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Fragment molecular orbital (FMO) method is a powerful computational tool for structure-based drug design, in which protein-ligand interactions can be described by the inter-fragment interaction energy (IFIE) and its pair interaction energy decomposition analysis (PIEDA). Here, we introduced a dynamically averaged (DA) FMO-based approach in which molecular dynamics simulations were used to generate multiple protein-ligand complex structures for FMO calculations. To assess this approach, we examined the correlation between the experimental binding free energies and DA-IFIEs of six CDK2 inhibitors whose net charges are zero. The correlation between the experimental binding free energies and snapshot IFIEs for X-ray crystal structures was R

Grain size is one of the essential determinants of rice yield. Our previous studies revealed that ethylene plays an important role in grain-size control; however, the precise mechanism remains to be determined. Here, we report that the ethylene response factor OsERF115 functions as a key downstream regulator for ethylene-mediated grain development. OsERF115 encodes an AP2/ERF-type transcriptional factor that is specifically expressed in young spikelets and developing caryopses. Overexpression of OsERF115 significantly increases grain length, width, thickness and weight by promoting longitudinal elongation and transverse division of spikelet hull cells, as well as enhancing grain-filling activity, whereas its knockout mutations lead to the opposite effects, suggesting that OsERF115 positively regulates grain size and weight. OsERF115 transcription is strongly induced by ethylene, and OsEIL1 directly binds to the promoter to activate its expression. OsERF115 acts as a transcriptional repressor to directly or indirectly modulate a set of grain-size genes during spikelet growth and endosperm development. Importantly, haplotype analysis reveals that the SNP variations in the EIN3-binding sites of OsERF115 promoter are significantly associated with the OsERF115 expression levels and grain weight, suggesting that natural variations in the OsERF115 promoter contribute to grain-size diversity. In addition, the OsERF115 orthologues are identified only in grass species, implying a conserved and unique role in the grain development of cereal crops. Our results provide insights into the molecular mechanism of ethylene-mediated grain-size control and a potential strategy based on the OsEIL1-OsERF115-target gene regulatory module for genetic improvement of rice yield.

A rhodium(III)-catalyzed domino annulation of simple olefins with diazo oxindoles to give spirooxindole pyrrolone products is described. This reaction can be formally viewed as the result of an anomalous tandem C-H activation, carbene insertion, Lossen rearrangement, and a nucleophilic addition process. The potential utility of this reaction was further demonstrated by the late-stage diversification of drug molecules.

WRKY transcription factors play important roles in response to various abiotic stresses. Previous study have proved that soybean GmWRKY54 can improve stress tolerance in transgenic Arabidopsis. Here, we generated soybean transgenic plants and further investigated roles and biological mechanisms of GmWRKY54 in response to drought stress. We demonstrated that expression of GmWRKY54, driven by either a constitutive promoter (pCm) or a drought-induced promoter (RD29a), confers drought tolerance. GmWRKY54 is a transcriptional activator and affects a large number of stress-related genes as revealed by RNA sequencing. Gene ontology (GO) enrichment and co-expression network analysis, together with measurement of physiological parameters, supported the idea that GmWRKY54 enhances stomatal closure to reduce water loss, and therefore confers drought tolerance in soybean. GmWRKY54 directly binds to the promoter regions of genes including PYL8, SRK2A, CIPK11 and CPK3 and activates them. Therefore GmWRKY54 achieves its function through abscisic acid (ABA) and Ca2+ signaling pathways. It is valuable that GmWRKY54 activates an ABA receptor and an SnRK2 kinase in the upstream position, unlike other WRKY proteins that regulate downstream genes in the ABA pathway. Our study revealed the role of GmWRKY54 in drought tolerance and further manipulation of this gene should improve growth and production in soybean and other legumes/crops under unfavorable conditions.

Protein-drug binding mode prediction from the apo-protein structure is challenging because drug binding often induces significant protein conformational changes. Here, the authors report a computational workflow that incorporates a novel pocket generation method. First, the closed protein pocket is expanded by repeatedly filling virtual atoms during molecular dynamics (MD) simulations. Second, after ligand docking toward the prepared pocket structures, binding mode candidates are ranked by MD/Molecular Mechanics Poisson-Boltzmann Surface Area. The authors validated our workflow using CDK2 kinase, which has an especially-closed ATP-binding pocket in the apo-form, and several inhibitors. The crystallographic pose coincided with the top-ranked docking pose for 59% (34/58) of the compounds and was within the top five-ranked ones for 88% (51/58), while those estimated by a conventional prediction protocol were 9% (5/58) and 50% (29/58), respectively. Our study demonstrates that the prediction accuracy is significantly improved by preceding pocket expansion, leading to generation of conformationally-diverse binding mode candidates. © 2018 Wiley Periodicals, Inc.

Background Annexin A2 (ANXA2) plays a crucial role in acute pancreatitis (AP). However, its potential mechanism remains unclear. Methods In the present study, we used caerulein-treated AR42J rat pancreatic acinar cells as cell model of AP to investigate the potential functions of ANXA2 and its predicted long noncoding RNA (lncRNA) FOXF1 adjacent noncoding developmental regulatory RNA (lncRNA Fendrr). Cell apoptosis was evaluated by flow cytometry using annexinV-fluorescein isothiocyanate/propidium iodide staining. The expressions of ANAX2 and lncRNA Fendrr were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, Western blot analysis was performed to determine the protein levels of ANXA2, Bcl-2, and Bax. The association between lncRNA Fendrr and ANXA2 was disclosed by RNA pull-down, RNA immunoprecipitation, and electrophoretic mobility shift assays. Results ANXA2 was elevated in caerulein-induced AP model and promoted apoptosis of AR42J cells. LncRNA Fendrr was also upregulated in AP cell model and directly bound ANXA2 protein. Further studies indicated that the interaction between ANXA2 and lncRNA Fendrr contributed to the apoptosis of AR42J cells in AP cell model. Conclusion Our study demonstrated that ANXA2 promoted AP progression via interacting with lncRNA Fendrr in vitro, which will provide a novel insight into the therapeutic target for AP.

Cultivated soybean has undergone many transformations during domestication. In this paper we report a comprehensive assessment of the evolution of gene co-expression networks based on the analysis of 40 transcriptomes from developing soybean seeds in cultivated and wild soybean accessions. We identified 2680 genes that are differentially expressed during seed maturation and established two cultivar-specific gene co-expression networks. Through analysis of the two networks and integration with quantitative trait locus data we identified two potential key drivers for seed trait formation, GA20OX and NFYA. GA20OX encodes an enzyme in a rate-limiting step of gibberellin biosynthesis, and NFYA encodes a transcription factor. Overexpression of GA20OX and NFYA enhanced seed size/weight and oil content, respectively, in seeds of transgenic plants. The two genes showed significantly higher expression in cultivated than in wild soybean, and the increases in expression were associated with genetic variations in the promoter region of each gene. Moreover, the expression of GA20OX and NFYA in seeds of soybean accessions correlated with seed weight and oil content, respectively. Our study reveals transcriptional adaptation during soybean domestication and may identify a mechanism of selection by expression for seed trait formation, providing strategies for future breeding practice.

There has been a surge of interest in the molecular biology of RNA. We describe a simple, fast, and convenient protocol for isolating total RNA from rice (Oryza sativa) seedlings and young tissues/organs. RNA obtained using this method can be used for reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), northern blot analysis, RNA-seq, microarray analysis, cDNA library construction, etc. Technical parameters critical to the success of the extraction are presented. © 2016 by John Wiley & Sons, Inc.

Plant homeodomain (PHD) finger proteins affect processes of growth and development by changing transcription and reading epigenetic histone modifications, but their functions in abiotic stress responses remain largely unclear. Here we characterized seven Arabidopsis thaliana Alfin1-like PHD finger proteins (ALs) in terms of the responses to abiotic stresses. ALs localized to the nucleus and repressed transcription. Except AL6, all the ALs bound to G-rich elements. Mutations of the amino acids at positions 34 and 35 in AL6 caused loss of ability to bind to G-rich elements. Expression of the AL genes responded differentially to osmotic stress, salt, cold and abscisic acid treatments. AL5-over-expressing plants showed higher tolerance to salt, drought and freezing stress than Col-0. Consistently, al5 mutants showed reduced stress tolerance. We used ChIP-Seq assays to identify eight direct targets of AL5, and found that AL5 binds to the promoter regions of these genes. Knockout mutants of five of these target genes exhibited varying tolerances to stresses. These results indicate that AL5 inhibits multiple signaling pathways to confer stress tolerance. Our study sheds light on mechanisms of AL5-mediated signaling in abiotic stress responses, and provides tools for improvement of stress tolerance in crop plants.

WRKY-type transcription factors are involved in multiple aspects of plant growth, development and stress response. WRKY genes have been found to be responsive to abiotic stresses; however, their roles in abiotic stress tolerance are largely unknown especially in crops. Here, we identified stress-responsive WRKY genes from wheat (Triticum aestivum L.) and studied their functions in stress tolerance. Forty-three putative TaWRKY genes were identified and two multiple stress-induced genes, TaWRKY2 and TaWRKY19, were further characterized. TaWRKY2 and TaWRKY19 are nuclear proteins, and displayed specific binding to typical cis-element W box. Transgenic Arabidopsis plants overexpressing TaWRKY2 exhibited salt and drought tolerance compared with controls. Overexpression of TaWRKY19 conferred tolerance to salt, drought and freezing stresses in transgenic plants. TaWRKY2 enhanced expressions of STZ and RD29B, and bound to their promoters. TaWRKY19 activated expressions of DREB2A, RD29A, RD29B and Cor6.6, and bound to DREB2A and Cor6.6 promoters. The two TaWRKY proteins may regulate the downstream genes through direct binding to the gene promoter or via indirect mechanism. Manipulation of TaWRKY2 and TaWRKY19 in wheat or other crops should improve their performance under various abiotic stress conditions.

BACKGROUND: Viper snake venoms contain a great variety of toxic proteins. These components mediate their toxicity by either stimulating or inhibiting the haemostatic system of human victims or experimental animals, resulting in common clinical complications of blood clotting or uncontrolled haemorrhage. Therefore it is deemed important to isolate the active component(s) from snake venom with kallikrein-like activity. RESULTS: A kallikrein-like proteinase of Agkistrodon halys pallas snake venom, designated AHP-Ka, was purified by anion exchange chromatography and affinity chromatography. Physicochemical studies showed that the purified enzyme was a 34 kDa monomeric glycoprotein, the molecular weight of which decreased to 26 kDa after deglycosylation with peptide N-glycosidase F (PNGase F). Sequence studies on the NH2-terminal region of the protein indicated that AHP-Ka shared a high degree of sequence homology with other serine proteinases from snake venoms. AHP-Ka showed high catalytic activity and kallikrein-like activity on substrates such as arginine esterase BAEE and chromogenic H-D-Pro-Phe-Arg-pNA·2HCl (S-2302) and was inhibited by protease inhibitor phenylmethylsulfonyl fluoride (PMSF). CONCLUSION: The results showed that AHP-Ka isolated from A. halys pallas snake venom and purified by anion exchange chromatography and affinity chromatography is in fact a kallikrein-like enzyme. c � 2011 Society of Chemical Industry

The NIMA-related kinases (NEKs) are a family of serine/threonine kinases involved largely in cell cycle control in fungi, mammals and other eukaryotes. In Arabidopsis, NEK6 is involved in the regulation of epidermal cell morphogenesis. However, other roles of NEK6 in plants are less well understood. Here we report functions of NEK6 in plant growth, development and stress responses in Arabidopsis. NEK6 transcripts and proteins are induced by ethylene precursor ACC and salt stress. Expression of other NEK genes except NEK5 is also responsive to the two treatments. Overexpression and mutant analysis disclose that the NEK6 gene increases rosette growth, seed yield and lateral root formation. However, NEK6 appears to play a negative role in the control of seed size. The gene also promotes plant tolerance to salt stress and osmotic stress in its overexpressing plants. The NEK6 gene may achieve its function through suppression of ethylene biosynthesis and activation of CYCB1;1 and CYCA3;1 expression. Our present study reveals new functions of the NEK6 gene in plant growth and stress tolerance, and manipulation of NEK6 may improve important agronomic traits in crop plants.

NAC transcription factors play important roles in plant growth, development and stress responses. Previously, we identified multiple NAC genes in soybean (Glycine max). Here, we identify the roles of two genes, GmNAC11 and GmNAC20, in stress responses and other processes. The two genes were differentially induced by multiple abiotic stresses and plant hormones, and their transcripts were abundant in roots and cotyledons. Both genes encoded proteins that localized to the nucleus and bound to the core DNA sequence CGT[G/A]. In the protoplast assay system, GmNAC11 acts as a transcriptional activator, whereas GmNAC20 functions as a mild repressor; however, the C-terminal end of GmANC20 has transcriptional activation activity. Over-expression of GmNAC20 enhances salt and freezing tolerance in transgenic Arabidopsis plants; however, GmNAC11 over-expression only improves salt tolerance. Over-expression of GmNAC20 also promotes lateral root formation. GmNAC20 may regulate stress tolerance through activation of the DREB/CBF-COR pathway, and may control lateral root development by altering auxin signaling-related genes. GmNAC11 probably regulates DREB1A and other stress-related genes. The roles of the two GmNAC genes in stress tolerance were further analyzed in soybean transgenic hairy roots. These results provide a basis for genetic manipulation to improve the agronomic traits of important crops.

Ethylene signalling regulates plant growth and development. However, its roles in salt stress response are less known. Here we studied functions of EIN2, a central membrane protein of ethylene signalling, and its interacting protein ECIP1 in salt stress responses. Mutation of EIN2 led to extreme salt sensitivity as revealed by phenotypic and physiological changes, and overexpression of C-terminus of EIN2 suppressed salt sensitivity in ein2-5, indicating that EIN2 is required for salt tolerance. Downstream components EIN3 and EIL1 are also essential for salt tolerance because ein3-1eil1-1 double mutant showed extreme salt-sensitive phenotype. A MA3 domain-containing protein ECIP1 was further identified to interact with EIN2 in yeast two-hybrid assay and GST pull-down assay. Loss-of-function of ECIP1 resulted in enhanced ethylene response but altered salt response during seed germination and plant growth. Double mutant analysis revealed that ein2-1 was epistatic to ecip1, and ecip1 mutation partially suppressed ethylene-insensitivity of etr2-1 and ein4-1. These studies strengthen that interactions between ECIP1 and EIN2 or ethylene receptors regulate ethylene response and stress response.

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 Mn(2+), 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 H(2)O(2) 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 H(2)O(2) 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.

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.