Auxin plays a role in the adaptation of rice to anaerobic germination and seedling establishment.

Auxin is well known to stimulate coleoptile elongation and rapid seedling growth in the air. However, its role in regulating rice germination and seedling establishment under submergence is largely unknown. Previous studies revealed that excessive levels of indole-3-acetic acid(IAA) frequently cause the inhibition of plant growth and development. In this study, the high-level accumulation of endogenous IAA is observed under dark submergence, stimulating rice coleoptile elongation but limiting the root and primary leaf growth during anaerobic germination (AG). We found that oxygen and light can reduce IAA levels, promote the seedling establishment and enhance rice AG tolerance. miRNA microarray profiling and RNA gel blot analysis results show that the expression of miR167 is negatively regulated by submergence; it subsequently modulates the accumulation of free IAA through the miR167-ARF-GH3 pathway. The OsGH3-8 encodes an IAA-amido synthetase that functions to prevent free IAA accumulation. Reduced miR167 levels or overexpressing OsGH3-8 increase auxin metabolism, reduce endogenous levels of free IAA and enhance rice AG tolerance. Our studies reveal that poor seed germination and seedling growth inhibition resulting from excessive IAA accumulation would cause intolerance to submergence in rice, suggesting that a certain threshold level of auxin is essential for rice AG tolerance.

Effects of Hypoxia-Hyperoxia Preconditioning on Indicators of Muscle Damage After Acute Resistance Exercise in Male Athletes.

Purpose: The purpose of this study was to investigate the effects of acute repeated hypoxia-hyperoxia preconditioning on resistance exercise (RE)-induced muscle damage in male athletes. Methods: Eleven young male athletes participated in this randomized double-blind counter-balanced crossover study, and were divided into Normoxia (N) and Hypoxia-Hyperoxia (HH) trials. Subjects of the respective trials were supplied with normoxic (FiO2 = 0.21), or alternating hypoxic/hyperoxic air (FiO2 = 0.10/0.99, 5 min each) for 60 min. Thirty minutes after preconditioning, subjects performed acute bouts of RE consisting of bench press, deadlift, and squats. Each exercise included 6 sets of 10 repetitions at 75% one-repetition maximum (1RM) with 2 min rest between sets. After a 2-week washout period, subjects changed trials and completed the same study procedure after the alternate preconditioning. Muscle soreness, maximal voluntary contraction (MVC), and circulating biochemical markers were tested before preconditioning (baseline) and during recovery at 0, 24, and 48 h after exercise. Results: Acute RE significantly increased levels of muscle soreness, creatine kinase (CK) and myoglobin (Mb), and decreased levels of peak knee extension torque in the N trial. Muscle soreness, CK, and Mb levels of the HH trial were significantly lower than that of the N trial after exercise. Interestingly, interleukin-6 (IL-6) levels of the HH trial increased significantly 0 h after exercise compared to baseline and were significantly higher than that of the N trial 0 and 24 h after exercise. However, no significant differences of thiobarbituric acid reactive substances (TBARS), cortisol, testosterone, peak torque, and average power levels were found between N and HH trials during recovery. Conclusion: Our data suggest that pre-exercise treatment of alternating hypoxic/hyperoxic air could attenuate muscle damage and pain after acute RE, but has no effect on muscle strength recovery in young male athletes.

Toward automated segmentation for acute ischemic stroke using non-contrast computed tomography.

PURPOSE: Non-contrast computed tomography (NCCT) is a first-line imaging technique for determining treatment options for acute ischemic stroke (AIS). However, its poor contrast and signal-to-noise ratio limit the diagnosis accuracy for radiologists, and automated AIS lesion segmentation using NCCT also remains a challenge. In this paper, we propose R2U-RNet, a novel model for AIS lesion segmentation using NCCT. METHODS: We used an in-house retrospective NCCT dataset with 261 AIS patients with manual lesion segmentation using follow-up diffusion-weighted images. R2U-RNet is based on an R2U-Net backbone with a novel residual refinement unit. Each input image contains two image channels from separate preprocessing procedures. The proposed model incorporates multiscale focal loss to mitigate the class imbalance problem and to leverage the importance of different levels of details. A proposed noisy-label training scheme is utilized to account for uncertainties in the manual annotations. RESULTS: The proposed model outperformed several iconic segmentation models in AIS lesion segmentation using NCCT, and our ablation study demonstrated the efficacy of the proposed model. Statistical analysis of segmentation performance revealed significant effects of regional stroke occurrence and side of the stroke, suggesting the importance of region-specific information for automated segmentation, and the potential influence of the hemispheric difference in clinical data. CONCLUSION: This study demonstrated the potentials of R2U-RNet model for automated NCCT AIS lesion segmentation. The proposed model can serve as a tool for accelerating AIS diagnoses and improving the treatment quality of AIS patients.

Influence of the Intention to Lean the Body Forward on Kinematics and Kinetics of Sprinting for Active Adults.

This study investigated the influence of the intention to lean the body forward on spatiotemporal and ground reaction force variables during the acceleration phase of a sprint. Fourteen active adults performed two 50 m sprints (with and without the intention to lean), during which spatiotemporal variables and impulses were obtained using a long force platform system. Effect size (Cohen's d) was used to examine the differences between the two trials. We found that running speed and net anteroposterior impulse did not change by the intention for all steps. However, step frequency increased in the initial two steps through decreases in support time and flight time by the intention. Moreover, these shorter support and flight times were caused by a decrease in the vertical impulse. The propulsive impulse did not change during the initial part of acceleration phase, but the braking impulse decreased at the first step. This study demonstrates that an intention to lean the body forward leads to a smaller braking impulse and a higher step frequency through shorter support and flight times and a smaller vertical impulse during the initial part of the acceleration phase of a sprint.

Identification of sugar response complex in the metallothionein OsMT2b gene promoter for enhancement of foreign protein production in transgenic rice.

KEY MESSAGE: A 146-bp sugar response complex MTSRC is identified in the promoter of rice metallothionein OsMT2b gene conferring high-level expression of luciferase reporter gene and bioactive recombinant haFGF in transgenic rice. A rice subfamily type 2 plant metallothionein (pMT) gene, OsMT2b, encoding a reactive oxygen species (ROS) scavenger protein, has been previously shown to exhibit the most abundant gene expression in young rice seedling. Expression of OsMT2b was found to be regulated negatively by ethylene and hydrogen peroxide in rice stem node under flooding stress, but little is known about its response to sugar depletion. In this study, transient expression assay and transgenic approach were employed to characterize the regulation of the OsMT2b gene expression in rice. We found that the expression of OsMT2b gene is induced by sugar starvation in both rice suspension cells and germinated embryos. Deletion analysis and functional assay of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides - 351 and - 121, which contains the sugar response complex (- 266 to - 121, designated MTSRC) is responsible for high-level promoter activity under sugar starvation. It was also found that MTSRC significantly enhances the Act1 promoter activity in transgenic rice cells and seedlings. The modified Act1 promoter, Act1-MTSRC, was used to produce the recombinant human acidic fibroblast growth factor (haFGF) in rice cells. Our result shows that the bioactive recombinant haFGF is stably produced in transformed rice cell culture and yields are up to 2% of total medium proteins. Our studies reveal that MTSRC serves as a strong transcriptional activator and the Act1-MTSRC promoter can be applicable in establishing an efficient expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings.

Design, synthesis and evaluation of novel diaryl-1,5-diazoles derivatives bearing morpholine as potent dual COX-2/5-LOX inhibitors and antitumor agents.

In this paper, 41 hybrid compounds containing diaryl-1,5-diazole and morpholine structures acting as dual COX-2/5-LOX inhibitors have been designed, synthesized and biologically evaluated. Most of them showed potent antiproliferative activities and COX-2/5-LOX inhibitory in vitro. Among them, compound A33 displayed the most potency against cancer cell lines (IC50 = 6.43-10.97 µM for F10, HeLa, A549 and MCF-7 cells), lower toxicity to non-cancer cells than celecoxib (A33: IC50 = 194.01 µM vs.celecoxib: IC50 = 97.87 µM for 293T cells), and excellent inhibitory activities on COX-2 (IC50 = 0.17 µM) and 5-LOX (IC50 = 0.68 µM). Meanwhile, the molecular modeling study was performed to position compound A33 into COX-2 and 5-LOX active sites to determine the probable binding models. Mechanistic studies demonstrated that compound A33 could block cell cycle in G2 phase and subsequently induced apoptosis of F10 cells. Furthermore, compound A33 could significantly inhibit tumor growth in F10-xenograft mouse model, and pharmacokinetic study of compound A33 indicated that it showed better stability in vivo. In general, compound A33 could be a promising candidate for cancer therapy.

Suppressive effect of microRNA319 expression on rice plant height.

KEY MESSAGE: miR319 was identified as a dwarf-inducing gene from Shiokari and its dwarf near isogenic line, and its transgenic rice showed a reduced plant height. This finding reveals the potential application of miR319 in future molecular breeding. It is well known that microRNAs (miRNAs) play important roles in plant physiology, especially in development and stress responses. However, little is known about the role of miRNAs in plant height. In this study, the rice cultivar Shiokari and its dwarf near isogenic line Shiokari-d6 were analysed to identify and characterize plant height-associated miRNAs. This anatomic and morphological investigation revealed that the major cause of the shorter height of Shiokari-d6 is the significantly dis-elongated internodes, particularly the second internode and those underneath it. The results of miRNA microarray profiling and real-time RT-PCR indicated that miR319 is expressed at a significantly higher level in Shiokari-d6 than in Shiokari. Transgenic rice overexpressing miR319 in Oryza sativa L. cv. Tainung 67 generated through Agrobacterium-mediated transformation had a stable dwarf phenotype regardless of whether the plants were from the T1 or T2 generation. We also found that the internodes of miR319-overexpressing rice are shortened, particularly the third internode and those underneath it. Furthermore, we identified three putative miR319 target genes that were previously uncharacterized with expression levels that were negatively correlated with the expression of miR319. In conclusion, miR319 is the first miRNA proposed to be involved in plant height regulation, and its function may influence the elongation of internodes, which leads to decreased plant height.

Metabolic adaptation to sugar/O2 deficiency for anaerobic germination and seedling growth in rice.

Rice is characterized by a broad range of metabolic and morphological adaptations to flooding, such as germination and mobilization of stored nutrients under submergence until seedlings reach the water surface to carry out photosynthesis, and sustainable growth of mature plants for long durations under partial submergence. The underlying mechanisms of the molecular basis of adaptation to anaerobic germination and seedling growth in rice are being uncovered. Induction of an ensemble of hydrolases to mobilize endosperm nutrient reserves is one of the key factors for successful germination and coleoptile elongation in rice under submergence. To compensate for reduced efficiency of Tricarboxylic Acid cycle and oxidative respiration in mitochondria under O2 deficient conditions, α-amylases play a central role in the hydrolysis of starch to provide sugar substrates for glycolysis and alcohol fermentation for generating ATP. We review the progress on the molecular mechanism regulating α-amylase expression that involves the integration of signals generated by the hormone gibberellin (GA), sugar starvation and O2 deprivation that results in germination and sustainable seedling growth in rice under anaerobic conditions. Comparisons are also made between dicots and monocots for the molecular mechanism of induction of genes involved in alcohol fermentation and sugar/O2 deficiency sensing system.

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings.

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O2 deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8% of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8% of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.

The promoter and the 5'-untranslated region of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos.

KEY MESSAGE: Critical regions within the rice metallothionein OsMT2b gene promoter are identified and the 5'-untranslated region (5'-UTR) is found essential for the high-level promoter activity in germinated transgenic rice embryos. Many metallothionein (MT) genes are highly expressed in plant tissues. A rice subfamily p2 (type 2) MT gene, OsMT2b, has been shown previously to exhibit the most abundant gene expression in young rice seedling. In the present study, transient expression assays and a transgenic approach were employed to characterize the expression of the OsMT2b gene in rice. We found that the OsMT2b gene is strongly and differentially expressed in germinated rice embryos during seed germination and seedling development. Histochemical staining analysis of transgenic rice carrying OsMT2b::GUS chimeric gene showed that high-level GUS activity was detected in germinated embryos and at the meristematic part of other tissues during germination. Deletion analysis of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides -351 and -121 relative to the transcriptional initiation site is important for promoter activity in rice embryos, and this region contains the consensus sequences of G box and TA box. Our study demonstrates that the 5'-untranslated region (5'-UTR) of OsMT2b gene is not only necessary for the OsMT2b promoter activity, but also sufficient to augment the activity of a minimal promoter in both transformed cell cultures and germinated transgenic embryos in rice. We also found that addition of the maize Ubi intron 1 significantly enhanced the OsMT2b promoter activity in rice embryos. Our studies reveal that OsMT2b351-ubi(In) promoter can be applied in plant transformation and represents potential for driving high-level production of foreign proteins in transgenic rice.

Convergent starvation signals and hormone crosstalk in regulating nutrient mobilization upon germination in cereals.

Germination is a unique developmental transition from metabolically quiescent seed to actively growing seedling that requires an ensemble of hydrolases for coordinated nutrient mobilization to support heterotrophic growth until autotrophic photosynthesis is established. This study reveals two crucial transcription factors, MYBS1 and MYBGA, present in rice (Oryza sativa) and barley (Hordeum vulgare), that function to integrate diverse nutrient starvation and gibberellin (GA) signaling pathways during germination of cereal grains. Sugar represses but sugar starvation induces MYBS1 synthesis and its nuclear translocation. GA antagonizes sugar repression by enhancing conuclear transport of the GA-inducible MYBGA with MYBS1 and the formation of a stable bipartite MYB-DNA complex to activate the α-amylase gene. We further discovered that not only sugar but also nitrogen and phosphate starvation signals converge and interconnect with GA to promote the conuclear import of MYBS1 and MYBGA, resulting in the expression of a large set of GA-inducible but functionally distinct hydrolases, transporters, and regulators associated with mobilization of the full complement of nutrients to support active seedling growth in cereals.

Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture.

Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

Coordinated responses to oxygen and sugar deficiency allow rice seedlings to tolerate flooding.

Flooding is a widespread natural disaster that leads to oxygen (O(2)) and energy deficiency in terrestrial plants, thereby reducing their productivity. Rice is unusually tolerant to flooding, but the underlying mechanism for this tolerance has remained elusive. Here, we show that protein kinase CIPK15 [calcineurin B-like (CBL)-interacting protein kinase] plays a key role in O(2)-deficiency tolerance in rice. CIPK15 regulates the plant global energy and stress sensor SnRK1A (Snf1-related protein kinase 1) and links O(2)-deficiency signals to the SnRK1-dependent sugar-sensing cascade to regulate sugar and energy production and to enable rice growth under floodwater. Our studies contribute to understanding how rice grows under the conditions of O(2) deficiency necessary for growing rice in irrigated lowlands.

Interaction between rice MYBGA and the gibberellin response element controls tissue-specific sugar sensitivity of alpha-amylase genes.

Expression of alpha-amylase genes during cereal grain germination and seedling growth is regulated negatively by sugar in embryos and positively by gibberellin (GA) in endosperm through the sugar response complex (SRC) and the GA response complex (GARC), respectively. We analyzed two alpha-amylase promoters, alphaAmy3 containing only SRC and alphaAmy8 containing overlapped SRC and GARC. alphaAmy3 was sugar-sensitive but GA-nonresponsive in both rice (Oryza sativa) embryos and endosperms, whereas alphaAmy8 was sugar-sensitive in embryos and GA-responsive in endosperms. Mutation of the GA response element (GARE) in the alphaAmy8 promoter impaired its GA response but enhanced sugar sensitivity, and insertion of GARE in the alphaAmy3 promoter rendered it GA-responsive but sugar-insensitive in endosperms. Expression of the GARE-interacting transcription factor MYBGA was induced by GA in endosperms, correlating with the endosperm-specific alphaAmy8 GA response. alphaAmy8 became sugar-sensitive in MYBGA knockout mutant endosperms, suggesting that the MYBGA-GARE interaction overrides the sugar sensitivity of alphaAmy8. In embryos overexpressing MYBGA, alphaAmy8 became sugar-insensitive, indicating that MYBGA affects sugar repression. alpha-Amylase promoters active in endosperms contain GARE, whereas those active in embryos may or may not contain GARE, confirming that the GARE and GA-induced MYBGA interaction prevents sugar feedback repression of endosperm alpha-amylase genes. We demonstrate that the MYBGA-GARE interaction affects sugar feedback control in balanced energy production during seedling growth and provide insight into the control mechanisms of tissue-specific regulation of alpha-amylase expression by sugar and GA signaling interference.

Rice alpha-amylase transcriptional enhancers direct multiple mode regulation of promoters in transgenic rice.

Expression of alpha-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. In a transient expression assay, a 105-bp sugar response sequence (SRS) in the promoter of a sugar starvation highly inducible rice alpha-amylase gene, alphaAmy3, was shown previously to confer sugar response and to enhance the activity of the rice Act1 promoter in rice protoplasts. A 230-bp SRS-like sequence was also found in the promoter of another sugar starvation highly inducible rice alpha-amylase gene, alphaAmy8. The alphaAmy8 SRS contains a GA response sequence and was designated as alphaAmy8 SRS/GARS. In the present study, a transgenic approach was employed to characterize the function of the alpha-amylase gene SRSs in rice. We found that the alphaAmy3 SRS significantly enhances the endogenous expression pattern of the Act1 promoter in various rice tissues throughout their developmental stages. By contrast, the alphaAmy8 SRS/GARS significantly enhances Act1 promoter activity only in embryos and endosperms of germinating rice seeds. A minimal promoter fused to the alphaAmy8 SRS/GARS is specifically active in rice embryo and endosperm and is subject to sugar repression and GA induction in rice embryos. This sugar repression was found to override GA induction of alphaAmy8 SRS/GARS activity. Our study demonstrates that the alpha-amylase transcriptional enhancers contain cis-acting elements capable of enhancing endogenous expression patterns or activating sugar-sensitive, hormone-responsive, tissue-specific, and developmental stage-dependent expression of promoters in transgenic rice. These enhancers may facilitate the design of highly active and tightly regulated composite promoters for monocot transformation and gene expression. Our study also reveals the existence of cross-talk between the sugar and GA signaling pathways in cereals and provides a system for analyzing the underlying molecular mechanisms involved.