Electrophoretic Method Research Articles

Evaluation of tribological and biological properties of Ti6Al4V coated with Si3N4/ND nanoparticles for orthopedic applications: a comprehensive analysis

In this study, the biocompatibility and tribological properties of Ti6Al4V coated with silicon nitride (Si3N4)/nanodiamond using the electrophoretic deposition method were investigated. Suspensions of various aqueous and alcoholic solutions were prepared in the presence of CTAB and SDS dispersers. The most stable suspension system for the electrophoresis process was selected (aqueous media/ SDS disperser). Four different voltages (20, 30, 40 and 50 V) were applied to study the effect of voltage on the coating property. One could find that processing with 40 V obtained the best coating. The nano-composite coating was characterized using scanning electron microscopy equipped with energy dispersive spectroscopy, mapping analysis and x-ray diffraction after the coating process. The samples were then subjected to two nanoindentation and nano-scratching tests to evaluate their tribological properties. Biocompatibility was assessed in an ex vivo environment using two cell culture tests to evaluate survival and cellular adhesion. The results showed that the hardness and modulus elasticity of the coated sample increased from 85 to 124 GPa and 1.14–3.55 GPa, respectively, compared to the non-coated sample. Additionally, the MTT test results indicated that cellular survival and proliferation of MG63 cells increased from 86% for the non-coated sample to 92% for the Ti6Al4V/Si3N4 /ND sample. These findings have implications for orthopedic implant applications.

PLASMA PROTEIN ELECTROPHORESIS IN THE WHITE STORK (CICONIA CICONIA): AGREEMENT BETWEEN AGAROSE GEL VERSUS CAPILLARY ZONE METHODS AND DEVELOPMENT OF REFERENCE INTERVALS.

The white stork (Ciconia ciconia) is a ciconiiform species widely represented in zoological institutions. Plasma protein electrophoresis is widely used in avian patients for assessment of inflammatory conditions, but reference intervals for this testing modality are lacking for the white stork. The two main electrophoretic methods are agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE). This study assessed fresh plasma samples of healthy adult white storks (n = 30). Statistical analyses were performed to evaluate agreement between AGE and CZE. Typical electrophoretic fractions were obtained from both methods (prealbumin, albumin, α1, α2, β, γ1, and γ2). The AGE and CZE methods were not equivalent for determining major electrophoretic fractions (except β-globulins) and albumin:globulin ratio on plasma samples. An additional prealbumin fraction was seen with CZE. Reference intervals were established for each method as the smallest n group was 27 individuals for a given value; most values had normal distribution, and robust or parametric methods were used on the data.

Electrophoretic method and assessing the formation of an RNA-interfering complex with miR775A in corn leaves under the conditions of normoxia and hypoxia

Oxidative processes in cells, in particular mitochondrial respiration, are most sensitive to a lack of oxygen, therefore, it requires cell metabolism coordination. MicroRNAs are one of the tools of the molecular epigenetic regulation. These are a class of non-coding small RNA molecules (20-22 nt) that are able to regulate target genes expression at the post-transcriptional level by inhibiting the translation or cleavage of their mRNAs. It is known that microRNAs can act by RNA interference or can bind to an RNA-induced transcriptional silencing (RITS) complex. The study of the role of microRNAs in the adaptive response of cell metabolism to hypoxic stress as a mechanism of molecular control is of particular interest. Phenol-chloroform extraction with a specific precipitant for LiCl ribonucleic acids made it possible to obtain the total cellular RNA from corn leaves. The resulting RNA preparations were of good quality, which was indicated by the presence of 2 distinct bands of 28S and 18S rRNA and the absence of traces of degradation. The resulting preparations of total cellular RNA were used as a matrix to assess the formation of RNA interfering complexes which formed as RNA:RNA duplexes based on complementarity between microRNA775A and the target mRNA. The method of analytical agarose gel electrophoresis with a specific fluorescent probe with ROX was used to assess the formation of an RNA interfering complex with miR775A. This method made it possible to identify the ratio of the number of formed RNA-interfering complexes of cell mRNA with miR775A separated on the basis of their molecular masses in the samples under hypoxic conditions. Electrophoresis in agarose gel followed by densitometry revealed the presence of only one interfering complex with an electrophoretic mobility of 0.23-0.25 units. The results showed that miR775A interacted with a single target mRNA in corn leaf cells under normal conditions and under the conditions of hypoxia. Quantitative assessment of the fluorescence intensity of the formed complex of mRNA-miR775A-probe by ROX emission indicated an increase in the analysed indicator in samples isolated from corn leaves under hypoxic stress.

Study of Nanohydroxyapatite Coatings Prepared by the Electrophoretic Deposition Method at Various Voltage and Time Parameters.

The aim of the work is to compare the properties of nanohydroxyapatite coatings obtained using the electrophoretic deposition method (EDP) at 10 V, 20 V, and 30 V, and with deposit times of 2 and 5 min. The primary sedimentation was used to minimize the risk of the formation of particle agglomerates on the sample surface. Evaluation of the coating was performed by using a Scanning Electron Microscope (SEM), Energy-Dispersive Spectroscopy (EDS), Atomic Force Microscopy (AFM), optical profilometer, drop shape analyzer, and a nanoscratch tester. All of the coatings are homogeneous without any agglomerates. When low voltage (10 V) was used, the coatings were uniform and continuous regardless of the deposition time. The increase in voltage resulted in the formation of cracks in the coatings. The wettability test shows the hydrophilic behavior of the coatings and the mean contact angle values are in the range of 20-37°. The coatings showed excellent adhesion to the substrate. The application of a maximum force of 400 mN did not cause delamination in most coatings. It is concluded that the optimal coating for orthopedic implants (such as hip joint implants, knee joint implants or facial elements) is obtained at 10 V and 5 min because of its homogeneity, and a contact angle that promotes osseointegration and great adhesion to the substrate.

Electrochemical Impedance Spectroscopy Analysis of 45S5 Bioglass Coating on After Oxidation of CoCrW Alloy

Implants and prostheses, which are used to replace a missing or damaged structure in living organisms, must show all the necessary mechanical, tribological, electrochemical and biocompatibility properties together. CoCr alloys are often preferred biometals for their good mechanical strength and wear resistance, especially in dental and orthopedic implants. Although these alloys show good corrosion resistance in terms of electrochemical behavior as well as other good properties, when CoCr alloys come into contact with bone tissue, their surfaces show bioinert properties in terms of tissue formation between the implant and bone tissue. Therefore, both their corrosion behavior and biocompatibility properties need to be improved. In this study, CoCrW alloys produced by selective laser melting were coated with commercial 45S5 bioglass powder, a bioactive material, by electrophoretic deposition method. In order to improve the adhesion after coating, CoCrW alloys were subjected to electrochemical etching process during coating. After the coating process was completed, untreated, oxidized, untreated-coated and oxidized-coated samples were examined by electrochemical impedance spectroscopy (EIS) after open circuit potential measurements to investigate their corrosion behavior. As a result of the corrosion tests, it was determined that the oxidized-coated sample showed the best condition compared to the other samples in EIS analyzes.

Production, purification, and determination of the biochemical properties of β-glucosidase in Trichoderma koningii via solid substrate fermentation.

The β-glucosidase enzyme was obtained from Trichoderma koningii Oudem. NRRL 54330 under optimal conditions by solid substrate fermentation (SSF) using corn cobs as substrate. The enzyme was purified by two-step procedures, ammonium sulphate precipitation and cefarose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography, followed by biochemical and kinetic characterisation. The β-glucosidase was obtained from T.koningii using ground corn cob as substrate and Na2HPO4, pH 9, as humidification medium. The optimum conditions for enzyme production by SSF were 30 °C and 6days. The purification efficiency of the obtained β-glucosidase was calculated to be 22.56-fold with a yield of 73.51 %. In the determination of β-glucosidase activity, p-nitrophenyl-β-d-glucopyranoside (pNPG) substrate was used, and the optimum pH and temperature values at which β-glucosidase showed high activity were determined to be pH 3.0 and 75 °C. The purity of the enzyme and the presence/number of subunits were checked using two different electrophoretic methods, SDS-PAGE and NATIVE-PAGE electrophoretic methods. The K m and V max values of the purified enzyme were determined to be 0.16 mM and 2000 EU respectively. Itwas also found that d-(+)-glucose and δ-gluconolactone inhibitors exhibited competitive inhibition of β-glucosidase in the presence of pNPG.

A Flexible One‐dimensional Woven Supercapacitor for Low‐power Smart Electronic Textiles

Flexible energy storage devices are attracting a great attention nowadays in the rapidly growing area of implantable/wearable electronics. This bloom has generated great demands on flexible wire‐shaped supercapacitors (WSC) as power sources in wearable electronic systems that can offer tunable sizes, shapes, and versatile designs. Herein, a flexible WSC is fabricated using MgMn2O4 nanofibers (MMO‐NFs) over a flexible carbon yarn substrate via the electrophoretic deposition method in the first report. The binder‐free deposition of highly porous and one‐dimensionally aligned MMO‐NFs over carbon yarn offers excellent ionic/charge transport into the fabricated device. The assembled WSC device at 0.1 mA/cm current rate shows capacitances of 386 mF/cm2 and 45.5 mF/cm. A capacitance retention of 88% for a WSC device after 10,000 cycles indicates its good cyclability. Further, the WSC exhibits good flexibility, retaining 94% of capacitance at 0.5 mA/cm after 2000 bending cycles. Furthermore, the flexible WSC device shows power and energy density of 744 μW/cm and 14.2 μWh/cm, respectively. Additionally, WSC devices demonstrate their practical energy storage capabilities in a video that shows three similar devices being knitted into a wearable glove and glowing a red light‐emitting diode (LED) for more than five minutes in various bending positions.

Evaluation of measured and calculated small dense low-density lipoprotein in capillary blood and association with the metabolic syndrome

Background and aimsSmall-dense-low-density-lipoprotein cholesterol (sdLDL-C) is proatherogenic and not commonly measured. The aims were to evaluate capillary blood and its stability for sdLDL-C measurement and measure sdLDL-C in patients with metabolic syndrome (MS). Methods182 patients were studied (49 with MS). sdLDL-C was measured by electrophoresis (LipoPrint®), direct measurement (Roche Diagnostics) and Sampson equation. Intima-media thickness (IMT) and presence of atheroma was evaluated. sdLDL-C was compared in paired venous and capillary blood according to CLSI-EP09c (n = 40). sdLDL-C stability was studied after 24 h at room temperature (RT). ResultssdLDL-C in capillary blood and venous blood showed agreement with the direct measurement (bias: 4.17 mg/dL, LOA 95 %:−5.66; 13.99) and estimation (bias:8.12 mg/dL, LOA 95 %:−8.59; 24.82). sdLDL-C is stable in capillary blood for 24 h at RT. The electrophoretic method yielded lower (p < 0.05) sdLDL-C than the equation or direct measurement. Patients with MS had (p < 0.05) higher sdLDL-C (%) than patients without MS. Patients with atheroma plaques had higher sdLDL-C (p < 0.05). Estimated sdLDL-C correlated with IMT (r = 0.259, p < 0.001). ConclusionsCapillary blood is an alternative to venous blood for sdLDL-C measurement and is stable for 24 h after collection. Estimated and directly measured sdLDL-C associate with the MS being accessible tools for cardiovascular risk assessment.

CdSe nanostructured thin film by electrophoretic deposition for quantum dots sensitized solar cell.

Cadmium selenide (CdSe) quantum dots (QDs) with different size, 2.5 and 3.2 nm, were successfully deposited on mesoporous titanium dioxide (TiO2) (Degussa-P25) nanostructures by electrophoretic deposition method (EPD) at the applied voltage 100 V for 120 s deposition time. In this study, the morphology of CdSe films deposited by EPD and the performance of the film when assembled into a solar cell were investigated. From the field emission scanning electron microscopy cross-section, the thickness of the CdSe nanoparticles with size 2.5 nm films were 3.4 and 3.0μm for CdSe 3.2 nm nanoparticles film. The structure of 2.5 nm is denser than compare of 3.2 nm CdSe nanoparticles. From UV visible spectroscopy, the band gap calculated for 2.5 nm CdSe nanoparticles is 2.28 eV and for 3.2 nm is 2.12 eV. Photovoltaic characterization was performed under an illumination of 100 mW cm-2. A photovoltaic conversion efficiency of 1.81% was obtained for 2.5 nm CdSe and 2.1% was obtained for 3.2 nm CdSe nanoparticles. This result shows that the photovoltaic efficiency is dependent on CdSe nanoparticle size.

Identification and quantification of chain-pairing variants or mispaired species of asymmetric monovalent bispecific IgG1 monoclonal antibody format using reverse-phase polyphenyl chromatography coupled electrospray ionization mass spectrometry

Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product.

Enhanced CMAS and hot corrosion degradation of YSZ thermal barrier coating with nano powders

Thermal barrier coatings are used to protect hot section parts of gas turbine engines. These coatings are subject to corrosion due to impurity elements in the fuel and dust absorbed from the atmosphere into the engine. The rough and porous surface nature of plasma-sprayed coatings facilitates the penetration of these factors into the coating. Coatings are damaged prematurely as a result of corrosion. In this study, nano-structure YSZ, YSZ + Al2O3, YSZ + TiO2, and YSZ + Al2O3 + TiO2 coatings were deposited on plasma sprayed YSZ coating by electrophoretic deposition method. Hot corrosion and CMAS resistance were investigated. The coatings were characterized by SEM, XRD, AFM, and microhardness before corrosion tests. The changes in the coatings after corrosion tests were characterized by SEM and XRD. The penetration behavior of corrosive factors was examined by elemental mapping with EDS. Nanostructure coatings deposited on APS YSZ made the surfaces of as-sprayed coatings more stable. The ability of these nanolayers to prevent pure penetration into coatings during hot corrosion and CMAS is limited. However, the Al2O3 + TiO2-doped nanostructure YSZ reduced the bond-layer oxidation. In addition, the addition of Al2O3 + TiO2 to the EPD deposited layer also reduced the tetragonal-monoclinic phase transformation that occurred in YSZ as a result of corrosion. Al2O3 and TiO2 additives alone are not effective in improving the corrosion behavior of coatings. Nanostructured coatings deposited with the EPD technique can help protect plasma-sprayed thermal barrier coatings against corrosion.

Biogenic amines detection in meat and meat products: the mechanisms, applications, and future trends

Biogenic amine is one of the main categories of hazardous compounds in meat and meat products, making its detection methods vital for the assurance of edible safety. In this sense, many biogenic amine detection techniques such as chromatographic, electrophoretic, and electrochemical methods have been developed, which play an irreplaceable role in ensuring the safety of meat and meat products. Due to the increasing demand for fast and on-site detection techniques, visual detection methods have been gradually developed compared with non-visual methods such as chromatography and electrophoresis. Herein, we comprehensively review the mechanism and the latest progress of these biogenic amine detection methods; besides, we put forward the prospects for the future development of biogenic amine detection techniques, with a view to providing support for the establishment of more accurate and efficient detection, prevention and control strategies of biogenic amines.

FABRICATION OF A NEW CERAMIC COATING BY ELECTROPHORETIC METHOD FOR GRAPHITE ELECTRODES AT HIGH TEMPERATURE

A large part of the cost of steel production in the world is related to the consumption of graphite electrodes. Electrode is the only material that maintains its electrical conductivity at high temperatures, and hence it is used as a conductor of electric current in EAF furnaces. Considering the high costs of graphite electrodes, any action that leads to reducing the consumption of graphite electrode can lead to increasing the productivity of steelmaking operations. In this study and as its novelty, the electrophoretic method was used to generate a ceramic coating for the protection of graphite electrodes. Electrophoretic deposition (EPD) is a two-step process in which charged particles suspended in a suspension move towards the oppositely charged electrode under the influence of an electric field which combine as a dense film on the surface. In the EPD technique, the particle size limit is not very important if a stable suspension is formed and micro and nano-sized particles can be used in this method. The numerical simulation of the thermal contour on the graphite electrode was done in ABAQUS software. Moreover, the surface morphology of graphite samples has been investigated via scanning electron microscopy (SEM). The created coating was a combination of Al2O3 and SiO2, which was placed on the surface of test samples by EPD treatment. The obtained results have the properties of resistance to thermal shock and show the increase in the life of coated graphite samples at different temperatures and times.

Linear DNA-driven recombination in mammalian mitochondria.

Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic due to its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we took advantage of a cell model with two different types of mitochondrial genomes and impaired its ability to degrade broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot and by long range PacBio® HiFi sequencing approach. Besides utilizing different electrophoretic methods, we also directly observed molecular complexes between different mtDNA haplotypes and recombination intermediates using transmission electron microscopy. We propose that the known copy-choice recombination by mitochondrial replisome could be sufficient for the needs of the small genome, thus removing the requirement for a specialized mitochondrial recombinase. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.

CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols

Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) of monoclonal antibodies (mAbs). The implemented reduction often leaves the intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on a neutral-coated capillary for the separation of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by mass spectrometry (MS), MS/MS identification, and trapped ion mobility mass spectrometry (timsTOF). Our CE approach enables the separation of (i) different subunit moieties, (ii) various reduction states, and (iii) positional isomers of these partly reduced subunit moieties. The location of the remaining disulfide bridges can be determined by middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All these CE-separated variants show differences in ion mobility in the timsTOF measurements. Applying the presented CE-MS/MS method, reduction parameters such as the use of chaotropic salts were studied. For the investigated antibodies, urea improved the subunit reduction significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals of the same subunit in the CE separation. The presented CE-MS method is a powerful tool for the disulfide-variant characterization of mAbs on the subunit level. It enables understanding disulfide bridge reduction processes in antibodies and potentially other proteins.

Functionalization of the NiTi Shape Memory Alloy Surface through Innovative Hydroxyapatite/Ag-TiO2 Hybrid Coatings.

The objective of this research was to develop a surface modification for the NiTi shape memory alloy, thereby enabling its long-term application in implant medicine. This was achieved through the creation of innovative multifunctional hybrid layers comprising a nanometric molecular system of silver-rutile (Ag-TiO2), known for its antibacterial properties, in conjunction with bioactive submicro- and nanosized hydroxyapatite (HAp). The multifunctional, continuous, crack-free coatings were produced using the electrophoretic deposition method (EPD) at 20 V/1 min. Structural and morphological analyses through Raman spectrometry and scanning electron microscopy (SEM) provided comprehensive insights into the obtained coating. The silver within the layer existed in the form of nanometric silver carbonates (Ag2CO3) and metallic nanosilver. Based on DTA/TG results, dilatometric measurements, and high-temperature microscopy, the heat treatment temperature for the deposited layers was set at 800 °C for 2 h. The procedures applied resulted in the creation of a new generation of materials with a distinct structure compared with the initial nanopowders. The resulting composite layer, measuring 2 μm in thickness, comprised hydroxyapatite (HAp), apatite carbonate (CHAp), metallic silver, silver oxides, Ag@C, and rutile exhibiting a defective structure. This structural characteristic contributes significantly to its heightened activity, influencing both bioactivity and biocompatibility properties.

Research of chitosan coatings deposited by electrophoretic deposition method at various voltage and time parameters

The aim of this research is to estimate the electrophoretic deposition main parameters, such as voltage and time duration, that will provide optimal characteristics of the surface layer. Chitosan coatings were synthesized on biomedical Ti13Zr13Nb alloys at 20 V and 30 V and with deposit times of 2min, 5 min, 10 min, and 15 min. Evaluation of the coating was performed by using Scanning Electron Microscope (SEM), Energy-Dispersive Spectroscopy (EDS), Laser-Induced Breakdown Spectroscopy (LIBS), Atomic Force Microscopy (AFM), drop shape analyzer and nanoscratch tester. Homogeneous and continuous coatings are obtained for 2 min and 5 min, regardless of the voltage. Defects in form of bubbles are observed only for 20 V during 10 min, 20 V during 15 min, and 30 V during 10 min. All of the coatings show hydrophilic behavior. The critical loads for delamination range are between 100 mN and 300 mN. It is concluded, that the optimal coating for orthopedic implants is obtained at 20 V, 5 min, because of homogeneity, a contact angle supporting osseointegration and the best adhesion. The optimal one for implant in contact with blood is obtained at 20 V, 2 min, because it is homogenous and shows the lowest hydrophilicity, which should limit the thrombogenicity of the coating.

Mechanical properties of SiCf/SiC composites with h‐BN interphase formed by the electrophoretic deposition method

AbstractSilicon carbide fiber–reinforced silicon carbide (SiCf/SiC) composites have been expected to be used as next‐generation heat resistant structural materials with high reliability. The fiber/matrix interface for SiCf/SiC composites plays an important role for toughening and strengthening, and it is important to form the optimal interphase for SiCf/SiC composites. In this study, hexagonal‐boron nitride (h‐BN), which shows better oxidation resistance than carbon, was selected as the interphase for SiCf/SiC composites, and h‐BN coating was formed on low‐conductive SiC fibers by the electrophoretic deposition (EPD) method using flaked h‐BN suspension prepared by wet‐jet milling. Unidirectional SiCf/SiC composites with the BN interphase formed by the EPD method were prepared by polymer impregnation and pyrolysis (PIP) method, and their mechanical properties were evaluated. The uniform h‐BN interphase was successfully formed on the low‐conductive SiC fibers by EPD when thin polypyrrole (Ppy) coating, that is, conductive polymer, was formed on the SiC fibers. The h‐BN coating became denser by heat‐treatment at 1000°C, and the h‐BN coating deposited tightly on the SiC fibers was confirmed. It was demonstrated that the SiCf/SiC composites with the uniform and relatively dense h‐BN interphase formed by EPD using flaked h‐BN particles exhibited pseudo‐ductile fracture behavior with large amount of fiber pullout and higher fracture energy.

Preparation and Characterization of Acrylic and Methacrylic Phospholipid-Mimetic Polymer Hydrogels and Their Applications in Optical Tissue Clearing.

The 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers are mimetic to phospholipids, being widely used as biocompatible polymers. In our previous study, MPC polymer hydrogels proved more effective for optical tissue clearing compared to acrylamide (AAm) polymer hydrogels. In the present study, 2-acryloyloxyethyl phosphorylcholine (APC) was synthesized and employed to create hydrogels for a comparative analysis with methacrylic MPC-based hydrogels. APC, an acrylic monomer, was copolymerized with AAm in a similar reactivity. In contrast, MPC, as a methacrylic monomer, demonstrated higher copolymerization reactivity than AAm, leading to a spontaneously delayed two-step polymerization behavior. This suggests that the polymer sequences and network structures became heterogeneous when both methacrylic and acrylic monomers, as well as crosslinkers, were present in the copolymerization system. The molecular weight of the APC polymers was considerably smaller than that of the MPC polymers due to the formation of mid-chain radicals and subsequent β-scission during polymerization. The swelling ratios in water and strain sweep profiles of hydrogels prepared using acrylic and methacrylic compounds differed from those of hydrogels prepared using only acrylic compounds. This implies that copolymerization reactivity influences the polymer network structures and crosslinking density in addition to the copolymer composition. APC-based hydrogels are effective for the optical clearing of tumor tissues and are applicable to both passive and electrophoretic methods.

Expression, purification and folding of native like mitochondrial carrier proteins in lipid membranes

Mitochondrial Carrier Family proteins (MCFs) are located in the mitochondrial inner membrane and play essential roles in various cellular processes. Due to the relatively low abundance of many members of the family, in vitro structure and function determination of most MCFs require over-expression and purification of recombinant versions of these proteins. In this study, we report on a new method for overexpression of MCFs in Escherichia coli (E. coli) membranes, efficient purification of native-like proteins, and their reconstitution in mitochondrial inner membrane lipid mimics. cDNAs of Uncoupling Protein 4 (UCP4), Adenine Nucleotide Translocase (ANT) and Phosphate Translocase (PiT) were subcloned into the pET26b (+) expression vector such that fusion proteins with a short N-terminal pelB leader sequence and a six-histidine tag were produced to target the proteins toward the inner membrane of E. coli and facilitate affinity purification, respectively. Utilizing a modified autoinduction method, these proteins were overexpressed and extracted from the membrane of E. coli BL21 (DE3) and two modified strains, E. coli BL21 C43 (DE3) and E. coli BL21 Lobstr (DE3), in high yields. The proteins were then purified by immobilized metal affinity chromatography as monomers. Purity, identity, and concentration of the eluted monomers were determined by semi-native SDS-PAGE, Western blotting and mass spectrometry, and a modified Lowry assay, respectively. Cleavage of the pelB leader sequence from proteins was verified by mass spectrometric analysis. The purified proteins, surrounded by a shell of bacterial membrane lipids, were then reconstituted from the mild non-denaturing octyl glucoside (OG) detergent into phospholipid liposomes. Monomeric UCP4 spontaneously self-associated to form stable tetramers in lipid membranes, which is consistent with our previous studies. However, PiT and ANT remained dominantly monomeric in both detergent and liposome milieus, as detected by a combination of spectroscopic and electrophoretic methods. Native-like helical conformations of proteins were then confirmed by circular dichroism spectroscopy. Overall, this study demonstrates that targeting mitochondrial carrier family proteins to E. coli membranes provides an effective expression system for producing this family of proteins for biophysical studies.