Diversification in the inositol tris/tetrakisphosphate kinase (ITPK) family: crystal structure and enzymology of the outlier AtITPK4.

Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Šresolution that, along with a description of the enantiospecificity of the enzyme, affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a KM for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologues in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology.

Structural basis of outer membrane protein insertion by the BAM complex.

All Gram-negative bacteria, mitochondria and chloroplasts have outer membrane proteins (OMPs) that perform many fundamental biological processes. The OMPs in Gram-negative bacteria are inserted and folded into the outer membrane by the ß-barrel assembly machinery (BAM). The mechanism involved is poorly understood, owing to the absence of a structure of the entire BAM complex. Here we report two crystal structures of the Escherichia coli BAM complex in two distinct states: an inward-open state and a lateral-open state. Our structures reveal that the five polypeptide transport-associated domains of BamA form a ring architecture with four associated lipoproteins, BamB-BamE, in the periplasm. Our structural, functional studies and molecular dynamics simulations indicate that these subunits rotate with respect to the integral membrane ß-barrel of BamA to induce movement of the ß-strands of the barrel and promote insertion of the nascent OMP.

Structural basis for outer membrane lipopolysaccharide insertion.

Lipopolysaccharide (LPS) is essential for most Gram-negative bacteria and has crucial roles in protection of the bacteria from harsh environments and toxic compounds, including antibiotics. Seven LPS transport proteins (that is, LptA-LptG) form a trans-envelope protein complex responsible for the transport of LPS from the inner membrane to the outer membrane, the mechanism for which is poorly understood. Here we report the first crystal structure of the unique integral membrane LPS translocon LptD-LptE complex. LptD forms a novel 26-stranded ß-barrel, which is to our knowledge the largest ß-barrel reported so far. LptE adopts a roll-like structure located inside the barrel of LptD to form an unprecedented two-protein 'barrel and plug' architecture. The structure, molecular dynamics simulations and functional assays suggest that the hydrophilic O-antigen and the core oligosaccharide of the LPS may pass through the barrel and the lipid A of the LPS may be inserted into the outer leaflet of the outer membrane through a lateral opening between strands ß1 and ß26 of LptD. These findings not only help us to understand important aspects of bacterial outer membrane biogenesis, but also have significant potential for the development of novel drugs against multi-drug resistant pathogenic bacteria.

BamA ß16C strand and periplasmic turns are critical for outer membrane protein insertion and assembly.

Outer membrane (OM) ß-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli, the ß-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a 'top hat'-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel ß1C-ß6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the ß16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique ß16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA ß1C-ß6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts.

An ultrathin terahertz quarter-wave plate using planar babinet-inverted metasurface.

Metamaterials promise an exotic approach to artificially manipulate the polarization state of electromagnetic waves and boost the design of polarimetric devices for sensitive detection, imaging and wireless communication. Here, we present the design and experimental demonstration of an ultrathin (0.29λ) terahertz quarter-wave plate based on planar babinet-inverted metasurface. The quarter-wave plate consisting of arrays of asymmetric cross apertures reveals a high transmission of 0.545 with 90 degrees phase delay at 0.870 THz. The calculated ellipticity indicates a high degree of polarization conversion from linear to circular polarization. With respect to different incident polarization angles, left-handed circular polarized light, right-handed circular polarized light and elliptically polarized light can be created by this novel design. An analytical model is applied to describe transmitted amplitude, phase delay and ellipticitiy, which are in good agreement with the measured and simulated results. The planar babinet-inverted metasurface with the analytical model opens up avenues for new functional terahertz devices design.

De Novo Transcriptome Sequencing of Oryza officinalis Wall ex Watt to Identify Disease-Resistance Genes.

Oryza officinalis Wall ex Watt is one of the most important wild relatives of cultivated rice and exhibits high resistance to many diseases. It has been used as a source of genes for introgression into cultivated rice. However, there are limited genomic resources and little genetic information publicly reported for this species. To better understand the pathways and factors involved in disease resistance and accelerating the process of rice breeding, we carried out a de novo transcriptome sequencing of O. officinalis. In this research, 137,229 contigs were obtained ranging from 200 to 19,214 bp with an N50 of 2331 bp through de novo assembly of leaves, stems and roots in O. officinalis using an Illumina HiSeq 2000 platform. Based on sequence similarity searches against a non-redundant protein database, a total of 88,249 contigs were annotated with gene descriptions and 75,589 transcripts were further assigned to GO terms. Candidate genes for plant-pathogen interaction and plant hormones regulation pathways involved in disease-resistance were identified. Further analyses of gene expression profiles showed that the majority of genes related to disease resistance were all expressed in the three tissues. In addition, there are two kinds of rice bacterial blight-resistant genes in O. officinalis, including two Xa1 genes and three Xa26 genes. All 2 Xa1 genes showed the highest expression level in stem, whereas one of Xa26 was expressed dominantly in leaf and other 2 Xa26 genes displayed low expression level in all three tissues. This transcriptomic database provides an opportunity for identifying the genes involved in disease-resistance and will provide a basis for studying functional genomics of O. officinalis and genetic improvement of cultivated rice in the future.

Transcriptome analysis to identify putative floral-specific genes and flowering regulatory-related genes of sweet potato.

Sweet potato flowers were collected for a transcriptome analysis to identify the putative floral-specific and flowering regulatory-related genes by using the RNA-sequencing technique. Pair-end short reads were de novo assembled by an integrated strategy, and then the floral transcriptome was carefully compared with several published vegetative transcriptomes. A total of 2595 putative floral-specific and 2928 putative vegetative-specific transcripts were detected. We also identified a large number of transcripts similar to the key genes in the flowering regulation network of Arabidopsis thaliana.

Structure of a cereal purple acid phytase provides new insights to phytate degradation in plants.

Grain phytate, a mixed metal ion salt of inositol hexakisphosphate, accounts for 60%-80% of stored phosphorus in plants and is a potent antinutrient of non-ruminant animals including humans. Through neofunctionalization of purple acid phytases (PAPhy), some cereals such as wheat and rye have acquired particularly high mature grain phytase activity. As PAPhy activity supplies phosphate, liberates metal ions necessary for seedling emergence, and obviates antinutrient effects of phytate, its manipulation and control are targeted crop traits. Here we show the X-ray crystal structure of the b2 isoform of wheat PAPhy induced during germination. This high-resolution crystal structure suggests a model for phytate recognition that, validated by molecular dynamics simulations, implicates elements of two sequence inserts (termed PAPhy motifs) relative to a canonical metallophosphoesterase (MPE) domain in forming phytate-specific substrate specificity pockets. These motifs are well conserved in PAPhys from monocot cereals, enzymes which are characterized by high specificity for phytate. Tested by mutagenesis, residues His229 in PAPhy motif 4 and Lys410 in the MPE domain, both conserved in PAPhys, are found to strongly influence phytase activity. These results explain the observed phytase activity of cereal PAPhys and open the way to the rational engineering of phytase activity in planta.

Giant photoluminescence enhancement in tungsten-diselenide-gold plasmonic hybrid structures.

Impressive properties arise from the atomically thin nature of transition metal dichalcogenide two-dimensional materials. However, being atomically thin limits their optical absorption or emission. Hence, enhancing their photoluminescence by plasmonic nanostructures is critical for integrating these materials in optoelectronic and photonic devices. Typical photoluminescence enhancement from transition metal dichalcogenides is 100-fold, with recent enhancement of 1,000-fold achieved by simultaneously enhancing absorption, emission and directionality of the system. By suspending WSe2 flakes onto sub-20-nm-wide trenches in gold substrate, we report a giant photoluminescence enhancement of ∼20,000-fold. It is attributed to an enhanced absorption of the pump laser due to the lateral gap plasmons confined in the trenches and the enhanced Purcell factor by the plasmonic nanostructure. This work demonstrates the feasibility of giant photoluminescence enhancement in WSe2 with judiciously designed plasmonic nanostructures and paves a way towards the implementation of plasmon-enhanced transition metal dichalcogenide photodetectors, sensors and emitters.

Trapped lipopolysaccharide and LptD intermediates reveal lipopolysaccharide translocation steps across the Escherichia coli outer membrane.

Lipopolysaccharide (LPS) is a main component of the outer membrane of Gram-negative bacteria, which is essential for the vitality of most Gram-negative bacteria and plays a critical role for drug resistance. LptD/E complex forms a N-terminal LPS transport slide, a hydrophobic intramembrane hole and the hydrophilic channel of the barrel, for LPS transport, lipid A insertion and core oligosaccharide and O-antigen polysaccharide translocation, respectively. However, there is no direct evidence to confirm that LptD/E transports LPS from the periplasm to the external leaflet of the outer membrane. By replacing LptD residues with an unnatural amino acid p-benzoyl-L-phenyalanine (pBPA) and UV-photo-cross-linking in E.coli, the translocon and LPS intermediates were obtained at the N-terminal domain, the intramembrane hole, the lumenal gate, the lumen of LptD channel, and the extracellular loop 1 and 4, providing the first direct evidence and "snapshots" to reveal LPS translocation steps across the outer membrane.

Transcriptomic Analysis and the Expression of Disease-Resistant Genes in Oryza meyeriana under Native Condition.

Oryza meyeriana (O. meyeriana), with a GG genome type (2n = 24), accumulated plentiful excellent characteristics with respect to resistance to many diseases such as rice shade and blast, even immunity to bacterial blight. It is very important to know if the diseases-resistant genes exist and express in this wild rice under native conditions. However, limited genomic or transcriptomic data of O. meyeriana are currently available. In this study, we present the first comprehensive characterization of the O. meyeriana transcriptome using RNA-seq and obtained 185,323 contigs with an average length of 1,692 bp and an N50 of 2,391 bp. Through differential expression analysis, it was found that there were most tissue-specifically expressed genes in roots, and next to stems and leaves. By similarity search against protein databases, 146,450 had at least a significant alignment to existed gene models. Comparison with the Oryza sativa (japonica-type Nipponbare and indica-type 93-11) genomes revealed that 13% of the O. meyeriana contigs had not been detected in O. sativa. Many diseases-resistant genes, such as bacterial blight resistant, blast resistant, rust resistant, fusarium resistant, cyst nematode resistant and downy mildew gene, were mined from the transcriptomic database. There are two kinds of rice bacterial blight-resistant genes (Xa1 and Xa26) differentially or specifically expressed in O. meyeriana. The 4 Xa1 contigs were all only expressed in root, while three of Xa26 contigs have the highest expression level in leaves, two of Xa26 contigs have the highest expression profile in stems and one of Xa26 contigs was expressed dominantly in roots. The transcriptomic database of O. meyeriana has been constructed and many diseases-resistant genes were found to express under native condition, which provides a foundation for future discovery of a number of novel genes and provides a basis for studying the molecular mechanisms associated with disease resistance in O. meyeriana.

Color generation via subwavelength plasmonic nanostructures.

Recent developments in color filtering and display technologies have focused predominantly on high resolution, color vibrancy, high efficiency, and slim dimensions. To achieve these goals, metallic nanostructures have attracted extensive research interest due to their abilities to manipulate the properties of light through surface plasmon resonances. In this paper, we review recent representative developments in plasmonic color engineering at the nanoscale using subwavelength nanostructures, demonstrating their great potential in high-resolution and high-fidelity color rendering, spectral filtering applications, holography, three-dimensional stereoscopic imaging, etc.

Lipopolysaccharide is inserted into the outer membrane through an intramembrane hole, a lumen gate, and the lateral opening of LptD.

Lipopolysaccharide (LPS) is essential for the vitality of most Gram-negative bacteria and plays an important role in bacterial multidrug resistance. The LptD/E translocon inserts LPS into the outer leaflet, the mechanism of which is poorly understood. Here, we report mutagenesis, functional assays, and molecular dynamics simulations of the LptD/E complex, which suggest two distinct pathways for the insertion of LPS. The N-terminal domain of LptD comprises a hydrophobic slide that injects the acyl tails of LPS directly into the outer membrane through an intramembrane hole, while the core oligosaccharide and O-antigen pass a lumen gate that triggers the unzipping of the lateral opening between strands ß1C and ß26C of the barrel of LptD, to finalize LPS insertion. Mutation of the LPS transport related residues or block of the LPS transport pathways results in the deaths of Escherichia coli. These findings are important for the development of novel antibiotics.

Switchable Ultrathin Quarter-wave Plate in Terahertz Using Active Phase-change Metasurface.

Metamaterials open up various exotic means to control electromagnetic waves and among them polarization manipulations with metamaterials have attracted intense attention. As of today, static responses of resonators in metamaterials lead to a narrow-band and single-function operation. Extension of the working frequency relies on multilayer metamaterials or different unit cells, which hinder the development of ultra-compact optical systems. In this work, we demonstrate a switchable ultrathin terahertz quarter-wave plate by hybridizing a phase change material, vanadium dioxide (VO2), with a metasurface. Before the phase transition, VO2 behaves as a semiconductor and the metasurface operates as a quarter-wave plate at 0.468 THz. After the transition to metal phase, the quarter-wave plate operates at 0.502 THz. At the corresponding operating frequencies, the metasurface converts a linearly polarized light into a circularly polarized light. This work reveals the feasibility to realize tunable/active and extremely low-profile polarization manipulation devices in the terahertz regime through the incorporation of such phase-change metasurfaces, enabling novel applications of ultrathin terahertz meta-devices.

Density functional theory and ab initio studies of geometry, electronic structure and vibrational spectra of novel benzothiazole and benzotriazole herbicides.

The ground-state geometries, electronic structures and vibrational wavenumbers of S-1,3-benzothiazolyl-4-bromobenzenecarbothioate and S-(5,7-dimethyl-3H-4lambda-5-[1,2,4]triazolo[1,5-a]pyridinyl)4-chlorobenzenecarbothioate were studied by DFT-B3LYP, BLYP and ab initio RHF method with different basis sets. The comparison was performed for optimized geometries, thermodynamic parameters and electronic structures at different levels of theory. Because of the larger repulsion effect in triazole ring, the vibrational wavenumbers of skeleton vibration of triazole ring are significantly lower than that of thiazole ring.

[Effects of hypoxia and hyperoxia on the regulation of the expression and activity of matrix metalloproteinase-2 in hepatic stellate cell].

OBJECTIVE: To study the effects of hypoxia and hyperoxia on the expression and activity regulation of matrix metalloproteinase-2 (MMP-2) of the hepatic stellate cell (HSC). METHODS: The expression of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC under hypoxic or hyperoxic conditions were detected with immunocytochemistry (LSAB method), the contents of MMP-2, TIMP-2 in culture supernatant with ELISA and the activity of MMP-2 in supernatant with zymography. RESULTS: (1) In the situation of hypoxia for 12 h, the expression of MMP-2 increased (hypoxia group positive indexes: 5.7 +/- 2.0; control: 3.2 +/- 1.0; P < 0.01), while TIMP-2 decreased (hypoxia group positive indexes: 2.5 +/- 0.7; control: 3.6 +/- 1.0; P < 0.05) in HSC, and the activity of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922; control: 17.277 +/- 7.424; P < 0.01). At the different time courses of hypoxia, the change of expression and activity of MMP-2 was most notable at 6 h. (2) In the situation of hyperoxia for 12 h, the protein contents of MMP-2, TIMP-2 in supernatant were both higher than those of the control, especially the TIMP-2 (hyperoxia group A(450): 0.050 +/- 0.014; control: 0.022 +/- 0.010; P < 0.01), and so was the activity of MMP-2 (hyperoxia group total A: 5.252 +/- 0.771; control: 4.304 +/- 1.083; P < 0.05). The expression of MT1-MMP was also increased. CONCLUSIONS: The HSC is sensitive to the oxygen. Hypoxia accelerates the expression of MMP-2 and the effect is more marked at the early stage. Hyperoxia increases the activity of MMP-2.

Exploring the polyadenylated RNA virome of sweet potato through high-throughput sequencing.

BACKGROUND: Viral diseases are the second most significant biotic stress for sweet potato, with yield losses reaching 20% to 40%. Over 30 viruses have been reported to infect sweet potato around the world, and 11 of these have been detected in China. Most of these viruses were detected by traditional detection approaches that show disadvantages in detection throughput. Next-generation sequencing technology provides a novel, high sensitive method for virus detection and diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: We report the polyadenylated RNA virome of three sweet potato cultivars using a high throughput RNA sequencing approach. Transcripts of 15 different viruses were detected, 11 of which were detected in cultivar Xushu18, whilst 11 and 4 viruses were detected in Guangshu 87 and Jingshu 6, respectively. Four were detected in sweet potato for the first time, and 4 were found for the first time in China. The most prevalent virus was SPFMV, which constituted 88% of the total viral sequence reads. Virus transcripts with extremely low expression levels were also detected, such as transcripts of SPLCV, CMV and CymMV. Digital gene expression (DGE) and reverse transcription polymerase chain reaction (RT-PCR) analyses showed that the highest viral transcript expression levels were found in fibrous and tuberous roots, which suggest that these tissues should be optimum samples for virus detection. CONCLUSIONS/SIGNIFICANCE: A total of 15 viruses were presumed to present in three sweet potato cultivars growing in China. This is the first insight into the sweet potato polyadenylated RNA virome. These results can serve as a basis for further work to investigate whether some of the 'new' viruses infecting sweet potato are pathogenic.

Direct excitation of dark plasmonic resonances under visible light at normal incidence.

Dark plasmon resonance modes are optical modes that have small scattering cross-sections and are thus difficult to excite directly by light at normal incidence. In this paper, we propose to excite quadrupolar and higher-order modes with normal incident light (in the visible regime) on a continuous plasmonic metallic surface covering a dielectric pillar array, hence resulting in narrow-band perfect absorption. Different from the general electromagnetic means of inducing dark modes, our dark modes are due to charge densities that are electrically induced by the standing-wave resonance of current on the thin metal sidewall of pillars. This new means of exciting dark modes can significantly improve the excitation efficiency and also provides an easy way to excite strong higher-order modes.

Scanning of transposable elements and analyzing expression of transposase genes of sweet potato [Ipomoea batatas].

BACKGROUND: Transposable elements (TEs) are the most abundant genomic components in eukaryotes and affect the genome by their replications and movements to generate genetic plasticity. Sweet potato performs asexual reproduction generally and the TEs may be an important genetic factor for genome reorganization. Complete identification of TEs is essential for the study of genome evolution. However, the TEs of sweet potato are still poorly understood because of its complex hexaploid genome and difficulty in genome sequencing. The recent availability of the sweet potato transcriptome databases provides an opportunity for discovering and characterizing the expressed TEs. METHODOLOGY/PRINCIPAL FINDINGS: We first established the integrated-transcriptome database by de novo assembling four published sweet potato transcriptome databases from three cultivars in China. Using sequence-similarity search and analysis, a total of 1,405 TEs including 883 retrotransposons and 522 DNA transposons were predicted and categorized. Depending on mapping sets of RNA-Seq raw short reads to the predicted TEs, we compared the quantities, classifications and expression activities of TEs inter- and intra-cultivars. Moreover, the differential expressions of TEs in seven tissues of Xushu 18 cultivar were analyzed by using Illumina digital gene expression (DGE) tag profiling. It was found that 417 TEs were expressed in one or more tissues and 107 in all seven tissues. Furthermore, the copy number of 11 transposase genes was determined to be 1-3 copies in the genome of sweet potato by Real-time PCR-based absolute quantification. CONCLUSIONS/SIGNIFICANCE: Our result provides a new method for TE searching on species with transcriptome sequences while lacking genome information. The searching, identification and expression analysis of TEs will provide useful TE information in sweet potato, which are valuable for the further studies of TE-mediated gene mutation and optimization in asexual reproduction. It contributes to elucidating the roles of TEs in genome evolution.

Digital gene expression analysis based on integrated de novo transcriptome assembly of sweet potato [Ipomoea batatas (L.) Lam].

BACKGROUND: Sweet potato (Ipomoea batatas L. [Lam.]) ranks among the top six most important food crops in the world. It is widely grown throughout the world with high and stable yield, strong adaptability, rich nutrient content, and multiple uses. However, little is known about the molecular biology of this important non-model organism due to lack of genomic resources. Hence, studies based on high-throughput sequencing technologies are needed to get a comprehensive and integrated genomic resource and better understanding of gene expression patterns in different tissues and at various developmental stages. METHODOLOGY/PRINCIPAL FINDINGS: Illumina paired-end (PE) RNA-Sequencing was performed, and generated 48.7 million of 75 bp PE reads. These reads were de novo assembled into 128,052 transcripts (≥ 100 bp), which correspond to 41.1 million base pairs, by using a combined assembly strategy. Transcripts were annotated by Blast2GO and 51,763 transcripts got BLASTX hits, in which 39,677 transcripts have GO terms and 14,117 have ECs that are associated with 147 KEGG pathways. Furthermore, transcriptome differences of seven tissues were analyzed by using Illumina digital gene expression (DGE) tag profiling and numerous differentially and specifically expressed transcripts were identified. Moreover, the expression characteristics of genes involved in viral genomes, starch metabolism and potential stress tolerance and insect resistance were also identified. CONCLUSIONS/SIGNIFICANCE: The combined de novo transcriptome assembly strategy can be applied to other organisms whose reference genomes are not available. The data provided here represent the most comprehensive and integrated genomic resources for cloning and identifying genes of interest in sweet potato. Characterization of sweet potato transcriptome provides an effective tool for better understanding the molecular mechanisms of cellular processes including development of leaves and storage roots, tissue-specific gene expression, potential biotic and abiotic stress response in sweet potato.