Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel small-intelligent strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.

No abstract

Triple-negative breast cancers (TNBCs), devoid of hormone receptors and human epidermal growth-factor receptor-2/Neu expression, bring about poor prognosis and induce a high rate of systematic metastases. The ineffectiveness of current therapies on TNBCs could be attributed to the lack of efficient targeted therapy. Paclitaxel (PTX) is considered one of first-line chemotherapeutics for TNBC treatment but, due to its low aqueous solubility and nonspecific accumulation, results in poor antitumor efficacy. The present study is aimed at enhancing the chemotherapeutic potency of PTX by improving the stability and targeting efficiency of PTX-loaded nanoparticulate drug carriers. Here, PTX was incorporated in nontoxic and endogenous material, human serum albumin (HSA), via an innovative disulfide reduction method to construct HSA-based PTX nanoparticle (HSA-PTX NP) to not only realize redox-responsive drug release but also improve in vivo stability. Besides, W peptide was selected as a target ligand to be conjugated with HSA-PTX NP for endowing active targeting ability. The resulting Wpep-HSA-PTX NP possessed a spherical structure (118 nm), 9.87% drug-loading content, and 86.3% entrapment efficiency. An in vitro drug release test showed that PTX release from Wpep-HSA-PTX NP was of a redox-responsive manner. Furthermore, cellular uptake of Wpep-HSA-PTX NP was significantly enhanced, exhibiting the improved antiproliferation and antitube formation effects of PTX in vitro. In comparison with those commercial formulations and conventional HSA NP, Wpep-HSA-PTX NP exhibited better pharmacokinetic behaviors and tumor homing characteristics. The antitumor efficacy of Wpep-HSA-PTX NP was further confirmed by the strong pro-apoptotic effect and reduced tumor burden. In a word, this evidence highlighted the proof of concept for Wpep-HSA NP as a promising conqueror to the ineffectiveness of TNBC therapy.

We evaluated the diagnostic usefulness of the 34betaE12-p63 cocktail, compared with 34betaE12 and p63 used alone, in 34 prostate needle biopsy (NBXs) and 3 transurethral resection specimens containing atypical glandular proliferations and in 18 NBXs containing unequivocal prostate carcinoma (PCa). Staining intensity; percentage of basal cells staining in benign, atypical, and malignant glands; number of benign glands lacking basal cell staining; and staining variance were analyzed. All NBXs with unequivocal PCa were negative with all 3 markers. Diagnoses were as follows for the atypical cases after staining for the 3 markers: PCa, 9; postatrophic hyperplasia, 12; high-grade prostatic intraepithelial neoplasia (HGPIN), 5; atypical adenomatous hyperplasia, 6; benign atypical proliferations, 4; and HGPIN with adjacent small atypical acinar proliferation suggestive of PCa, 1. The cocktail demonstrated consistently strong staining intensity and improved basal cell staining in morphologically benign and benign atypical glands compared with p63 and 34betaE12 alone; it had the smallest staining variance compared with 34betaE12 (F < 0.0001) and p63 (F = 0.31), although its advantage for resolving individual atypical cases was limited compared with 34betaE12 and p63 alone. Of 37 atypical cases, 1 (3%) additionally was resolved as benign using the cocktail and p63. Because the diagnosis of PCa is supported by lack of basal cell staining, the immunohistochemical analysis with highest possible sensitivity and lowest possible variability is critical to ensure that a negative reaction is true. The cocktail provides a simple, cost-effective improvement in basal cell immunohistochemical analysis of difficult prostate lesions.

Results showed that all 66 species investigated had unique COI sequences and no sequences were shared between the species. Our results were congruent with previous studies suggesting that the COI barcode permits distinguishing most of the closely related species. Furthermore, by using geographically distinct clusters, diagnostic characters, and threshold levels, deep genetic splits (>1.5%) were observed in three species, and we therefore suggest treating them as evolutionary significant units.

The impact strength of spot welds is a very important quality index in the automotive industry. However, impact testing is not routinely performed for weld quality testing because of its complexity, cost and relatively low reliability and repeatability. In this paper, the impact strength of steel spot welds was measured using a newly designed impact tester. Besides the impact energy of the welds, detailed impact process characteristics, such as impact force and displacement profiles, which may be important to understanding the impact performance of a weld for vehicle design and analysis, were also recorded. A brief description of the new impact tester and experimental results on welded joints using this device are presented. A sensitivity study of testing results to specimen size has been conducted, prior to experiments on the influence of weld size, shape and orientation on the impact strength of resistance spot-welded joints. Results of impact testing were also compared with those of static tensile shear tests. The correlation between weld attributes and impact performance also provides important information for the quality definition of spot welds. Initial tests using this impact tester show promise that it can be used to measure impact strength of a large variety of welds (spot welded, laser welded, brazed, etc.), and other joints (such as mechanical fasteners and adhesion).

Direct fabrication of a three-dimensional (3D) structure using soft materials has been challenging. The hybrid bilayer is a promising approach to address this challenge because of its programable shape-transformation ability when responding to various stimuli. The goals of this study are to experimentally and theoretically establish a rational design principle of a hydrogel/elastomer bilayer system and further optimize the programed 3D structures that can serve as substrates for multi-electrode arrays. The hydrogel/elastomer bilayer consists of a hygroscopic polyacrylamide (PAAm) layer cofacially laminated with a water-insensitive polydimethylsiloxane (PDMS) layer. The asymmetric volume change in the PAAm hydrogel can bend the bilayer into a curvature. We manipulate the initial monomer concentrations of the pre-gel solutions of PAAm to experimentally and theoretically investigate the effect of intrinsic mechanical properties of the hydrogel on the resulting curvature. By using the obtained results as a design guideline, we demonstrated stimuli-responsive transformation of a PAAm/PDMS flower-shaped bilayer from a flat bilayer film to a curved 3D structure that can serve as a substrate for a wide-field retinal electrode array.

Featured Application: This work is potential to apply in the design of optimum rotor speed and long-endurance helicopter. Abstract:The aerodynamic design of rotor blades is challenging, and is crucial for the development of helicopter technology. Previous aerodynamic optimizations that focused only on limited design points find it difficult to balance flight performance across the entire flight envelope. This study develops a global optimum envelope (GOE) method for determining blade parameters-blade twist, taper ratio, tip sweep-for optimum rotor speed helicopters (ORS-helicopters), balancing performance improvements in hover and various freestream velocities. The GOE method implements aerodynamic blade design by a bi-level optimization, composed of a global optimization step and a secondary optimization step. Power loss as a measure of rotor performance is chosen as the objective function, referred to as direct power loss (DPL) in this study. A rotorcraft comprehensive code for trim simulation with a prescribed wake method is developed. With the application of the GOE method, a DPL reduction of as high as 16.7% can be achieved in hover, and 24% at high freestream velocity.