Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing PDGFRA Variant in Cultured Human Glioblastoma Cell Lines

Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha (PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.     

Reuse of carwash water with a cellulose acetate ultrafiltration membrane aided by flocculation and activated carbon treatments

A reuse system for carwash water with a cellulose acetate (CA), hollow-fiber-type ultrafiltration membrane with the aid of flocculation and activated carbon treatments was proposed. The multi-blended flocculating agent containing bentonite, Al₂(SO₄)₃, sodium alginic acid and a cationic polyacrylamide showed higher removals of COD and turbidity for carwash wastewater compared with Al₂(SO₄)₃ or a water-soluble polymer individually. The effect of pure water permeability of the membrane on permeation flux in pretreated carwash wastewater by this agent was examined using three kinds of CA membranes whose molecular weight cut-offs were 150,000 Dalton. Permeation flux showed a higher value in the case of the membrane with higher pure water permeability. Practical scale experiments with a membrane area of 32 m² and 48 m² were conducted under a membrane pressure of 20 kPa. When carwash wastewater was pretreated with 50 mg/L of this multi-blended flocculating agent, permeation flux through the CA membrane with pure water permeability of 0.78 m³/(m²/h) at 100 kPa showed 1.0 m³/(m²/d) for more than 6 months. The COD, BOD and extract by n-hexane values of reuse water were 3.7-15.7 mg/L, 2.5-14.0 mg/L and below 0.5 mg/L, respectively.     

Use of cryogenic machining to improve the adhesion of sphene bioceramic coatings on titanium substrates for dental and orthopaedic applications

Rods of commercially pure titanium were machined using standard oil-based emulsion and cryogenic cooling, and were then coated with sphene (CaTiSiO₅) bioceramic by spray coating using an automatic airbrush. The sphene bioceramic was synthesized in-situ starting from a suspension of polysiloxane that used as SiO₂ precursor, CaCO₃ and TiO₂ nanoparticles. The suspension was deposited on the machined substrates, which were heat treated up to 950?°C in order to promote the formation of sphene ceramic. The produced coated prototypes were characterized to evaluate the effect of the machining conditions on surface roughness and microstructure of the substrate, and thereby their effect on coating adhesion. Nanoindentation tests were employed to determine the hardness and elastic modulus of the coating through its thickness. Results showed that the reduced amount of defects on the surface of the cryo-machined substrates, contributed to increase the hardness, elastic modulus and adhesion strength of the coating-substrate interfaces compared to standard machined samples, therefore improving adhesion of the coating to the underlying substrate.     

The effect of molding conditions on mechanical and morphological properties at the interface of film insert injection molded polypropylene‐film/polypropylene matrix

An extensive study on the peel strength between a polypropylene (PP) film and PP substrate fabricated using film insert injection molding technique was carried out through a 180° peel test. Injection molding conditions such as barrel temperature, injection speed and holding pressure were varied to gauge their effects on the mechanical and morphological properties. Morphological observations were made at the film‐substrate interfacial regions by means of transmission electron microscopy (TEM). The injection molded products, with the films still attached, were subjected to bending and impact tests to determine if there is any relationship between film‐substrate adhesion and bulk properties. Observation of the load‐displacement curves during the peel test revealed three unique and interesting curves, corresponding to different peeling and film fracture mechanisms. Increases in injection speed, barrel temperature and holding pressure lead to increased bonding between the film and substrate surfaces. The enhancement of bonding between these two polymer surfaces could be attributed to polymer‐polymer interdiffusion. Substantiating evidence from TEM, which shows the fading of the interface as the bond strengthens, further boosts the accuracy of this assumption. The hope that the films could contribute to enhancing bulk properties has been diminished since the bending properties appeared to be similar with or without the film attached. Polym. Eng. Sci. 44:2327–2334, 2004. © 2004 Society of Plastics Engineers.     

A flow cell for transient voltammetry and in situ grazing incidence X-ray diffraction characterization of electrocrystallized cadmium(II) tetracyanoquinodimethane

An easy to fabricate and versatile cell that can be used with a variety of electrochemical techniques, also meeting the stringent requirement for undertaking cyclic voltammetry under transient conditions in in situ electrocrystallization studies and total external reflection X-ray analysis, has been developed. Application is demonstrated through an in situ synchrotron radiation-grazing incidence X-ray diffraction (SR-GIXRD) characterization of electrocrystallized cadmium (II)-tetracyanoquinodimethane material, Cd(TCNQ)₂, from acetonitrile (0.1 mol dm⁻³ [NBu₄][PF₆]). Importantly, this versatile cell design makes SR-GIXRD suitable for almost any combination of total external reflection X-ray analysis (e.g., GIXRF and GIXRD) and electrochemical perturbation, also allowing its application in acidic, basic, aqueous, non-aqueous, low and high flow pressure conditions. Nevertheless, the cell design separates the functions of transient voltammetry and SR-GIXRD measurements, viz., voltammetry is performed at high flow rates with a substantially distended window to minimize the IR (Ohmic) drop of the electrolyte, while SR-GIXRD is undertaken using stop-flow conditions with a very thin layer of electrolyte to minimize X-ray absorption and scattering by the solution.     

Fabrication of core–shell nanocomposites with enhanced photocatalytic efficacy

The current study establishes the unprecedented involvement in the evolution and production of novel core–shell nanocomposites composed of nanosized titanium dioxide and aniline‐o‐phenylenediamine copolymer. TiO₂@copoly(aniline and o‐phenylenediamine) (TiO₂@PANI‐o‐PDA) core–shell nanocomposites were chemically synthesized in a molar ratio of 5:1 of the particular monomers and several weights of nano‐TiO₂ via oxidative copolymerization. The construction of the TiO₂@PANI‐o‐PDA core–shell nanocomposites was ascertained from Fourier transform IR spectroscopy, UV–visible spectroscopy and XRD. A reasonable thermal behavior for the original copolymer and the TiO₂@PANI‐o‐PDA core–shell nanocomposites was investigated. The bare PANI‐o‐PDA copolymer was thermally less stable than the TiO₂@PANI‐o‐PDA nanocomposites. The core–shell feature of the nanocomposites was found to have core and shell sizes of 17 nm and 19–26 nm, respectively. In addition, it was found that the addition of a high ratio of TiO₂ nanoparticles increases the electrical conductivity and consequently lowers the electrical resistivity of the TiO₂@PANI‐o‐PDA core–shell nanocomposites. The hybrid photocatalysts exhibit a dramatic photocatalytic efficacy of methylene blue degradation under solar light irradiation. A plausible interpretation of the photocatalytic degradation results of methylene blue is also demonstrated. Our setup introduces a facile, inexpensive, unique and efficient technique for developing new core–shell nanomaterials with various required functionalities and colloidal stabilities. © 2018 Society of Chemical Industry     

Preparation of nanocomposite dispersions based on cellulose whiskers and acrylic copolymer by miniemulsion polymerization: Effect of the silane content

A one‐step method was used to prepare stable aqueous nanocomposite dispersions based on cellulose whiskers extracted from the rachis of the date palm tree and a poly(styrene‐co‐2‐ethyl hexylacrylate) copolymer via miniemulsion polymerization. A reactive silane, i.e., methacryloxypropyl triethoxysilane was added to stabilize the dispersion and favor the anchoring of the whiskers on polymer particles. Dynamic light scattering was used to study the effect of the silane and whiskers contents on the average particle size of the polymer. Nanocomposites materials were prepared from these dispersions using a casting/evaporation method. The effect of the silane and whiskers contents on the thermal and mechanical properties were studied using differential scanning calorimetry and dynamic mechanical analysis. POLYM. ENG. SCI., 2011. © 2010 Society of Plastics Engineers     

Compatibilization of recycled poly(ethylene terephthalate) and polypropylene blends: Effect of polypropylene molecular weight on homogeneity and compatibility

The effectiveness of compatibilizers in enhancing the dispersion of polypropylene (PP) at various molecular weights in recycled polyethylene terephthalate (RPET) was elucidated. The idea of incorporating PP of different molecular weights evolved from the intention of simultaneously recycling the PET bottles together with the PP‐based bottle caps, which are often of low molecular weight (M_w). Three grades of PP with known molecular weights were blended with RPET at various loadings of compatibilizers. Morphological analyses suggest that the dispersion of the PP particles was more homogeneous, and the average particle size was smaller when low M_w PP was incorporated. This indicates that the interaction between the compatibilizer and PP particles was more intense with the presence of a large number but shorter PP molecular chains. Moreover, specimens containing low M_w PP were found to remain homogeneous regardless of compatibilizer and PP content in the RPET/PP blends. The homogeneity of the blends significantly affected their mechanical performance as well. Higher stiffness, yield strength, deformability, and toughness were observed when low M_w PP was incorporated, regardless of PP and compatibilizer loadings. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Inactivation of pathogenic Klebsiella pneumoniae by CuO/TiO2 nanofibers: A multifunctional nanomaterial via one-step electrospinning

The fabrication and characterization of one-dimensional CuO/TiO₂ nanofibers with high photocatalytic and antibacterial activities are presented. The CuO/TiO₂ nanofibers were prepared by electrospinning of colloid composed of titanium isopropoxide, poly(vinylpyrroliodine) (PVP) and copper nanoparticles and calcination at 700 °C in air for 1 h. The antibacterial activity was tested using Klebsiella pneumoniae as model organism by calculation of the minimum inhibitory concentration (MIC). The obtained CuO/TiO₂ nanofibers showed prominent photocatalytic activity under visible light to degrade reactive black5 and reactive orange16 dyes in aqueous solutions and effectively catalyze K. pneumoniae inactivation. The decomposition process of the cell wall and cell membrane was directly observed by TEM analysis after the exposure of the K. pneumoniae to the nanofibers. Interestingly, the introduced photocatalyst can be reused with the same photocatalytic activity. Overall, the combination of CuO and TiO₂ can be synergistic and resulted in CuO/TiO₂ composite nanofibers having superior photocatalytic and antimicrobial potential to impede K. pneumoniae growth which causes bacterium to die ultimately.     

Photocatalytic activities of electrospun tin oxide doped titanium dioxide nanofibers

SnO₂ doped TiO₂ electropsun nanofiber photocatalysts were successfully prepared by means of electrospinning process. The surface morphology, structure and optical properties of the resultant products were characterized by field-emission electron microscopy (FE-SEM), X-ray diffraction (XRD), high-resolution transmission electron microscopy (HR-TEM), X-ray photoelectron spectroscopy (XPS), UV–vis spectroscopy, photoluminescence (PL) and cathodoluminescence (CL) techniques. The utilized physiochemical analyses indicated that the introduced SnO₂ doped TiO₂ nanofibers have a smooth surface and uniform diameters along their lengths. The photocatalytic performance of the composite nanofibers was tested for degradation of methylene blue (MB) and methyl orange (MO) dye solution under ultraviolet (UV) irradiation. Under the UV irradiation, the photocatalytic reaction rate in case of utilizing SnO₂-doped TiO₂ nanofibers was rapidly increased than that of the pristine TiO₂ nanofibers. Overall, this study demonstrates cheap, stable and effective material for photocatalytic degradation at room temperature.