Anorectal malformation associated with a mutation in the P63 gene in a family with split hand–foot malformation

PURPOSE: The aims of this study were to identify the mutation gene of a Chinese family with anorectal malformation (ARM) associated with split hand–foot malformation and to determine the spatiotemporal expression of the mutated gene during hindgut and anorectum development in human embryos. METHOD: A Chinese family with intrafamilial clinically variable manifestation was analyzed and primers were designed for exons 3–14 of P63, DLX5, DLX6, DAC, and HOXD13 as candidate genes and direct sequence analysis of the exons was performed. Immunohistochemical study of mutated gene in the hindgut and anorectum of human embryos of 4th–10th weeks was performed. RESULT: Affected individuals were found to have an Arg227Gln P63 gene mutation. From the 4th–10th weeks of gestation of the human embryo, the P63-positive cells were mainly located on the epithelium of the apical urorectal septum, hindgut, and cloacal membrane. After the anorectum ruptured during the 8th week, the P63 remained strongly immunoreactive on the epithelium of the anal canal and urethra, but the mucous membrane of the rectum exhibited no reaction. CONCLUSIONS: The mutation identified strongly suggests a causal relationship between the ARM phenotype and P63. The expression of P63 was persistently active during the dynamic and incessant septation of the cloaca and hindgut, suggesting that P63 may play a pivotal role in the morphogenesis of the hindgut and anorectum

Tuning of Ionic Liquid–Solvent Electrolytes for High-Voltage Electrochemical Double Layer Capacitors: A Review

Electrochemical double-layer capacitors (EDLCs) possess extremely high-power density and a long cycle lifespan, but they have been long constrained by a low energy density. Since the electrochemical stability of electrolytes is essential to the operating voltage of EDLCs, and thus to their energy density, the tuning of electrolyte components towards a high-voltage window has been a research focus for a long time. Organic electrolytes based on ionic liquids (ILs) are recognized as the most commercially promising owing to their moderate operating voltage and excellent conductivity. Despite impressive progress, the working voltage of IL–solvent electrolytes needs to be improved to meet the growing demand. In this review, the recent progress in the tuning of IL- based organic electrolyte components for higher-voltage EDLCs is comprehensively summarized and the advantages and limitations of these innovative components are outlined. Furthermore, future trends of IL–solvent electrolytes in this field are highlighted

New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases

Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to thelonger side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered Km values from 0.32 (wild type PG8fn) to 0.75–4.74 mg/ml, all mutants displayed remarkably lowered kcat (~3–20,000 fold) and kcat/Km (~8–187,500 fold) values and significantly increased △(△G) values (5.92–33.47 kJ/mol). Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.Peer reviewe

The consensus sequence logo of the loop T3 regions of 1,000 GH28 endo-PGs generated using the WEBLOGO.

<p>The consensus sequence logo of the loop T3 regions of 1,000 GH28 endo-PGs generated using the WEBLOGO.</p

Illustration of the substrate pentagalacturonic acid docked to the wild type PG8fn catalytic pocket.

<p>The system was constructed using PyMOL. The protein surface is shown in transparent gray. The catalytic region forms a tunnel through which the substrate passes. Hydrogen bond is depicted as blue dashed lines. Asn94 is marked in green. Catalytic triads in the active center are marked in cyan. The key amino acids interacted with GalpA at –1/+1 subsites are marked in orange.</p

Hydrogen bonds^a existing in T3 loop (residues from 88 to 101) of wild type PG8fn and two mutants (N94Q, N94A), and their occupancies during the last 20 ns of MD simulations.

<p>^<i>a</i> Only H-bonds with occupancies >50% are shown.</p><p>^<i>b</i> Not observed.</p><p>Hydrogen bonds<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135413#t001fn001" target="_blank">^a</a> existing in T3 loop (residues from 88 to 101) of wild type PG8fn and two mutants (N94Q, N94A), and their occupancies during the last 20 ns of MD simulations.</p

Conformation analysis of the last 20 ns MD trajectory of the enzymes.

<p>(A) The RMSF value of each residue C_α atom of the wild type PG8fn (black) and mutants N94Q (red) and N94A (green) measured against the corresponding starting structures. (B) Superimpositions of the average structure of the wild type PG8fn and two mutants viewed from the C-terminal side.</p

Root mean square deviation (RMSD) analysis of the wild type PG8fn (black), N94Q (red) and N94A (green) during a 50-ns MD simulation.

<p>(A) The enzyme backbones. (B) The pentagalacturonic acid. Each simulation was repeated three times with the same initial configurations.</p

Structure analysis of wild type PG8fn.

<p>(A) Schematic structure of PG8fn with the N-terminus on the left and the C-terminus on the right viewed onto β-sheet PB1 (yellow). PB2a, PB2b, and PB3 are shown in orange, pink, and blue, respectively. The key residues, Asn94, Asp155, Asp176, and Asp177, are shown in stick models. The structure viewed from the N-terminal side shows the cleft that is formed by the loops T1 (right side) and T3 (left side). (B) Ribbon diagram of the cross section of PG8fn on the right panel; the color code indicates the secondary structure elements in a representative coil.</p