Modification of the thermoresistance to spray-drying of a cold-adapted subtilisin by genetic engineering

The thermoresistance of a cold-adapted subtilisin dried by spray-drying was studied. Proteolytic activity of this enzyme was measured before and after spray-drying. Without chemical additives, spray-drying yields ranged from 2–13%. The use of arabic gum and lactose in the composition of the enzyme solutions allowed the strengthening of the enzyme structures and increased water mobility in the product. Increase of water mobility led to a shorter residence time of the product in the spray-drier and a net yield increase was obtained (yield higher than 50%). The effect of two selective mutations on the thermoresistance to spray-drying of the cold-adapted subtilisin was also investigated. Mutation T85D (introduction of an additional link with an ion Ca²⁺ necessary for enzyme activity, by substitution of Asp for Thr 85) had no effect on the thermoresistance of the subtilisin to spray-drying. Mutation H121W (introduction of an additional aromatic link by substitution of Trp for His 121) reduced the drying yield from 66% (not modified subtilisin) to 52%. This higher thermosensitivity could be explained by an increase of the hygroscopic character of the modified subtilisin (mutation H121W).     

Synthesis and properties of isoviagra. A 2‐methyl‐2H‐pyrazolo[4,3‐d]pyrirnidin‐7‐one isomer of viagra®

The synthesis and spectral properties (ir, ms, nmr) of a substituted 2‐methyl‐2H‐pyrazolo[4,3‐d]‐pyrimidin‐7‐one ( 3 ), an isomer of Viagra^®, are described. The key synthon, 4‐amino‐1‐methyl‐5‐propyl‐3‐pyrazolecarboxamide ( 7 ), is prepared via the reaction of ethyl 2,4‐dioxoheptanoate with methylhydrazine, followed by cyclization, nitration, amidation, and nitro group reduction. Interaction of 7 with 2‐ethoxyben‐zoyl chloride yielded the respective bis‐amide ( 8 ) which was cyclized in polyphosphoric acid to the corresponding pyrazolo[4,3‐d]pyrimidin‐7‐one derivative 9 . Chlorosulfonylation of 9 , and subsequent treatment with 1‐methylpiperazine furnished iso Viagra ( 3 ).     

Computational Screening for the Anticancer Potential of Seed-Derived Antioxidant Peptides: A Cheminformatic Approach

Some seed-derived antioxidant peptides are known to regulate cellular modulators of ROS production, including those proposed to be promising targets of anticancer therapy. Nevertheless, research in this direction is relatively slow owing to the inevitable time-consuming nature of wet-lab experimentations. To help expedite such explorations, we performed structure-based virtual screening on seed-derived antioxidant peptides in the literature for anticancer potential. The ability of the peptides to interact with myeloperoxidase, xanthine oxidase, Keap1, and p47^phox was examined. We generated a virtual library of 677 peptides based on a database and literature search. Screening for anticancer potential, non-toxicity, non-allergenicity, non-hemolyticity narrowed down the collection to five candidates. Molecular docking found LYSPH as the most promising in targeting myeloperoxidase, xanthine oxidase, and Keap1, whereas PSYLNTPLL was the best candidate to bind stably to key residues in p47^phox. Stability of the four peptide-target complexes was supported by molecular dynamics simulation. LYSPH and PSYLNTPLL were predicted to have cell- and blood-brain barrier penetrating potential, although intolerant to gastrointestinal digestion. Computational alanine scanning found tyrosine residues in both peptides as crucial to stable binding to the targets. Overall, LYSPH and PSYLNTPLL are two potential anticancer peptides that deserve deeper exploration in future.     

Global robust stability analysis of neural networks with discrete time delays

Global robust convergence properties of continuous-time neural networks with discrete delays are studied. By using a Lyapunov functional, we derive a delay independent stability condition for the existence uniqueness and global robust asymptotic stability of the equilibrium point. The condition is in terms of the network parameters only and can be easily verified. It is also shown that the obtained result improves and generalizes a previously published result.     

Organically Templated Vanadium Oxide Phases: Synthesis and Structures of [H3NCH2CH2NH3][V2O6] and [HN(CH2CH2)3NH][VV2VIV4O14]·H2O

The synthesis and crystal structures of [H₃NCH₂CH₂NH₃][V₂O₆] (1) and [HN(CH₂CH₂)₃NH][V^V ₂V^IV ₄O₁₄]·H₂O (2) are described. The structure of the oxidized compound 1 consists of parallel stacks of vanadium oxide chains of corner sharing {VO₄} tetrahedra. The chains are stabilized by extensive hydrogen bonding involving oxide ligands of the chains and ethylenediammonium ions which fill the space between the stacks of chains. The structure of compound 2 consists of vanadium oxide layers separated by doubly protonated 1,4-diazabicyclo[2.2.2]octane and lattice water. The vanadium oxide layers, containing mixed-valence vanadium (V^V and V^IV) centers, are composed of zigzag ribbons of edge-sharing {VO₅} square pyramids interconnected by {VO₄} tetrahedra. Crystal data. C₂H₁₀N₂O₆V₂ , 1: monoclinic, space group P2₁/c (No. 14), a=5.5359(5), b=12.9430(12), c=5.6856(5) Å, =90, =97.460(2), =90°, V=403.93(6) ų, Z=2. A total of 2506 reflections ( _max=27.89°) was collected, of which 954 were used to resolve the structure. The structure was solved by direct methods and least-squares refinement converged at R=0.0592. C₆H₁₆N₂O₁₅V₆, 2: monoclinic, space group C2 (No. 5), a=19.303(4), b=6.667(2), c=7.579(2) Å, =90, =111.31(2), =90°, V=908.4(4) ų, Z=2. A total of 1779 reflections was collected, of which 1591 unique reflections were used for structural elucidation. The structure was solved by direct methods and least-squares refinement converged at R=0.0314.     

Development of a novel microfluidic immunoassay for the detection of Helicobacter pylori infection

The miniaturization of laboratory processes offers substantial advantages over traditional techniques in terms of cost, speed, and potential for multistage automation. To date, only a few studies have reported successful microfluidics-based immunoassays, most of which rely on fluorescence detection technologies. The goal of this study was to develop a poly(dimethylsiloxane) microfluidics-based immunoassay methodology and a versatile colorimetric quantification scheme for the detection of visual colour changes resulting from immune reactions in microchannels. The novel immunoassay technique was applied towards the detection of Helicobacter pylori infection using 20 human serum samples of known infection status, and results compared with conventional nitrocellulose membrane-based dot-ELISA. The microchannel immunoassay reliably detected H. pylori antigens in quantities on the order of 10 ng, which provides a sensitivity of detection comparable to conventional dot-blot assays. Sensitivity was 100%, specificity was 90%, positive predictive value 91%, and negative predictive value 100%, with an overall accuracy of 95%. The software developed generated results that were consistent with visual observations and by automatically taking into account background intensity changes, the software minimized subjectivity. Volumes of solutions used were 100-fold less compared with conventional immunoassays. Miniaturization of the ELISA using this technique provides a means for the accurate diagnosis of microbial infection while minimizing waste production.     

Synthesis of pyrazole‐fused azepino[5,4,3‐cd]indoles

The synthesis of some new pyrazolo[3′,4′:6,7]azepino[5,4,3‐cd] indoles (10a‐c) was achieved via regios‐elective cyclization of the respective 3‐(4‐acylaminopyrazol‐5‐yl)indoles (9a‐c) under Bischler‐Napieralski reaction conditions. The latter compounds were obtained by acylation of the corresponding 3‐(4‐aminopyra‐zol‐5‐yl)indoles (8a,b) which, in turn, were prepared by reduction of the 3‐(4‐nitropyrazol‐5‐yl)indoles precursors (7a,b) . The latter synthons were accessible from the reaction of indolylzinc chlorides (5a,b) with 5‐chloro‐1,3‐dimethyl‐4‐nitropyrazole. Ms and nmr spectral data of 10a‐c are in agreement with the assigned azepino‐indole structure as determined for 10a by X‐ray crystal measurements which demonstrate that the azepine ring is almost completely planar with the indole and pyrazole rings.