A transcriptomic analysis of the kidney tissue of Tra catfish (pangasianodon hypophthalmus) reared in saline condition: De novo assembly, annotation, SNP discovery

Pangasianodon hypophthalmus is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The current study using Ion Torrent technology generated EST resources from the kidney for Tra catfish reared at a salinity level of 9 ppt. We obtained 2,623,929 reads after trimming and processing with an average length of 104 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 29,940 contigs, and allowing identification of 5,710 putative genes when comppared with NCBI non-redundant database. A large number of single nucleotide polymorphisms (SNPs) were also detected. The sequence collection generated in our study represents the most comprehensive transcriptomic resource for P. hypophthalmus available to date.

Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

A phosphorylation-induced turn defines the Alzheimer's disease AT8 antibody epitope on the tau protein

Post mortem biochemical staging of Alzheimer’s disease is currently based on immunochemical analysis of brain slices with the AT8 antibody. The epitope of AT8 is described around the pSer202/pThr205 region of the hyperphosphorylated form of the neuronal protein tau. In this study, NMR spectroscopy was used to precisely map the AT8 epitope on phosphorylated tau, and derive its defining structural features by a combination of NMR analyses and molecular dynamics. A particular turn conformation is stabilized by a hydrogen bond of the phosphorylated Thr205 residue to the amide proton of Gly207, and is further stabilized by the two Arg residues opposing the pSer202/pThr205.

Free energy calculations of glycosaminoglycan - Protein interactions

Glycosaminoglycans (GAGs) are complex highly charged linear polysaccharides that have a variety of roles in biological processes. We report the first use of molecular dynamics (MD) free energy calculations using the MM/PBSA method to investigate the binding of GAGs to protein molecules, namely the platelet endothelial cell adhesion molecule 1 (PECAM-1) and annexin A2. Calculations of the free energy of the binding of heparin fragments of different sizes reveal the existence of a region of low GAG-binding affinity in domains 5-6 of PECAM-1 and a region of high affinity in domains 2-3, consistent with experimental data and ligand-protein docking studies. A conformational hinge movement between domains 2 and 3 was observed, which allows the binding of heparin fragments of increasing size (pentasaccharides to octasaccharides) with an increasingly higher binding affinity. Similar simulations of the binding of a heparin fragment to annexin A2 reveal the optimization of electrostatic and hydrogen bonding interactions with the protein and protein-bound calcium ions. In general, these free energy calculations reveal that the binding of heparin to protein surfaces is dominated by strong electrostatic interactions for longer fragments, with equally important contributions from van der Waals interactions and vibrational entropy changes, against a large unfavorable desolvation penalty due to the high charge density of these molecules.

Molecular modeling of Bt Cry1Ac (DI–DII)–ASAL (Allium sativum lectin)–fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI–DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI–DII domains of Cry1Ac and lectin has been identified using protein–protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI–DII–lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI–DII–DIII) protein. Molecular mechanics/Poisson–Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein–protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic–aromatic, aromatic–sulphur, cation–pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac–APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac–APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.

Free energy calculations of the interactions of c-Jun-based synthetic peptides with the c-Fos protein

The c-Fos–c-Jun complex forms the activator protein 1 transcription factor, a therapeutic target in the treatment of cancer. Various synthetic peptides have been designed to try to selectively disrupt the interaction between c-Fos and c-Jun at its leucine zipper domain. To evaluate the binding affinity between these synthetic peptides and c-Fos, polarizable and nonpolarizable molecular dynamics (MD) simulations were conducted, and the resulting conformations were analyzed using the molecular mechanics generalized Born surface area (MM/GBSA) method to compute free energies of binding. In contrast to empirical and semiempirical approaches, the estimation of free energies of binding using a combination of MD simulations and the MM/GBSA approach takes into account dynamical properties such as conformational changes, as well as solvation effects and hydrophobic and hydrophilic interactions. The predicted binding affinities of the series of c-Jun-based peptides targeting the c-Fos peptide show good correlation with experimental melting temperatures. This provides the basis for the rational design of peptides based on internal, van der Waals, and electrostatic interactions.

Evaluating viewpoint entropy for ribbon representation of protein structure

While many measures of viewpoint goodness have been proposed in computer graphics, none have been evaluated for ribbon representations of protein secondary structure. To fill this gap, we conducted a user study on Amazon’s Mechanical Turk platform, collecting human viewpoint preferences from 65 participants for 4 representative su- perfamilies of protein domains. In particular, we evaluated viewpoint entropy, which was previously shown to be a good predictor for human viewpoint preference of other, mostly non-abstract objects. In a second study, we asked 7 molecular biology experts to find the best viewpoint of the same protein domains and compared their choices with viewpoint entropy. Our results show that viewpoint entropy overall is a significant predictor of human viewpoint preference for ribbon representations of protein secondary structure. However, the accuracy is highly dependent on the complexity of the structure: while most participants agree on good viewpoints for small, non-globular structures with few secondary structure elements, viewpoint preference varies considerably for complex structures. Finally, experts tend to choose viewpoints of both low and high viewpoint entropy to emphasize different aspects of the respective structure.

Defects in the IFT-B component IFT172 cause Jeune and Mainzer-Saldino syndromes in humans

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A.

Unravelling the self-assembly of hydrogen bonded NDI semiconductors in 2D and 3D

Supramolecular ordering of organic semiconductors is the key factor defining their electrical characteristics. Yet, it is extremely difficult to control, particularly at the interface with metal and dielectric surfaces in semiconducting devices. We have explored the growth of n-type semiconducting films based on hydrogen-bonded monoalkylnaphthalenediimide (NDI-R) from solution and through vapor deposition on both conductive and insulating surfaces. We combined scanning tunneling and atomic force microscopies with X-ray diffraction analysis to characterize, at the submolecular level, the evolution of the NDI-R molecular packing in going from monolayers to thin films. On a conducting (graphite) surface, the first monolayer of NDI-R molecules adsorbs in a flat-lying (face-on) geometry, whereas in subsequent layers the molecules pack edge-on in islands (Stranski–Krastanov-like growth). On SiO2, the NDI-R molecules form into islands comprising edge-on packed molecules (Volmer–Weber mode). Under all the explored conditions, self-complementary H bonding of the imide groups dictates the molecular assembly. The measured electron mobility of the resulting films is similar to that of dialkylated NDI molecules without H bonding. The work emphasizes the importance of H bonding interactions for controlling the ordering of organic semiconductors, and demonstrates a connection between on-surface self-assembly and the structural parameters of thin films used in electronic devices.

Two-step self-assembly of hierarchically-ordered nanostructures

Due to their unique size- and shape-dependent physical and chemical properties, highly hierarchically-ordered nanostructures have attracted great attention with a view to application in emerging technologies, such as novel energy generation, harvesting, and storage devices. The question of how to get controllable ensembles of nanostructures, however, still remains a challenge. This concept paper first summarizes and clarifies the concept of the two-step self-assembly approach for the synthesis of hierarchically-ordered nanostructures with complex morphology. Based on the preparation processes, two-step self-assembly can be classified into two typical types, namely, two-step self-assembly with two discontinuous processes and two-step self-assembly completed in one-pot solutions with two continuous processes. Compared to the conventional one-step self-assembly, the two-step self-assembly approach allows the combination of multiple synthetic techniques and the realization of complex nanostructures with hierarchically-ordered multiscale structures. Moreover, this approach also allows the self-assembly of heterostructures or hybrid nanomaterials in a cost-effective way. It is expected that widespread application of two-step self-assembly will give us a new way to fabricate multifunctional nanostructures with deliberately designed architectures. The concept of two-step self-assembly can also be extended to syntheses including more than two chemical/physical reaction steps (multiple-step self-assembly).

Generalized self-assembly of scalable two-dimensional transition metal oxide nanosheets

Two-dimensional (2D) transition metal oxide systems present exotic electronic properties and high specific surface areas, and also demonstrate promising applications ranging from electronics to energy storage. Yet, in contrast to other types of nanostructures, the question as to whether we could assemble 2D nanomaterials with an atomic thickness from molecules in a general way, which may give them some interesting properties such as those of graphene, still remains unresolved. Herein, we report a generalized and fundamental approach to molecular self-assembly synthesis of ultrathin 2D nanosheets of transition metal oxides by rationally employing lamellar reverse micelles. It is worth emphasizing that the synthesized crystallized ultrathin transition metal oxide nanosheets possess confined thickness, high specific surface area and chemically reactive facets, so that they could have promising applications in nanostructured electronics, photonics, sensors, and energy conversion and storage devices.

Synthesis of mesoporous TiO2/SiO2 hybrid films as an efficient photocatalyst by polymeric micelle assembly

Thermally stable mesoporous TiO2/SiO2 hybrid films with pore size of 50 nm have been synthesized by adopting the polymeric micelle-assembly method. A triblock copolymer, poly(styrene-b-2-vinyl pyridine-b-ethylene oxide), which serves as a template for the mesopores, was utilized to form polymeric micelles. The effective interaction of titanium tetraisopropoxide (TTIP) and tetraethyl orthosilicate (TEOS) with the polymeric micelles enabled us to fabricate stable mesoporous films. By changing the molar ratio of TEOS and TTIP, several mesoporous TiO2/SiO2 hybrid films with different compositions can be synthesized. The presence of amorphous SiO2 phase effectively retards the growth of anatase TiO2 crystal in the pore walls and retains the original mesoporous structure, even at higher temperature (650 °C). These TiO2/SiO2 hybrid films are of very high quality, without any cracks or voids. The addition of SiO2 phase to mesoporous TiO2 films not only adsorbs more organic dyes, but also significantly enhances the photocatalytic activity compared to mesoporous pure TiO2 film without SiO2 phase.

Robust superhydrophobicity of hierarchical ZnO hollow microspheres fabricated by two-step self-assembly

Superhydrophobic and superhydrophilic surfaces have been extensively investigated due to their importance for industrial applications. It has been reported, however, that superhydrophobic surfaces are very sensitive to heat, ultraviolet (UV) light, and electric potential, which interfere with their long-term durability. In this study, we introduce a novel approach to achieve robust superhydrophobic thin films by designing architecture-defined complex nanostructures. A family of ZnO hollow microspheres with controlled constituent architectures in the morphologies of 1D nanowire networks, 2D nanosheet stacks, and 3D mesoporous nanoball blocks, respectively, was synthesized via a two-step self-assembly approach, where the oligomers or the constituent nanostructures with specially designed structures are first formed from surfactant templates, and then further assembled into complex morphologies by the addition of a second co-surfactant. The thin films composed of two-step synthesized ZnO hollow microspheres with different architectures presented superhydrophobicities with contact angles of 150°-155°, superior to the contact angle of 103° for one-step synthesized ZnO hollow microspheres with smooth and solid surfaces. Moreover, the robust superhydrophobicity was further improved by perfluorinated silane surface modification. The perfluorinated silane treated ZnO hollow microsphere thin films maintained excellent hydrophobicity even after 75 h of UV irradiation. The realization of environmentally durable superhydrophobic surfaces provides a promising solution for their long-term service under UV or strong solar light irradiations.

Nano-confined synthesis of fullerene mesoporous carbon (C60-FMC) with bimodal pores: XRD, TEM, structural properties, NMR, and protein immobilization

Nanoconfined synthesized crystalline fullerene mesoporous carbon (C60-FMC) with bimodal pore architectures of 4.95 nm and 10-15 nm pore sizes characterized by XRD, TEM, nitrogen adsorption/ desorption isotherm and solid-state NMR, and the material was used for protein immobilization. The solid-state 13C NMR spectrum of C60-FMC along with XRD, BET and TEM confirms the formation of fullerene mesoporous carbon structure C60-FMC. The immobilization of albumin (from bovine serum, BSA) protein biomolecule in a buffer solution at pH 4.7 was used to determine the adsorption properties of the C60-FMC material and its structural changes investigated by FT-IR. We demonstrated that the C60-FMC with high surface area and pore volumes have excellent adsorption capacity towards BSA protein molecule. Protein adsorption experiments clearly showed that the C60-FMC with bimodal pore architectures (4.95 nm and 10-15 nm) are suitable material to be used for protein adsorption