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The calcium-activated chloride channel TMEM16A is a potential drug target to treat hypertension, secretory diarrhea, and several cancers. However, all reported TMEM16A structures are either closed or desensitized, and direct inhibition of the open state by drug molecules lacks a reliable structural basis. Therefore, revealing the druggable pocket of TMEM16A exposed in the open state is important for understanding protein-ligand interactions and facilitating rational drug design. Here, we reconstructed the calcium-activated open conformation of TMEM16A using an enhanced sampling algorithm and segmental modeling. Furthermore, we identified an open-state druggable pocket and screened a potent TMEM16A inhibitor, etoposide, which is a derivative of a traditional herbal monomer. Molecular simulations and sitedirected mutagenesis showed that etoposide binds to the open state of TMEM16A, thereby blocking the ion conductance pore of the channel. Finally, we demonstrated that etoposide can target TMEM16A to inhibit the proliferation of prostate cancer PC-3 cells. Together, these findings provide a deep understanding of the TMEM16A open state at an atomic level and identify pockets for the design of novel inhibitors with broad applications in chloride channel biology, biophysics, and medicinal chemistry. [ABSTRACT FROM AUTHOR]

Permeable asphalt mixtures are widely used to achieve good permeability of the pavements used for urban road surfaces. Based on the law of water migration in permeable road structures, the water-holding performance of permeable asphalt mixtures was analyzed and the evaluation indexes were determined. Open-graded asphalt friction course (OGFC-13) permeable asphalt mixture was selected, and the changes in the saturation, water retention, and water-holding rate of OGFC-13 permeable asphalt mixtures with different porosities were compared using water-holding equipment. The water-holding characteristics of permeable pavement materials under different seepage conditions were analyzed. A model of the relationship governing the water-holding capacity of the permeable asphalt mixture, water action conditions, and material porosity was established by a linear fitting method, and the reliability of the model was verified by comparison with laboratory test results, providing a foundation for related research on the water-holding and cooling behavior of permeable pavements. [ABSTRACT FROM AUTHOR]

Lung cancer has the highest mortality among cancers worldwide due to its high incidence and lack of the effective cures. We have previously demonstrated that the membrane ion channel TMEM16A is a potential drug target for the treatment of lung adenocarcinoma and have identified a pocket of inhibitor binding that provides the basis for screening promising new inhibitors. However, conventional drug discovery strategies are lengthy and costly, and the unpredictable side effects lead to a high failure rate in drug development. Therefore, finding new therapeutic directions for already marketed drugs may be a feasible strategy to obtain safe and effective therapeutic drugs. Here, we screened a library of over 1400 Food and Drug Administration-approved drugs through virtual screening and activity testing. We identified a drug candidate, Zafirlukast (ZAF), clinically approved for the treatment of asthma, that could inhibit the TMEM16A channel in a concentration-dependent manner. Molecular dynamics simulations and site-directed mutagenesis experiments showed that ZAF can bind to S387/N533/R535 in the nonselective inhibitor binding pocket, thereby blocking the channel pore. Furthermore, we demonstrate ZAF can target TMEM16A channel to inhibit the proliferation and migration of lung adenocarcinoma LA795 cells. In vivo experiments showed that ZAF can significantly inhibit lung adenocarcinoma tumor growth in mice. Taken together, we identified ZAF as a novel TMEM16A channel inhibitor with excellent anticancer activity, and as such, it represents a promising candidate for future preclinical and clinical studies. [ABSTRACT FROM AUTHOR]

As a calcium-activated chloride channel regulated by the intracellular Ca2+ concentration and membrane potential, TMEM16A has attracted considerable attention and has been proposed as a novel anticancer drug target. We have previously reported that the pocket above the ion conductance pore could be a nonselective inhibitor-binding pocket. However, whether this pocket is druggable remains unexplored. In this study, we performed virtual screening to target the presumed inhibitorbinding pocket and identified a highly effective TMEM16A inhibitor, theaflavin (TF: a tea polyphenol in black tea). Molecular dynamics simulations revealed that theaflavin adopts a “wedge insertion mode” to block the ion conduction pore and induces pore closure. Moreover, the binding mode showed that the TF pedestal plays an important role in pore blockade, and R515, R535, T539, K603, E623, and E633 were determined to be most likely to interact directly with the pedestal. Mutagenesis experiment results corroborated the mechanism through which TF binds to this pocket. Combined with the quantitative calculation results, our data indicated that the three hydroxyl groups on the pedestal may be the most crucial pharmacophores for TMEM16A inhibition by TF. Finally, antitumor experiments revealed that TF could target TMEM16A to inhibit the proliferation and migration of LA795 cells, indicating the potential therapeutic effect of TF on the growth of lung adenocarcinoma with high TMEM16A expression. The successful application of drug screening strategies based on this binding pocket highlights new directions for discovering superior modulators and contributes to the development of novel therapeutics for lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Understanding the molecular basis of male sterility and developing practical male-sterility systems are essential for heterosis utilization and commercial hybrid seed production in crops. Here, we report molecular regulation by genic male-sterility gene maize male sterility 7 (ZmMs7) and its application for developing a dominant male-sterility system in multiple species. ZmMs7 is specifically expressed in maize anthers, encodes a plant homeodomain (PHD) finger protein that functions as a transcriptional activator, and plays a key role in tapetal development and pollen exine formation. ZmMs7 can interact with maize nuclear factor Y (NF-Y) subunits to form ZmMs7-NF-YA6-YB2-YC9/12/15 protein complexes that activate target genes by directly binding to CCAAT box in their promoter regions. Premature expression of ZmMs7 in maize by an anther-specific promoter p5126 results in dominant and complete male sterility but normal vegetative growth and femalef ertility. Early expression of ZmMs7 downstream genes induced by prematurely expressed ZmMs7 leads to abnormal tapetal development and pollen exine formation in p5126-ZmMs7 maize lines. The p5126-ZmMs7 transgenic rice and Arabidopsis plants display similar dominant male sterility. Meanwhile, the mCherry gene coupled with p5126-ZmMs7 facilitates the sorting of dominant sterility seeds based on fluorescent selection. In addition, both the ms7-6007 recessive male-sterility line and p5126-ZmMs7M dominant male-sterility line are highly stable under different genetic germplasms and thus applicable for hybrid maize breeding. Together, our work provides insight into the mechanisms of anther and pollen development and a promising technology for hybrid seed production in crops. [ABSTRACT FROM AUTHOR]

The solid digestate from high solid anaerobic digestion was used as growth medium for seeding production. The garage-type dry fermentation system using bundled rice straw and swine manure was performed to obtain solid digestate. The addition of solid digestate addition greatly influenced the properties of the growth medium. The bulk density increased and the total porosity, pH, and electrical conductivity (EC) values were decreased with the reduction of solid digestate. The solid digestate-based media had a bulk density < 0.3 g/cm3, total porosity > 70%, air filled porosity ~ 3%, water holding porosity > 60%, EC < 3 mS/cm, and 6.5 < pH < 8. Those properties almost satisfied the essential requirements of nursery substrate. Also, the concentrations of nutrients and heavy metals of the substrate exhibited a positive relationship with solid digestate addition, and they are all within acceptable ranges for plant growth. When the addition of solid digestate was 50% (v/v), the germination rate of tomato seeding cultivated in that solid digestate-based growth medium reached 85%. These findings showed that the solid digestate from the high solid anaerobic digestion could be successfully applied in the seeding nursery and merit consideration for industrial applications. [ABSTRACT FROM AUTHOR]

Road adhesion coefficient is an important parameter in vehicle active safety control system. Many researchers estimate road adhesion coefficient by total tire self-aligning torque (SAT, also called front-axle aligning torque), which obtains the average road adhesion coefficient of front wheels, thus leading large estimation error. In this paper, a novel estimation of road adhesion coefficient based on single tire SAT, which is obtained by tire aligning torque distribution, is brought forward. Due to the use of SAT, the proposed estimation method is available in steering only condition. The main idea of the proposed method is that road adhesion coefficient is estimated by single tire SAT instead of total tire SAT. The single tire SAT is closer to real tire torque state, and it can be obtained by aligning torque distribution, which makes use of the ratio for the aligning torque of front-left wheel and front-right wheel. Tire sideslip angle used in torque distribution is estimated by unscented Kalman filter (UKF). Two coefficients, including front-left and front-right tire-road friction coefficients, are estimated by iteration algorithm form single tire SAT. The final road adhesion coefficient is determined by a coefficient identification rule, which is designed to determine which tire-road friction coefficient as the final road adhesion coefficient. Both simulations and tests that use gyroscope/lateral accelerometer/global position system (GPS)/strain gauge are conducted, to validate the proposed methodology that can provide accurate road adhesion coefficient to vehicle active safety control. [ABSTRACT FROM AUTHOR]

Plant hormones ethylene and auxin synergistically regulate plant root growth and development. Ubiquitin-mediated proteolysis of Aux/IAA transcriptional repressors by the E3 ubiquitin ligase SCFTIR1/AFB triggers a transcription-based auxin signaling. Here we show that rice (Oryza sativa L.) soil-surface rooting 1 (SOR1), which is a RING finger E3 ubiquitin ligase identified from analysis of a rice ethylene-insensitive mutant mhz2/sor1-2, controls root-specific ethylene responses by modulating Aux/IAA protein stability. SOR1 physically interacts with OsIAA26 and OsIAA9, which are atypical and canonical Aux/IAA proteins, respectively. SOR1 targets OsIAA26 for ubiquitin/26S proteasome-mediated degradation, whereas OsIAA9 protects the OsIAA26 protein from degradation by inhibiting the E3 activity of SOR1. Auxin promotes SOR1-dependent degradation of OsIAA26 by facilitating SCFOsTIR1/AFB2-mediated and SOR1-assisted destabilization of OsIAA9 protein. Our study provides a candidate mechanism by which the SOR1-OsIAA26 module acts downstream of the OsTIR1/AFB2-auxin-OsIAA9 signaling to modulate ethylene inhibition of root growth in rice seedlings. [ABSTRACT FROM AUTHOR]

The phytohormone ethylene regulates many aspects of plant growth and development. EIN2 is the central regulator of ethylene signaling, and its turnover is crucial for triggering ethylene responses. Here, we identified a stabilizer of OsEIN2 through analysis of the rice ethyleneresponse mutant mhz3. Loss-of-function mutations lead to ethylene insensitivity in etiolated rice seedlings. MHZ3 encodes a previously uncharacterized membrane protein localized to the endoplasmic reticulum. Ethylene induces MHZ3 gene and protein expression. Genetically, MHZ3 acts at the OsEIN2 level in the signaling pathway. MHZ3 physically interacts with OsEIN2, and both the N- and C-termini of MHZ3 specifically associate with the OsEIN2 Nramp-like domain. Loss of mhz3 function reduces OsEIN2 abundance and attenuates ethylene-induced OsEIN2 accumulation, whereas MHZ3 overexpression elevates the abundance of both wild-type and mutated OsEIN2 proteins, suggesting that MHZ3 is required for proper accumulation of OsEIN2 protein. The association of MHZ3 with the Nramp-like domain is crucial for OsEIN2 accumulation, demonstrating the significance of the OsEIN2 transmembrane domains in ethylene signaling. Moreover, MHZ3 negatively modulates OsEIN2 ubiquitination, protecting OsEIN2 from proteasome-mediated degradation. Together, these results suggest that ethylene-induced MHZ3 stabilizes OsEIN2 likely by binding to its Nramp-like domain and impeding protein ubiquitination to facilitate ethylene signal transduction. Our findings provide insight into the mechanisms of ethylene signaling. [ABSTRACT FROM AUTHOR]

Vehicle sideslip angle is one of the important indicators to determine whether vehicles are stable and is also a significant parameter for vehicle stability control. However, it is almost impossible to be measured directly without complex and expensive sensors or devices. Soft measurements are therefore generally adopted to estimate vehicle sideslip angle on the basis of easily observed physical quantities. This paper proposes an estimation method of sideslip angle based on steering torque instead of steering angle, for the fact that steering torque signal has more rapid and direct response compared with that obtained from steering angle. The frequency is analyzed between steering torque-sideslip angle and steering angle-sideslip angle transfer functions, and the extended Kalman filter (EKF) for vehicle sideslip angle based on steering torque is proposed. Both simulation results and vehicle tests prove the validity of our estimating method, indicating that the proposed method based on steering torque exhibits better accuracy and more rapid response in extreme region than the one based on steering angle. [ABSTRACT FROM AUTHOR]

Pyrolysis of four different crop residues (wheat straw, maize straw, rice straw and rice husk) was carried out at 400 °C in a designed mobile bench-scale pyrolyser. Proximate analysis and elemental analysis were performed together with determination of pH, nutrients, surface area, and functional groups of the produced biochars. Significant variations of the biochar characteristics were related to type and source of the feedstock. Biochar yield was found to vary from 33.4% to 43.8%, whereas the liquid and syn-gas yield varied from 21.2% to 27.5% and 30.8% to 39.1%, respectively. Carbon and major nutrients of N, P and K were enriched but hydrogen, oxygen and sulfur were lower in the biochars due to the pyrolysis process. All of the biochars were alkaline, ranging from a pH 7.3 to pH 10.7. Among the feedstock sources, biochar from rice straw was high in pH, cation exchange capacity as well as in nutrient contents, indicating a greater potential use of rice straw biochar as amendment to acid, nutrient-poor rice soils. As tested in this study, the crop residues available in China currently could potentially produce 160.2 Mt of biochar per year, with a potential to sequester 0.30 Pg CO2 year-1. In addition, syngas produced via pyrolysis process could be used to provide process heat. [ABSTRACT FROM AUTHOR]

A full-scale multi-disc clutch test bench was set up and some sliding experiments were conducted to investigate the temperature evolution processes in low and high lubrication regimes. Friction discs with single friction lining were used and arranged back-to-back in order to preserve possible evidences of buckling. Temperatures were measured with thermocouples from four different radii on the mid-plane of the separator disc. Two different kinds of temperature variation processes with obvious critical points of cone shape buckling were obtained. These temperatures can be divided into three effective stages that represent different deformation status of the discs in these experiments. The temperature fields in the contacting separator disc and friction disc were studied through a transient heat conduction model, and the results show that the temperatures measured by the thermocouples from the separator disc can represent the average temperatures in both of the separator disc and friction disc for a long sliding time. By comparing the computational critical moments of the friction components with the experimental results, the capability of the curved beam model for predicting the critical moments of cone buckling was validated. [ABSTRACT FROM PUBLISHER]

Ethylene plays important roles in plant growth, development, and stress responses. The ethylene signaling pathway has been studied extensively, mainly in Arabidopsis (Arabidopsis thaliana). However, the molecular mechanism of ethylene signaling is largely unknown in rice (Oryza sativa). Previously, we have isolated a set of rice ethylene-response mutants. Here, we characterized the mutant maohuzi6 (mhz6). Through map-based cloning, we found that MHZ6 encodes ETHYLENE INSENSITIVE3-LIKE1 (OsEIL1), a rice homolog of ETHYLENE INSENSITIVE3 (EIN3), which is the master transcriptional regulator of ethylene signaling in Arabidopsis. Disruption of MHZ6/OsEIL1 caused ethylene insensitivity mainly in roots, whereas silencing of the closely related OsEIL2 led to ethylene insensitivity mainly in coleoptiles of etiolated seedlings. This organ-specific functional divergence is different from the functional features of EIN3 and EIL1, both of which mediate the incomplete ethylene responses of Arabidopsis etiolated seedlings. In Arabidopsis, EIN3 and EIL1 play positive roles in plant salt tolerance. In rice, however, lack of MHZ6/OsEIL1 or OsEIL2 functions improves salt tolerance, whereas the overexpressing lines exhibit salt hypersensitivity at the seedling stage, indicating that MHZ6/OsEIL1 and OsEIL2 negatively regulate salt tolerance in rice. Furthermore, this negative regulation by MHZ6/OsEIL1 and OsEIL2 in salt tolerance is likely attributable in part to the direct regulation of HIGH-AFFINITY K+ TRANSPORTER2;1 expression and Na+ uptake in roots. Additionally, MHZ6/OsEIL1 overexpression promotes grain size and thousand-grain weight. Together, our study provides insights for the functional diversification of MHZ6/OsEIL1 and OsEIL2 in ethylene response and finds a novel mode of ethylene-regulated salt stress response that could be helpful for engineering salt-tolerant crops. [ABSTRACT FROM AUTHOR]

The aim of this study was to predict the kinase inhibitors that may regulate autophagy. A total of 62 kinases were obtained through text mining by importing the keyword 'autophagy' and a 'protein kinase' Excel file to PubMed. Subsequently, 146 kinases were derivated through screening in the PubMed database by importing the 'autophagy-associated gene' and 'protein kinase' files. Following intersection of the above two methods, 54 candidate autophagy-associated kinases were obtained. Enrichment analysis indicated that these candidate autophagy-associated kinases were mainly enriched in pathways such as the calcium, Wnt, HIF-1 and mTOR signaling pathways. Among the 54 kinases, 24 were identified through text mining to have specific kinase inhibitors that regulate the corresponding functions; a total of 56 kinase inhibitors were found to be involved in the regulation of these 24 kinases. In total, nine of these 56 kinase inhibitors identified had been widely reported in autophagy regulation studies, 23 kinase inhibitors had been seldom reported and 24 had never been reported. Therefore, introducing these kinases into autophagy regulation analysis in subsequent studies may produce important results. [ABSTRACT FROM AUTHOR]

Accurate deterioration models play a critical role in designing and managing transportation infrastructure. Regular models just consider loading factor and its relative uncertainties. However, climate and environment impacts are not considered or just taken as certain variables. Thermal cracks and moisture distresses are principal distress forms in cold regions, where early damage is more significant than general regions. In this paper, Freeze-Thaw (F-T) cycle test was performed to investigate the impact of the cold climate and moisture resistance on paving mixtures, and the compressive strength and resilient modulus were studied. Then reliability method was applied to analyze the pavement reliability functions with various uncertainties. The analytical results showed that the resilient modulus of asphalt concrete mixture declined under F-T cycles. Consequently, pavement structure capacity was reduced. The results also illustrated that reliability method was capable of accommodating uncertainties in pavement parameters. The sensitivity analysis addressed that F-T cycles had a significant impact on estimating reliability, especially with the large coefficient of variance. The larger coefficient of variance, the faster reliability declined. Reliability analysis results indicate that decrease in variability of F-T cycles can significantly increase the estimated reliability. As a result, F-T cycles with uncertainty do harm to pavement loading capacity, which should not be neglected in actual engineering. This paper also proposes some instructions on simulating pavement performance models. [ABSTRACT FROM AUTHOR]

The yeast Candida glabrata is an opportunistic pathogen of humans. C. glabrata is a NAD+ auxotroph, and its growth depends on the availability of niacin (environmental vitamin precursors of NAD+). We have previously shown that a virulence-associated adhesin, encoded by EPA6, is transcriptionally induced in response to niacin limitation. Here we used transcript profiling to characterize the transcriptional response to niacin limitation and the roles of the sirtuins Hst1, Hst2, and Sir2 in mediating this response. The majority of genes transcriptionally induced by niacin limitation are regulated by Hst1, suggesting that it is the primary sensor of niacin limitation in C. glabrata. We show that three highly induced genes, TNA1, TNR1, and TNR2, encode transporters which are necessary and sufficient for high-affinity uptake of NAD+ precursors. Strikingly, if a tna1 tnr1 tnr2 mutant is starved for niacin, it exhibits an extended lag phase, suggesting a central role for the transporters in restoring NAD+ homeostasis after niacin limitation. Lastly, we had previously shown that the adhesin encoded by EPA6 is induced during experimental urinary tract infection (UTI); we show here that EPA6 transcriptional induction during UTI is strongly enhanced in the tna1 tnr1 tnr2 mutant strain, implicating the transporters in the growth of C. glabrata during infection. [ABSTRACT FROM AUTHOR]

The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD+) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD+. C. glabrata salvage pathways defined in this article allow NAD+ to be synthesized from three compounds – nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss–Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss–Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss–Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD+ via the nicotinamidase Pnc1 and the Preiss–Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD+ sources during disseminated infection. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5'end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5'end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5'phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, provide direct support for the 5'region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5'end of the guide RNA. [ABSTRACT FROM AUTHOR]

Short RNAs mediate gene silencing, a process associated with virus resistance, developmental control and heterochromatin formation in eukaryotes. RNA silencing is initiated through Dicer-mediated processing of double-stranded RNA into small interfering RNA (siRNA). The siRNA guide strand associates with the Argonaute protein in silencing effector complexes, recognizes complementary sequences and targets them for silencing. The PAZ domain is an RNA-binding module found in Argonaute and some Dicer proteins and its structure has been determined in the free state. Here, we report the 2.6?Å crystal structure of the PAZ domain from human Argonaute eIF2c1 bound to both ends of a 9-mer siRNA-like duplex. In a sequence-independent manner, PAZ anchors the 2-nucleotide 3' overhang of the siRNA-like duplex within a highly conserved binding pocket, and secures the duplex by binding the 7-nucleotide phosphodiester backbone of the overhang-containing strand and capping the 5'-terminal residue of the complementary strand. On the basis of the structure and on binding assays, we propose that PAZ might serve as an siRNA-end-binding module for siRNA transfer in the RNA silencing pathway, and as an anchoring site for the 3' end of guide RNA within silencing effector complexes. [ABSTRACT FROM AUTHOR]

Focuses on the preparation of calcium phosphate cements with phosphorylated chitins. Absorption of hydroxyl groups; Mixture of chitosan acetate and lactate; Composition of organic collagen.

Reports that the green fluorescent protein (GFP) system faithfully reports gene expression when it is driven by the glyceraldehyde-p-dehydrogenase or the mnp gene promoter from Phanerochaete chrysosporium. Construction of transformation vectors; Intracellular GFP determination; Expression plasmids; Transformation and characterization of transformants.

Examines the synthesis of polymeric beta-cyclodextrin supported by crosslinked poly(acrylamide-co-vinylamine) as an artificial analog of hydrolytic enzyme and its catalysis of the hydrolysis of p-nitrophenyl acetate (p-NPA). Hydrolysis of p-NPA catalyzed by free beta-cyclodextrin; Final conversion percentage of the p-NPA hydrolysis in different systems.

To solve the problems of high error on both sides and high coefficient of variation during liquid manure distribution, this work designed a distribution mechanism integrating conveying, stirring and distribution functions, combined with the physical properties of selected liquid manure. Taking rotor speed, inlet flow and moving cutter structure as test factors, the Design-Expert 8.0.6 software was used to design "three-factor three-level quadratic regression" orthogonal test and establish response surface regression model. Through observing relative error and coefficient of variation, uniform distribution characteristics test and parameter optimization of liquid manure were performed. The results showed that the primary and secondary order of influencing factors on the relative error is rotor speed> inlet flow > moving cutter structure; the primary and secondary order of influencing factors on the coefficient of variation is inlet flow > moving cutter structure> rotor speed. Further, the optimization test indicated that 170 r/min rotor speed, 80 m3/h inlet flow, combined with arc-shaped moving cutter structure could output 10.50% relative error and 9.30% variation rate, which was less than 5% relative to the model predicted value. [ABSTRACT FROM AUTHOR]

In order to study the mechanical properties of compression and stress relaxation of manure under drainage conditions, the experiment was carried out with cattle manure as the object, and the constitutive models were improved to describe the stress-strain curves of compression and stress relaxation. The defined constitutive model was determined by parameter identification, and the influence laws of moisture content and filling mass on the compression and relaxation process were investigated. The results show that the improved Nishihara model and the five-component generalized Maxwell model can better describe the compression and stress relaxation properties of cattle manure, and the coefficients of determination of the parameters of the fitted intrinsic model are all greater than 0.9, with high fitting accuracy. The compression process was divided into three stages: exhaust, drainage, and viscoelastic-plastic deformation. The stress decay rate tended to change quickly and then slowly, and there was a negative correlation between the compressed mass, moisture content, and stress decay rate. Duncan's mean comparisons revealed that the differences in elastic modulus between different moisture content / compressed mass groups in the parameters of the cattle manure compression principal model were all significant (P<0.05). The differences in elastic modulus and coefficient of viscous attenuation between different moisture content groups in the parameters of the stress relaxation model were all significant. The study's results can provide the theoretical basis for the study and simulation of CM's compression and dewatering mechanism and provide support for the solid-liquid separator for livestock and poultry manure. [ABSTRACT FROM AUTHOR]

NEK (NIMA-related kinase) is known as a family of serine/threonine kinases which mainly participate in microtubule-related mitotic events in fungi, mammals and other eukaryotes. Our previous studies found that Arabidopsis NEK6 plays an important role in plant response to abiotic stress. We further investigated roles of NEK family in soybean and found that at least eight members can respond to abiotic stresses. Among them, only GmNEK1, a novel NEK member which is distantly-related to Arabidopsis NEK6, enhanced plant growth and promoted salt and cold tolerance in transgenic Arabidopsis plants. The growth of soybean plants harboring GmNEK1-overexpressing hairy roots under saline condition was also improved. A series of stress-related genes including RH3, CORI3 and ALDH10A8 were found to be upregulated in GmNEK1-overexpressing Arabidopsis plants and soybean hairy roots. Moreover, soybean plants with GmRH3-overexpressing hairy roots exhibited increased salt tolerance, while soybean plants with GmRH3-RNAi roots were more sensitive to salt stress than the wild type plants. Our study uncovers a novel role of GmNEK1 in promoting plant adaptive growth under adverse conditions at least partially through upregulation of GmRH3. Manipulation of these genes in soybean or other crops may improve growth and production under stress conditions. [ABSTRACT FROM AUTHOR]

Identification and isolation of breast cancer stem cells (CSCs) based on CD44/CD24 expression and/or enzymatic activity of aldehyde dehydrogenase 1 (ALDH1). However, the differences among the CD44+/CD24-/low cells, ALDH1+ cells and the overlap between the sub-populations have not been frequently investigated. Thus, it is imperative to improve the understanding of breast CSC with different stem markers. CD44+/CD24-/low, ALDH1+ a nd A LDH1+CD44+/CD24-/low cell populations were isolated from fresh breast cancer tissues and analyzed by flow cytometry and immunofluorescence. Mammosphere formation, cell proliferation assay and Transwell experiments, were used to analyze self-renewal, proliferation and invasion, respectively, for each sub-population. Finally, in vivo experimentation in mice was performed to evaluate the tumorigenic abilities of the sub-populations. The sub-populations of CD44+/CD24-/low, ALDH1+ and ALDH1+CD44+/CD24-/low in human breast cancer cells, represented the 7.2, 4.6 and 1.5% of the total tumor cell population, respectively. ALDH1+CD44+/CD24-/low cells had the strongest ability of self-renewal, invasion, proliferation and tumorigenicity compared with the other sub-populations (P<0.05). In conclusion, different phenotypes of CD44+/CD24-/low, ALDH1+ and ALDH1+CD44+/CD24-/low were isolated and demonstrated that breast CSCs are heterogeneous, and they exhibit distinct biological characteristics. As ALDH1+CD44+/CD24-/low cells demonstrated the strongest stem-like properties, it may be a useful specific stem cell marker. The utilization of more reliable biomarkers to distinguish the breast CSC pool will be important for the development of specific target therapies for breast cancer. [ABSTRACT FROM AUTHOR]

A horizontal distributor for biogas slurry application was proposed to explore the distribution performance through CFD analysis and verified by field test. The rheological properties of biogas slurry were analyzed at first, and key parameters were obtained for the next simulation. The effects of distribution modes, inlet direction, and outlets number on the velocity distribution of flow field and mass flow rate of the horizonal distributor were investigated by CFD simulations. Results of rheological properties indicated that biogas slurry was a non-Newtonian fluid and exhibited shear-thinning behavior. It can be well described by power-law model. The simulation results showed that the geometry of rotor, especially the block numbers was the main factor that determining the fluid movement and trajectory of distribution and output. The mode rotor 1 with two blocks reached the lowest variable coefficient of mass flow rate (4.49%), indicating a higher degree of uniformity. The upward inlet direction would obtain less dead zone, and the distributor with an even outlets number would possess more uniform distribution and less dead zone. The field test of the distributor with rotor 1, upward inlet direction, and 24 outlets has been carried on to verify the simulation results, the variable coefficient of mass flow was 13.06%, which was slightly higher than the simulation (9.23%), but it still within the range of requirement (<15%). The proposed model and the findings of this work are of guiding significance for the study of the utilization technology and equipment of liquid biogas residue. [ABSTRACT FROM AUTHOR]

DBC1 has been characterized as a key regulator of physiological and pathophysiological activities, such as DNA damage, senescence, and tumorigenesis. However, the mechanism by which the functional stability of DBC1 is regulated has yet to be elucidated. Here, we report that the ubiquitination-mediated degradation of DBC1 is regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5 under hypoxic stress. Mechanistically, hypoxia promoted DBC1 to interact with SIAH2 but not OTUD5, resulting in the ubiquitination and subsequent degradation of DBC1 through the ubiquitin-proteasome pathway. SIAH2 knockout inhibited tumor cell proliferation and migration, which could be rescued by double knockout of SIAH2/CCAR2. Human tissue microarray analysis further revealed that the SIAH2/DBC1 axis was responsible for tumor progression under hypoxic stress. These findings define a key role of the hypoxia-mediated SIAH2-DBC1 pathway in the progression of human breast cancer and provide novel insights into the metastatic mechanism of breast cancer. [ABSTRACT FROM AUTHOR]

Ag recognition and Ab production in B cells are major components of the humoral immune response. In the current study, we found that the core fucosylation catalyzed by a1,6-fucosyltransferase (Fut8) was required for the Ag recognition of BCR and the subsequent signal transduction. Moreover, compared with the 3-83 B cells, the coalescing of lipid rafts and Ag-BCR endocytosis were substantially reduced in Fut8 -knockdown (3-83-KD) cells with p31 stimulation and then completely restored by reintroduction of the Fut8 gene to the 3-83-KD cells. Indeed, Fut8-null (Fut8-/-) mice evoked a low immune response following OVA immunization. Also, the frequency of IgG-producing cells was significantly reduced in the Fut8-/- spleen following OVA immunization. Our results clearly suggest an unexpected mode of BCR function, in which the core fucosylation of IgG-BCR mediates Ag recognition and, concomitantly, cell signal transduction via BCR and Ab production. [ABSTRACT FROM AUTHOR]

In this paper we obtain a recursive formula for the shuffle product and apply it to derive two restricted decomposition formulas for multiple zeta values (MZVs). The first formula generalizes the decomposition formula of Euler and is similar to the restricted formula of Eie and Wei for MZVs with one strings of 1's. The second formula generalizes the previous results to the product of two MZVs with one and two strings of 1's respectively. [ABSTRACT FROM AUTHOR]

We report on the NMR-based solution structure of the 93del d(GGGGTGGGAGGAGGGT) aptamer, a potent nanomolar inhibitor of HIV-1 integrase. This guanine-rich DNA sequence adopts an unusually stable dimeric quadruplex architecture in K+ solution. Within each 16-nt monomer subunit which contains one A(G.G.G.G) pentad sandwiched between two G.G.G.G tetrads, all G-stretches are parallel, and all guanines are anti with the exception of Gi, which is syn. Dimer formation is achieved through mutual pairing of GI of one monomer, with G2, G6, and G13 of the other monomer, to complete G.G.G.G tetrad formation. There are three single-nucleotide double-chain-reversal loops within each monomer fold, such that the first (T5) and third (A12) loops bridge three G-tetrad layers, whereas the second (A9) loop bridges two G-tetrad layers and participates in A'(G.G.G.G) pentad formation. Results of NMR and of integrase inhibition assays on loop-modified sequences allowed us to propose a strategy toward the potential design of improved HIV-1 integrase inhibitors. Finally, we propose a model, based on molecular docking approaches, for positioning the 93del dimeric DNA quadruplex within a basic channel/canyon formed between subunits of a dimer of dimers of HIV-1 integrase. [ABSTRACT FROM AUTHOR]

Next generation sequencing (NGS)-based tumor profiling identified an overwhelming number of uncharacterized somatic mutations, also known as variants of unknown significance (VUS). The therapeutic significance of EGFR mutations outside mutational hotspots, consisting of >50 types, in nonsmall cell lung carcinoma (NSCLC) is largely unknown. In fact, our pan-nation screening of NSCLC without hotspot EGFR mutations (n = 3,779) revealed that the majority (>90%) of cases with rare EGFR mutations, accounting for 5.5% of the cohort subjects, did not receive EGFR-tyrosine kinase inhibitors (TKIs) as a first-line treatment. To tackle this problem, we applied a molecular dynamics simulation-based model to predict the sensitivity of rare EGFR mutants to EGFR-TKIs. The model successfully predicted the diverse in vitro and in vivo sensitivities of exon 20 insertion mutants, including a singleton, to osimertinib, a third-generation EGFR-TKI (R2 = 0.72, P = 0.0037). Additionally, our model showed a higher consistency with experimentally obtained sensitivity data than other prediction approaches, indicating its robustness in analyzing complex cancer mutations. Thus, the in silico prediction model will be a powerful tool in precision medicine for NSCLC patients carrying rare EGFR mutations in the clinical setting. Here, we propose an insight to overcome mutation diversity in lung cancer. [ABSTRACT FROM AUTHOR]

Plant homeodomain (PHD) finger proteins are involved in various developmental processes and stress responses. They recognize and bind to epigenetically modified histone H3 tail and function as histone code readers. Here we report that GmPHD6 reads low methylated histone H3K4me0/1/2 but not H3K4me3 with its N-terminal domain instead of the PHD finger. GmPHD6 does not possess transcriptional regulatory ability but has DNA-binding ability. Through the PHD finger, GmPHD6 interacts with its coactivator, LHP1-1/2, to form a transcriptional activation complex. Using a transgenic hairy root system, we demonstrate that overexpression of GmPHD6 improves stress tolerance in soybean (Glycine max) plants. Knocking down the LHP1 expression disrupts this role of GmPHD6, indicating that GmPHD6 requires LHP1 functions during stress response. GmPHD6 influences expression of dozens of stress-related genes. Among these, we identified three targets of GmPHD6, including ABA-stress-ripening-induced CYP75B1 and CYP82C4. Overexpression of each gene confers stress tolerance in soybean plants. GmPHD6 is recruited to H3K4me0/1/2 marks and recognizes the G-rich elements in target gene promoters, whereas LHP1 activates expression of these targets. Our study reveals a mechanism involving two partners in a complex. Manipulation of the genes in this pathway should improve stress tolerance in soybean or other legumes/crops. [ABSTRACT FROM AUTHOR]

Background: Epithelial-mesenchymal transition (EMT) and dysregulated microRNAs (miRNAs) have important roles in driving chemoresistance. We previously reported that iASPP is a key EMT inducer and could increase cisplatin resistance in cervical cancer (CC) cells. Herein, we investigate the downstream mechanisms through which iASPP contributes to EMT and cisplatin resistance in CC. Methods: By using a lentiviral system, we investigated the effects of iASPP knockdown on CC cell growth and chemosensitivity of CC cells to cisplatin in vivo. We examined if miR-20a, which was up-regulated following iASPP overexpression, would influence metastatic phenotypes and cisplatin resistance in CC cells, and explored the possible molecular mechanisms involved. Results: Knockdown of iASPP suppressed CC cell proliferation and sensitized CC cells to cisplatin in vivo. iASPP promotes miR-20a expression in a p53-dependent manner. Upregulation of miR-20a induced EMT and the recovery of CC cell invasion and cisplatin chemoresistance that was repressed by iASPP knockdown. We identified FBXL5 and BTG3 as two direct miR-20a targets. Silencing of FBXL5 and BTG3 restored cell invasion and cisplatin chemoresistance, which was suppressed by iASPP or miR-20a knockdown. Reduced FBXL5 and BTG3 expression was found in CC samples and associated with poor prognosis in CC patients. Conclusions: iASPP promotes EMT and confers cisplatin resistance in CC via miR-20a-FBXL5/BTG3 signaling. [ABSTRACT FROM AUTHOR]

Seed oil is a momentous agronomical trait of soybean (Glycine max) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351, encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1, BIOTIN CARBOXYL CARRIER PROTEIN2, 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III, DIACYLGLYCEROL O-ACYLTRANSFERASE1, and OLEOSIN2 in transgenic Arabidopsis (Arabidopsis thaliana) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean (Glycine soja) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops. [ABSTRACT FROM AUTHOR]

Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation. [ABSTRACT FROM AUTHOR]

Ethylene is a gaseous hormone that regulates many processes involved in plant growth, development and stress responses. Previously, we found that the tobacco ethylene receptor NTHK1 (Nicotiana tabacum histidine kinase 1) promotes seedling growth and affects plant salt stress responses. In this study, NTHK1 ethylene receptor-interacting protein 2 (NEIP2) was identified and further characterized in relation to these processes. NEIP2 contains three ankyrin repeats that mediate an interaction with NTHK1 as demonstrated by yeast two-hybrid, glutathione S-transferase (GST) pull-down and co-immunoprecipitation assays. NTHK1 phosphorylates NEIP2 in vitro. Salt stress and ethylene treatment induce NEIP2 accumulation in the first few hours and then the NEIP2 can be phosphorylated in planta. The overexpression of NTHK1 enhances NEIP2 accumulation in the presence of ethylene and salt stress. NEIP2 overexpression promotes plant growth but reduces ethylene responses, which is consistent with the functions of NTHK1. Additionally, NEIP2 improves plant performance under salt and oxidative stress. These results suggest that ethylene-induced NEIP2 probably acts as a brake to reduce ethylene response but resumes growth through interaction with NTHK1. Manipulation of NEIP2 may be beneficial for crop improvement. [ABSTRACT FROM AUTHOR]

Background: Trihelix transcription factor family is plant-specific and plays important roles in developmental processes. However, their function in abiotic stress response is largely unclear. Results: We studied one member GT-4 from Arabidopsis in relation to salt stress response. GT-4 expression is induced by salt stress and GT-4 protein is localized in nucleus and cytoplasm. GT-4 acts as a transcriptional activator and its C-terminal end is the activation domain. The protein can bind to the cis-elements GT-3 box, GT-3b box and MRE4. GT-4 confers enhanced salt tolerance in Arabidopsis likely through direct binding to the promoter and activation of Cor15A, in addition to possible regulation of other relevant genes. The gt-4 mutant shows salt sensitivity. TEM2, a member of AP2/ERF family was identified to interact with GT-4 in yeast two-hybrid, BiFC and Co-IP assays. Loss-of-function of TEM2 exerts no significant difference on salt tolerance or Cor15A expression in Arabidopsis. However, double mutant gt-4/tem2 shows greater sensitivity to salt stress and lower transcript level of Cor15A than gt-4 single mutant. GT-4 plus TEM2 can synergistically increase the promoter activity of Cor15A. Conclusions: GT-4 interacts with TEM2 and then co-regulates the salt responsive gene Cor15A to improve salt stress tolerance. [ABSTRACT FROM AUTHOR]

Background Translocation of high-mobility group box 1 (HMGB1) from nucleus could trigger inflammation. Extracellular HMGB1 up-regulates inflammatory response in sepsis as a late mediator. However, little was known about its role in subarachnoid hemorrhage-inducible inflammation, especially in the early stage. This study aims to identify whether HMGB1 translocation occurred early after SAH and also to clarify the potential role of HMGB1 in brain injury following SAH. Methods Sprague-Dawley (SD) rats were randomly divided into sham group and SAH groups at 2 h, 12 h and on day 1, day 2. SAH groups suffered experimental subarachnoid hemorrhage by injection of 0.3 ml autoblood into the pre-chiasmatic cistern. Rats injected by recombinant HMGB1(rHMGB1) solution were divided into four groups according to different time points. Cultured neurons were assigned into control group and four hemoglobin (Hb) incubated groups. Mixed glial cells were cultured and stimulated in medium from neurons incubated by Hb. HMGB1 expression is measured by western blot analysis, real-time polymerase chain reaction (PCR), immunohistochemistry and immunofluorescence. Downstream nuclear factor kappa B (NF-κB) subunit P65 and inflammatory factor Interleukin 1β (IL-1β) were measured by western blot and real-time PCR, respectively. Brain injury was evaluated by cleaved caspase-3 staining. Results Our results demonstrated HMGB1 translocation occurred as early as 2 h after experimental SAH with mRNA and protein level increased. Immunohistochemistry and immunofluorescence results indicated cytosolic HMGB1 was mainly located in neurons while translocated HMGB1 could also be found in some microglia. After subarachnoid injection of rHMGB1, NF-κB, downstream inflammatory response and cleaved caspase-3 were up-regulated in the cortex compared to the saline control group. In-vitro, after Hb incubation, HMGB1 was also rapidly released from neurons to medium. Incubation with medium from neurons up-regulated IL-1β in mixed glial cells. This effect could be inhibited by HMGB1 specific inhibitor glycyrrhizic acid (GA) treatment. Conclusion HMGB1 was released from neurons early after SAH onset and might trigger inflammation as an upstream inflammatory mediator. Extracellular HMGB1 contributed to the brain injury after SAH. These results might have important implications during the administration of specific HMGB1 antagonists early in order to prevent or reduce inflammatory response following SAH. [ABSTRACT FROM AUTHOR]

Background Soybean is one of the most important oil crops. The regulatory genes involved in oil accumulation are largely unclear. We initiated studies to identify genes that regulate this process. Results One MYB-type gene GmMYB73 was found to display differential expression in soybean seeds of different developing stages by microarray analysis and was further investigated for its functions in lipid accumulation. GmMYB73 is a small protein with single MYB repeat and has similarity to CPC-like MYB proteins from Arabidopsis. GmMYB73 interacted with GL3 and EGL3, and then suppressed GL2, a negative regulator of oil accumulation. GmMYB73 overexpression enhanced lipid contents in both seeds and leaves of transgenic Arabidopsis plants. Seed length and thousand-seed weight were also promoted. GmMYB73 introduction into the Arabidopsis try cpc double mutant rescued the total lipids, seed size and thousand-seed weight. GmMYB73 also elevated lipid levels in seeds and leaves of transgenic Lotus, and in transgenic hairy roots of soybean plants. GmMYB73 promoted PLDα1 expression, whose promoter can be bound and inhibited by GL2. PLDα1 mutation reduced triacylglycerol levels mildly in seeds but significantly in leaves of Arabidopsis plants. Conclusions GmMYB73 may reduce GL2, and then release GL2-inhibited PLDα1 expression for lipid accumulation. Manipulation of GmMYB73 may potentially improve oil production in legume crop plants. [ABSTRACT FROM AUTHOR]

Receptor-like kinases play important roles in plant development and defense responses; however, their functions in other processes remain unclear. Here, we report that OsSIK2, an S-domain receptor-like kinase from rice (Oryza sativa), is involved in abiotic stress and the senescence process. OsSIK2 is a plasma membrane-localized protein with kinase activity in the presence of Mn2+. OsSIK2 is expressed mainly in rice leaf and sheath and can be induced by NaC1, drought, cold, dark, and abscisic acid treatment. Transgenic plants overexpressing OsSIK2 and mutant sik2 exhibit enhanced and reduced tolerance to salt and drought stress, respectively, compared with the controls. Interestingly, a truncated version of OsSIK2 without most of the extracellular region confers higher salt tolerance than the full-length OsSIK2, likely through the activation of different sets of downstream genes. Moreover, seedlings of OsSIK2-overexpressing transgenic plants exhibit early leaf development and a delayed dark- induced senescence phenotype, while mutant sik2 shows the opposite phenotype. The downstream PR-related genes specifically up-regulated by full-length OsSIK2 or the DREB-like genes solely enhanced by truncated OsSIK2 are all induced by salt, drought, and dark treatments. These results indicate that OsSIK2 may integrate stress signals into a developmental program for better adaptive growth under unfavorable conditions. Manipulation of OsSIK2 should facilitate the improvement of production in rice and other crops. [ABSTRACT FROM AUTHOR]

α1,6-Fucosyltransferase (Fut8) knock-out (Fut8-/-) mice showed an abnormality in pre-B cell generation. Membrane assembly of pre-BCR is a crucial checkpoint for pre-B cell differentiation and proliferation in both humans and mice. The assembly of pre-BCR on the cell surface was substantially blocked in the Fut8-knockdown pre-B cell line, 70Z/3-KD cells, and then completely restored by re-introduction of the Fut8 gene to 70Z/3-KD (70Z/3-KD-re) cells. Moreover, loss of α1,6-fucosylation (also called core fucosylation) of μHC was associated with the suppression of the interaction between μHC and λ5. In contrast to Fut8-/- CD19+CD43- cells, the subpopulation expressing the μHCαλ5 complex in the Fut8-/- CD19+CD43- cell fraction was decreased. The pre-BCR-mediated tyrosine phosphorylation of CD79a and activation of Btk were attenuated in Fut8-KD cells, and restored in 70Z/3-KD-re cells. The frequency of CD19kw- CD43- cells (pre-B cell enriched fraction) was also reduced in Fut8+/+ bone marrow cells, and then the levels of IgM, IgG, and IgA of 12-week-old Fut8-/- mice sera were significantly lower than those of Fut8+/+ mice. Our results suggest that the core fucosylation of μHC mediates the assembly of pre-BCR to regulate pre-BCR intracellular signaling and pre-B cell proliferation. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in higher plants. miRNAs and related target genes have been widely studied in model plants such as Arabidopsis and rice; however, the number of identified miRNAs in soybean (Glycine max) is limited, and global identification of the related miRNA targets has not been reported in previous research. Results: In our study, a small RNA library and a degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. GO analysis indicated that many of the identified miRNA targets may function in soybean seed development. Additionally, a soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3 (AtSGS3) was detected as a target of the newly identified miRNA Soy•25, suggesting the presence of feedback control of miRNA biogenesis. Conclusions: We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library and a degradome library. Our study provides more information about the regulatory network of miRNAs in soybean and advances our understanding of miRNA functions during seed development. [ABSTRACT FROM AUTHOR]

Receptor-like kinases (RLKs) play essential roles in plant growth, development and responses to environmental stresses. A putative RLK gene, OsSIK1, with extracellular leucine-rich repeats was cloned and characterized in rice ( Oryza sativa). OsSIK1 exhibits kinase activity in the presence of Mn2+, and the OsSIK1 kinase domain has the ability to autophosphorylate and phosphorylate myelin basic protein (MBP). OsSIK1 promoter-GUS analysis revealed that OsSIK1 is expressed mainly in the stem and spikelet in rice. The expression of OsSIK1 is mainly induced by salt, drought and H2O2 treatments. Transgenic rice plants with overexpression of OsSIK1 show higher tolerance to salt and drought stresses than control plants. On the contrary, the knock-out mutants sik1-1 and sik1-2, as well as RNA interference (RNAi) plants, are sensitive to drought and salt stresses. The activities of peroxidase, superoxide dismutase and catalase are enhanced significantly in OsSIK1-overexpressing plants. Also, the accumulation of H2O2 in leaves of OsSIK1-overexpressing plants is much less than that of the mutants, RNAi plants and control plants, as measured by 3,3′-diamino benzidine (DAB) staining. We also show that OsSIK1 affects stomatal density in the abaxial and adaxial leaf epidermis of rice. These results indicate that OsSIK1 plays important roles in salt and drought stress tolerance in rice, through the activation of the antioxidative system. [ABSTRACT FROM AUTHOR]

RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of ∼20–30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2′-O-methylation on the 3′ terminal nucleotide. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 Å crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2′ and 3′ hydroxyls on the 3′-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg2+-dependent 2′-O-methylation mechanism. [ABSTRACT FROM AUTHOR]

Ethylene receptor is the first component of ethylene signaling that regulates plant growth, development and stress responses. Previously, we have demonstrated that tobacco subfamily 2 ethylene receptor NTHK1 had Ser/Thr kinase activity, and overexpression of NTHK1 caused large rosette, reduced ethylene sensitivity, and increased salt sensitivity in transgenic Arabidopsis plants. Here we found that N-box mutation in the NTHK1 kinase domain abolished the kinase activity and led to disruption of NTHK1 roles in conferring reduced ethylene sensitivity and salt sensitive response in transgenic Arabidopsis plants. However, N-box mutation had partial effects on NTHK1 regulation of rosette growth and expression of salt- and ethylene-responsive genes AtNAC2, AtERF1 and AtCor6.6. Mutation of conserved residues in the H box did not affect kinase activity, seedling growth, ethylene sensitivity or salt-induced epinasty in transgenic plants but did influence NTHK1 function in control of specific salt- and ethylene-responsive gene expression. Compared with NTHK1, the tobacco subfamily 1 ethylene receptor NtETR1 had His kinase activity and played a weak role in regulation of rosette growth, triple response and salt response. Mutation of the conserved His residue in the NtETR1 H box eliminated phosphorylation and altered the effect of Ntetr1-1 on reporter gene activity. These results imply that the Ser/Thr kinase activity of NTHK1 is differentially required for various responses, and NTHK1 plays a larger role than NtETR1. [ABSTRACT FROM PUBLISHER]

Background: Soybean [Glycine max (L.) Merr.] is one of the most important crops for oil and protein resource. Improvement of stress tolerance will be beneficial for soybean seed production. Principal Findings: Six GmPHD genes encoding Alfin1-type PHD finger protein were identified and their expressions differentially responded to drought, salt, cold and ABA treatments. The six GmPHDs were nuclear proteins and showed ability to bind the cis-element ''GTGGAG''. The N-terminal domain of GmPHD played a major role in DNA binding. Using a protoplast assay system, we find that GmPHD1 to GmPHD5 had transcriptional suppression activity whereas GmPHD6 did not have. In yeast assay, the GmPHD6 can form homodimer and heterodimer with the other GmPHDs except GmPHD2. The N-terminal plus the variable regions but not the PHD-finger is required for the dimerization. Transgenic Arabidopsis plants overexpressing the GmPHD2 showed salt tolerance when compared with the wild type plants. This tolerance was likely achieved by diminishing the oxidative stress through regulation of downstream genes. Significance: These results provide important clues for soybean stress tolerance through manipulation of PHD-type transcription regulator. [ABSTRACT FROM AUTHOR]

Background: Trihelix transcription factors play important roles in light-regulated responses and other developmental processes. However, their functions in abiotic stress response are largely unclear. In this study, we identified two trihelix transcription factor genes GmGT-2A and GmGT-2B from soybean and further characterized their roles in abiotic stress tolerance. Findings: Both genes can be induced by various abiotic stresses, and the encoded proteins were localized in nuclear region. In yeast assay, GmGT-2B but not GmGT-2A exhibits ability of transcriptional activation and dimerization. The N-terminal peptide of 153 residues in GmGT-2B was the minimal activation domain and the middle region between the two trihelices mediated the dimerization of the GmGT-2B. Transactivation activity of the GmGT-2B was also confirmed in plant cells. DNA binding analysis using yeast one-hybrid assay revealed that GmGT-2A could bind to GT-1bx, GT-2bx, mGT-2bx-2 and D1 whereas GmGT-2B could bind to the latter three elements. Overexpression of the GmGT-2A and GmGT-2B improved plant tolerance to salt, freezing and drought stress in transgenic Arabidopsis plants. Moreover, GmGT-2B-transgenic plants had more green seedlings compared to Col-0 under ABA treatment. Many stress-responsive genes were altered in GmGT-2A- and GmGT-2B-transgenic plants. Conclusion: These results indicate that GmGT-2A and GmGT-2B confer stress tolerance through regulation of a common set of genes and specific sets of genes. GmGT-2B also affects ABA sensitivity. [ABSTRACT FROM AUTHOR]

Ethylene regulates multiple aspects of plant growth and development in dicotyledonous plants; however, its roles in monocotyledonous plants are po.orly known. Here, we characterized a subfamily II ethylene receptor, ETHYLENE RESPONSE2 (ETR2), in rice (Oryza sativa). The ETR2 receptor with a diverged His kinase domain is a Ser/Thr kinase, but not a His kinase, and can phosphorylate its receiver domain. Mutation of the N box of the kinase domain abolished the kinase activity of ETR2. Overexpression of ETR2 in transgenic rice plants reduced ethylene sensitivity and delayed floral transition. Conversely, RNA interference (RNAi) plants exhibited early flowering and the ETR2 T-DNA insertion mutant etr2 showed enhanced ethylene sensitivity and early flowering. The effective panicles and seed-setting rate were reduced in the ETR2-overexpressing plants, while thousand-seed weight was substantially enhanced in both the ETR2-RNAi plants and the etr2 mutant compared with controls. Starch granules accumulated in the internodes of the ETR2-overexpressing plants, but not in the etr2 mutant. The GIGANTEA and TERMINAL FLOWER1/CENTRORADIALIS homolog (RCN1) that cause delayed flowering were upregulated in ETR2-overexpressing plants but downregulated in the ett'2 mutant. Conversely, the α-amylase gene RAmy3D was suppressed in ETR2-overexpressing plants but enhanced in the etr'2 mutant. Thus, ETR2 may delay flowering and cause starch accumulation in stems by regulating downstream genes. [ABSTRACT FROM AUTHOR]