mechanical properties. The microstructure, segregation, and mechanical properties of the samples were investigated and compared with those of conventional HPDC. The results indicated that the GISS and SEED can produce semi-solid slurries containing a spherical α-Al primary phase, as opposed to the dendritic structure commonly found in conventional castings. The liquid fraction had a significant effect on the flow behavior, resulting in variations in liquid segregation and mechanical properties. It was observed that a higher solid fraction (>75%) had a suppressing effect on surface liquid segregation. In addition, the tendency for liquid segregation gradually increased along the filling direction due to the special flow behavior of the semi-solid slurry with a low solid fraction. Furthermore, under the same die-casting process parameters, the conventional HPDC samples exhibit higher yield stress (139 ± 3 MPa) compared to SEED-HPDC and GISS-HPDC samples, which may be attributed to the small grain size and the distribution of eutectic phases. After undergoing the T6 treatment, both SEED-HPDC and GISS-HPDC samples showed a significant improvement in yield and tensile strength. These improvements are a result of solution and precipitation strengthening effects as well as the spheroidization of the eutectic Si phase. Moreover, the heat-treated SEED-HPDC samples demonstrate higher ultimate strength (336 ± 5 MPa) and elongation (13.7 ± 0.3%) in comparison to the GISS-HPDC samples (307 ± 4 MPa, 8.8 ± 0.2%) after heat treatment, mainly due to their low porosity density. These findings suggest that both GISS-HPDC and SEED-HPDC processes can be utilized to produce parts with favorable mechanical properties by implementing appropriate heat treatments. However, further investigation is required to control the porosities of GISS-HPDC samples during heat treatment.

HbCIPK2, a novel CBL-interacting protein kinase from halophyte Hordeum brevisubulatum, confers salt and osmotic stress tolerance.

Protein kinases play an important role in regulating the response to abiotic stress in plant. CIPKs are plant-specific signal transducers, and some members have been identified. However, the precise functions of novel CIPKs still remain unknown. Here we report that HbCIPK2 is a positive regulator of salt and osmotic stress tolerance. HbCIPK2 was screened out of the differentially expressed fragments from halophyte Hordeum brevisubulatum by cDNA-AFLP technique, and was a single-copy gene without intron. Expression of HbCIPK2 was increased by salt, drought and ABA treatment. HbCIPK2 is mainly localized to the plasma membrane and nucleus. Ectopic expression of 35S:HbCIPK2 not only rescued the salt hypersensitivity in Arabidopsis mutant sos2-1, but also enhanced salt tolerance in Arabidopsis wild type, and exhibited tolerance to osmotic stress during germination. The HbCIPK2 contributed to the ability to prevent K(+) loss in root and to accumulate less Na(+) in shoot resulting in K(+) /Na(+) homeostasis and protection of root cell from death, which is consistent with the gene expression profile of HbCIPK2-overexpressing lines. These findings imply possible novel HbCIPK2-mediated salt signalling pathways or networks in H. brevisubulatum.

Genome-wide analysis and identification of HAK potassium transporter gene family in maize (Zea mays L.).

The high-affinity K(+) (HAK) transporter gene family constitutes the largest family that functions as potassium transporter in plant and is important for various cellular processes of plant life. In spite of their physiological importance, systematic analyses of ZmHAK genes have not yet been investigated. In this paper, we indicated the isolation and characterization of ZmHAK genes in whole-genome wide by using bioinformatics methods. A total of 27 members (ZmHAK1-ZmHAK27) of this family were identified in maize genome. ZmHAK genes were distributed in all the maize 10 chromosomes. These genes expanded in the maize genome partly due to tandem and segmental duplication events. Multiple alignment and motif display results revealed major maize ZmHAK proteins share all the three conserved domains. Phylogenetic analysis indicated ZmHAK family can be divided into six subfamilies. Putative cis-elements involved in Ca(2+) response, abiotic stress adaption, light and circadian rhythms regulation and seed development were observed in the promoters of ZmHAK genes. Expression data mining suggested maize ZmHAK genes have temporal and spatial expression pattern. In all, these results will provide molecular insights into the potassium transporter research in maize.

[Highly sensitive determination of three kinds of amanitins in urine and plasma by ultra performance liquid chromatography-triple quadrupole mass spectrometry coupled with immunoaffinity column clean-up].

Cases of toxic mushroom poisoning occur frequently in China every year. In particular, mushrooms containing amanitins can cause acute liver damage, with high mortality rates. The symptoms of acute liver damage are experienced 9-72 h after consumption of the mushrooms. At this time, the concentration of amanitins in blood and urine is too low to be detected even by the highly sensitive ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS), thus rendering clinical diagnosis and treatment difficult. To this end, a method was developed for the determination of α-amanitin, ß-amanitin and γ-amanitin in urine and plasma by UPLC-MS/MS. Urine and plasma samples were extracted and cleaned up by using an immunoaffinity column. A sample of 2.00 mL urine or 1.00 mL of plasma was diluted with 8.00 mL of phosphate buffer solution (PBS) and then loaded onto the immunoaffinity column at a flow rate of 0.5-1.0 mL/min. After washing the column with 10 mL of PBS and 13 mL of water successively, the bound amanitins were eluted with 3.00 mL of methanol-acetone (1∶1, v/v). The eluent was dried under nitrogen at 55 ℃. The residue was dissolved in 100 µL of 10% (v/v) methanol aqueous solution. The amanitins in urine were concentrated 20 times, while those in plasma were concentrated 10 times. Chromatographic separation was performed on a Kinetex Biphenyl column (100 mm × 2.1 mm, 1.7 µm) with gradient elution using methanol and 0.005% (v/v) formic acid aqueous solution as mobile phases. The three amanitins were detected by negative electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode and quantified by the solvent standard curve external standard method. Method validation was performed as recommended by the European Drug Administration (EMEA). Four levels of quality control (QC) samples were prepared, which covered the calibration curve range, viz., the limit of quantification (LOQ), within three times the LOQ (low QC), medium QC, and at 85% of the upper calibration curve range (high QC), and used to test the accuracy, precision, matrix effect, extraction recovery, and stability. The calibration curves for the three amanitins showed good linear relationships in the range of 0.1-200 ng/mL, and the correlation coefficients (r) were greater than 0.999. The matrix effects and extraction efficiencies of the three amanitins in urine and plasma were 92%-108% and 90%-103%, respectively, and the coefficients of variation were less than 13%. The accuracies of the three amanitins in urine were within -9.4%-8.0%. The repeatability and intermediate accuracies were 3.0%-14% and 3.5%-18%, respectively. When the sampling volume was 2.00 mL, the limits of detection of the three amanitins in urine were 0.002 ng/mL. The accuracies of the three amanitins in plasma were within -13%-8.0%. The repeatability and intermediate accuracies were 3.9%-9.7% and 5.5%-12%, respectively. When the sampling volume was 1.00 mL, the limits of detection of the three amanitins in plasma were 0.004 ng/mL. The developed method is simple, sensitive, and accurate. During toxic mushroom poisoning detection, 0.0067 ng/mL of α-amanitin and 0.0059 ng/mL of ß-amanitin were detected in the urine of poisoned patients 138 h after ingesting poisonous mushrooms. This method has successfully solved the problem of detecting ultra-trace levels of amanitins in the urine and plasma of poisoned patients. It has important practical significance for the early diagnosis, early treatment, and mortality reduction of suspected poisoning patients. This method can also provide reliable technical support for future research on the toxicological effects and in vivo metabolism of these toxins.

A Novel Transcriptional Regulator HbERF6 Regulates the HbCIPK2-Coordinated Pathway Conferring Salt Tolerance in Halophytic Hordeum brevisubulatum.

Halophytic Hordeum brevisubulatum is a perennial grass which has evolved many distinctive salt-adaptive mechanisms. Our previous studies indicated it could thrive under salt stress through maintaining better K+ and Na+ homeostasis. Stress-responsive HbCIPK2 can phosphorylate K+ channel HbVGKC1 and Na+ transporter HbSOS1L to prevent Na+ accumulation and K+ reduction, hence pathway was not detected in glycophytic plants. In this study, we cloned the inducible promoter of HbCIPK2 by genome-walking, and identified a novel transcriptional regulator HbERF6 through yeast one-hybrid screening. HbERF6 functioned as a transcription factor which can bind to the GCC-box of the HbCIPK2 promoter to activate its expression. HbERF6 transgenic lines in Arabidopsis improved salt tolerance compared with wild type, and especially induced AtCIPK24 (SOS2) expression, resulting in K+/Na+ homeostasis to enhance salt tolerance. All the results confirmed the inducible function of HbERF6 for CIPK genes during salt tolerance. This regulatory network that integrates transcriptional regulation and post-translation modification will unravel a novel salt stress-responsive mechanism, highlighting the value and utilization of the halophytic resource.