Background: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. Results: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. Conclusions: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.

Background: The microstructure of trabecular bone is a composite trait governed by a complex interaction of multiple genetic determinants. Identifying these genetic factors should significantly improve our ability to predict of osteoporosis and its associated risks. Genetic mapping using collaborative cross mice (CC), a genetically diverse recombinant inbred mouse reference panel, offers a powerful tool to identify causal loci at a resolution under one mega base-pairs, with a relatively small cohort size.Here, we utilized 31 CC lines (160 mice of both sexes in total) to perform genome-wide haplotype mapping across 77,808 single-nucleotide polymorphisms (SNPs). Haplotype scans were refined by imputation with the catalogue of sequence variation segregating in the CC to suggest potential candidate genes. Trabecular traits were obtained following microtomographic analysis, performed on 10-μm resolution scans of the femoral distal metaphysis. We measured the trabecular bone volume fraction (BV/TV), number (Tb.N), thickness (Tb.Th), and connectivity density (Conn.D). Results: Heritability of these traits ranged from 0.6 to 0.7. In addition there was a significant (P < 0.01) sex effect in all traits except Tb.Th. Our haplotype scans yielded six quantitative trait loci (QTL) at 1 % false discovery rate; BV/TV and Tb.Th produced two proximal loci each, on chromosome 2 and 7, respectively, and Tb.N and Conn.D yielded one locus on chromosomes 8 and 14, respectively. We identified candidate genes with previously-reported functions in bone biology, and implicated unexpected genes whose function in bone biology has yet to be assigned. Based on the literature, among the genes that ranked particularly high in our analyses (P < 10 -6 ) and which have a validated causal role in skeletal biology, are Avp, Oxt, B2m (associated with BV/TV), Cnot7 (with Tb.N), Pcsk6, Rgma (with Tb.Th), Rb1, and Cpb2 (with Conn.D). Other candidate genes strongly suggested by our analyses are Sgcz, Fgf20 (associated with Tb.N), and Chd2 (with Tb.Th). Conclusion: We have demonstrated for the first time genome-wide significant association between several genetic loci and trabecular microstructural parameters for genes with previously reported experimental observations, as well as proposing a role for new candidate genes with no previously characterized skeletal function.

Background: Pyropia haitanensis is an economically important marine crop grown in harsh intertidal habitats of southern China; it is also an excellent model system for studying mechanisms of stress tolerance. To understand the molecular mechanisms underlying osmotic tolerance and adaptation to intertidal environments, a comprehensive analysis of genome-wide gene expression profiles in response to dehydration and rehydration in Py. haitanensis was undertaken using digital gene expression profile (DGE) approaches combined with de novo transcriptome sequencing. Results: RNA-sequencing of the pooled RNA samples from different developmental phases and stress treatments was performed, which generated a total of 47.7 million clean reads. These reads were de novo assembled into 28,536 unigenes (≥200 bp), of which 18,217 unigenes (63.83 %) were annotated in at least one reference database. DGE analysis was performed on four treatments (two biological replicates per treatment), which included moderate dehydration, severe dehydration, rehydration, and normal conditions. The number of raw reads per sample ranged from 12.47 to 15.79 million, with an average of 14.69 million reads per sample. After quality filtering, the number of clean reads per sample ranged from 11.83 to 15.04 million. All distinct sequencing reads were annotated using the transcriptome of Py. haitanensis as reference. A total of 1,681 unigenes showed significant differential expression between moderate dehydration and normal conditions, in which 977 genes were upregulated, and 704 genes were downregulated. Between severe dehydration and normal conditions, 1,993 unigenes showed significantly altered expression, which included both upregulated (1,219) and downregulated genes (774). In addition, 1,086 differentially expressed genes were detected between rehydration and normal conditions, of which 720 genes were upregulated and 366 unigenes were downregulated. Most gene expression patterns in response to dehydration differed from that of rehydration, except for the synthesis of unsaturated fatty acids, several transcription factor families, and molecular chaperones that have been collectively implicated in the processes of dehydration and rehydration in Py. haitanensis. Conclusions: Taken together, these data provide a global high-resolution analysis of gene expression changes during osmotic stress that could potentially serve as a key resource for understanding the biology of osmotic acclimation in intertidal red seaweed.

The UV-induced photochemistry of HCFC-132b (CF2ClCH2Cl) was investigated by computing excited-state properties with time-dependent density functional theory (TDDFT), multiconfigurational second-order perturbation theory (CASPT2), and coupled cluster with singles, doubles, and perturbative triples (CCSD(T)). Excited states calculated with TDDFT show good agreement with CASPT2 and CCSD(T) results, correctly predicting the main excited-states properties. Simulations of ultrafast nonadiabatic dynamics in the gas phase were performed, taking into account 25 electronic states at TDDFT level starting in two different spectral windows (8.5 ± 0.25 and 10.0 ± 0.25 eV). Experimental data measured at 123.6 nm (10 eV) is in very good agreement with our simulations. The excited-state lifetimes are 106 and 191 fs for the 8.5 and 10.0 eV spectral windows, respectively. Internal conversion to the ground state occurred through several different reaction pathways with different products, where 2Cl, C-Cl bond breakage, and HCl are the main photochemical pathways in the low-excitation region, representing 95% of all processes. On the other hand, HCl, HF, and C-Cl bond breakage are the main reaction pathways in the higher excitation region, with 77% of the total yield. © 2015 Wiley Periodicals, Inc.

HCFC-132b is an important industrial compound, with a strong impact on health and environment. Upon UV irradiation, it decomposes into dozens of different photoproducts. In this article, nonadiabatic dynamics simulation is used to explain how photo-decomposition takes place through the competition between diverse reaction pathways in the subpicosecond time scale.

On page 78 (DOI: 10.1002/jcc.24021), Ramon Carbó-Dorca discusses aromaticity, quantummultimolecular polyhedral, and the quantumQSPR fundamental equation. First, a concise description of the Kekulé's historical origin of aromaticity and the actual state of the question is given. After this, it is argued that still room is left to the discussion about the quantummechanical foundation existence of aromaticity. In order to perform that, quantum multimolecular polyhedra (QMP) are defined: they are based onmolecular density functions sets attached to QMP vertices. Fromthere, collective QMP distances, QSPR fundamental equation and aromaticity descriptors are proposed as away to construct an equation, able to estimate aromaticity via expectation values of Hermitian operators.

On page 18 (DOI: 10.1002/jcc.23914), the Gibbs energies of association between primary alkyl ammonium ions and crown ethers in solution are measured and calculated by Andreas J. Achazi, Larissa K. S. von Krbek, Christoph A. Schalley, and Beate Paulus.Measurementswere carried out by isothermal titration calorimetry. Calculations were done as accurate as possible for the gas phasewith DFT-D3(BJ). The Gibbs energies to transfer the educts in the gas phase and the products back in solution were calculated with the solvation model COSMO-RS in order to get the Gibbs energies of association in solution. Calculated andmeasured Gibbs energies of association in solution agreewell and reveal a strong solvent-dependent ion pair effect.

The cover image shows the largestof a family of fullerenes used for extrapolating to the graphene limit, as presented by Lukas N. Wirz, Ralf Tonner, Andreas Hermann, Rebecca Sure, and Peter Schwerdtfeger on page 10 (DOI: 10.1002/jcc.23894). The structures were obtained from a newly developed force field treated subsequently by density functional theory. Our results confirm Paul von Ragué Schleyer's hypothesis that C60 is not especially stable – 60 is not amagic number – comparedwith other fullerenes.

ABSTRACT

The CASP experiment is a biannual benchmark for assessing protein structure prediction methods. In CASP11, RBO Aleph ranked as one of the top-performing automated servers in the free modeling category. This category consists of targets for which structural templates are not easily retrievable. We analyze the performance of RBO Aleph and show that its success in CASP was a result of its ab initio structure prediction protocol. A detailed analysis of this protocol demonstrates that two components unique to our method greatly contributed to prediction quality: residue–residue contact prediction by EPC-map and contact-guided conformational space search by model-based search (MBS). Interestingly, our analysis also points to a possible fundamental problem in evaluating the performance of protein structure prediction methods: Improvements in components of the method do not necessarily lead to improvements of the entire method. This points to the fact that these components interact in ways that are poorly understood. This problem, if indeed true, represents a significant obstacle to community-wide progress. Proteins 2015. © 2015 Wiley Periodicals, Inc.

ABSTRACT

The Sarcolipin (SLN) is a transmembrane protein that can form a self-assembled pentamer. In this work, the homology modeling and all-atom molecular dynamic (MD) simulation was performed to study the model of SLN pentamer in POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane. The potential of mean force (PMF) was calculated for transmembrane transportation of Na+, Cl− and water molecule along the pore channel of penta-SLN complex. The root mean square deviation (RMSD) of the SLN pentamer in POPC membrane showed that the stabilized SLN protein complex could exist in the membrane and that the Na+ and Cl− could not permeate through the channel when the pore was under the vacuum state, but the water could permeate through from cytoplasm to lumen. Under the aqueous state, our simulation demonstrated that hydrated state of Na+ and Cl− could pass through the channel. The PMF and radii of the pore showed that the channel had a gate at Leu21 that is a key hydrophobicity residue in the channel. Our simulations help to clarify and to understand better the SLN pentamer channel that had a hydrophobic gate and could switch Na+ and Cl− ion permeability by hydrated and vacuum states. Proteins 2015. © 2015 Wiley Periodicals, Inc.

ABSTRACT

Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethylene terephthalate) (PET), opening up new options for enzymatic routes for polymer recycling and surface modification reactions. Cutinase from Aspergillus oryzae (AoC) is promising owing to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temperature (Tg) of PET, that is, 70°C. Consequently, in this study we worked toward the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6°C improvement in the thermal unfolding temperature (Tm) and a 10-fold increase in the half-life of the enzyme activity at 60°C. Surprisingly, thermostabilization did not improve the rate or temperature optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding, and (c) the lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the Tm of the global structure. Proteins 2015. © 2015 Wiley Periodicals, Inc.

ABSTRACT

Iron acquisition is a complex, multicomponent process critical for most organisms' survival and virulence. Small iron chelating molecules, siderophores, mediate transport as key components of common pathways for iron assimilation in many microorganisms. The chemistry and biology of the extraordinary tight and specific metal binding siderophores is of general interest in terms of host/guest chemistry and is a potential target toward the development of therapeutic treatments for microbial virulence. The siderophore pathway of the moderate thermophile, Thermobifida fusca, is an excellent model system to study the process in Gram-positive bacteria. Here we describe the structure and characterization of the siderophore periplasmic binding protein, FscJ from the fuscachelin gene cluster of T. fusca. The structure shows a di-domain arrangement connected with a long α-helix hinge. Several X-ray structures detail ligand-free conformational changes at different pH values, illustrating complex interdomain flexibility of the siderophore receptors. We demonstrated that FscJ has a unique recognition mechanism and details the binding interaction with ferric-fuscachelin A through ITC and docking analysis. The presented work provides a structural basis for the complex molecular mechanisms of siderophore recognition and transportation. Proteins 2015. © 2015 Wiley Periodicals, Inc.

Morphological disintegration of neurons invariably triggers neural death and is a hallmark feature of neurodegeneration [1–6]. Photoreceptors neurons are the primary photon-capturing neurons of the retina. In particular, the outer segment (OS) structures of photoreceptors undergo degeneration owing to intrinsic (e.g. genetic mutations) or extrinsic (e.g. environmental) pathological stimuli [5,6]. Light-stress is a powerful and extrinsic deleterious stressor that promotes the dysplasia of the OS of photoreceptors and ultimately photoreceptor death [7–13].

Small non-coding RNA microRNAs (miRNAs) regulate gene expression at the post-transcriptional level by binding sequence-specific sites within the 3′ untranslated region of target mRNAs [1]. In addition, miRNAs can fine-tune various biological processes, including development, organogenesis, metabolism, and homeostasis [2]. miRNAs have been found in human body fluids despite the abundant presence of ribonucleases (RNases) [3]. This finding has led to the proposal of a scenario in which miRNAs could be packaged in certain RNase-resistant containers when they are secreted out of cells.

Phospholipases A2 (PLA2) are a large family of enzymes ubiquitously expressed that catalyze the breakdown of glycerophospholipids, releasing arachidonic acid (AA): cytosolic PLA2 (cPLA2), Ca2+-independent PLA2 (iPLA2), and Ca2+-dependent secretory PLA2 (sPLA2) differ from each other in terms of substrate specificity and Ca2+-requirement [1]. The cPLA2, present in many cell types, including pancreatic β cells (cPLA2β), requires phosphorylation at Ser505 and binding with Ca2+ for its activity. Activation of cPLA2β would cause translocation of the enzyme to the secretory granules and accumulation of AA and lysophospholipids in the membrane, leading to changes in membrane structure or fluidity [2].

Glycoside phosphorylases (GPs) are useful tools for efficient oligosaccharide synthesis because of the reversible nature of their reactions [1–3]. Although increasing numbers of GPs have been reported recently, their utilization for the production of various oligosaccharides remains limited by the extent of known and characterized enzymes. In the Carbohydrate-Active enZyme database [4], anomer-inverting GPs are all categorized into glycoside hydrolase (GH) families: GH65, GH94, GH112, and GH130.

EZH2 plays a major role in HIV-1 latency, the molecular linkage between Tat-induced HIV-1 transactivation and EZH2 activity, is not fully understood. It was shown Tat-induced HIV-1 transactivation through inhibiting EZH2 activity. Tat decreased the levels of H3K27me3 and EZH2 occupy at the long terminal repeat (LTR) of HIV-1. We further showed for the first time that transfected with Tat construct resulted in an increase in phosphorylated EZH2 (p-EZH2), mediated by active Akt. ROS/Akt-dependent p-EZH2 was correlated with Tat-induced transactivation.