High-yield Method Research Articles

Enhancing recombinant interleukin-6 production yield by fermentation optimization, two-step denaturing, and one-step purification.

Interleukin-6 a pleiotropic cytokine involved in a wide range of biological activities. So the large-scale production of biologically active recombinant human interleukin-6 is important for its structural and functional studies. Here, we report an optimized method for shake flask fermentation and a simplified high-yield purification procedure for the recombinant interleukin-6. This high-yield expression method not only involves the optimization of the fermentation condition but also the single step purification method as well as a two-step denaturing and one-step refolding process. This approach replaces the more conventional procedure of protein solubilization and refolding. Through applying these strategies, the final cell density and overall product yield of the recombinant human interleukin-6 were obtained as 20.4 g as cell biomass and 150 mg as purified active protein from the I-L of the culture. The purified protein was characterized by HPLC and SDS-PAGE. The results of the current work demonstrate that the described method may be used to develop the process for industrial-scale production of the biologically active recombinant interleukin-6 protein.

High-Quality Monolithic Graphene Films via Laterally Stitched Growth and Structural Repair of Isolated Flakes for Transparent Electronics

Exfoliation of graphene flakes in solution is a high-yield and low-cost synthesis method, but the quality of the obtained graphene flakes is not high, because of the presence of functional groups and structural defects. Therefore, the ability to synthesize high-quality graphene with excellent electrical properties is desirable for electronic applications. Here, we present a facile and rapid annealing approach with nickel for structural repair in isolated graphene flakes on rough insulating substrates, accompanied by lateral stitching of the isolated parts to form a continuous and monolithic film. This process involves the active carbon species being coalesced at the desaturation edge of graphene flakes. Meanwhile, the defects in graphene can be also repaired to improve its crystal quality and electrical properties. Significantly, the carrier mobility of graphene with excellent structural properties is >1000 cm2 V–1 s–1 on average, nearly 10 times higher than that of the process with copper or 100 times hi...

High-Yield Methods for Accurate Two-Alternative Visual Psychophysics in Head-Fixed Mice

SummaryResearch in neuroscience increasingly relies on the mouse, a mammalian species that affords unparalleled genetic tractability and brain atlases. Here, we introduce high-yield methods for probing mouse visual decisions. Mice are head-fixed, facilitating repeatable visual stimulation, eye tracking, and brain access. They turn a steering wheel to make two alternative choices, forced or unforced. Learning is rapid thanks to intuitive coupling of stimuli to wheel position. The mouse decisions deliver high-quality psychometric curves for detection and discrimination and conform to the predictions of a simple probabilistic observer model. The task is readily paired with two-photon imaging of cortical activity. Optogenetic inactivation reveals that the task requires mice to use their visual cortex. Mice are motivated to perform the task by fluid reward or optogenetic stimulation of dopamine neurons. This stimulation elicits a larger number of trials and faster learning. These methods provide a platform to accurately probe mouse vision and its neural basis.

Synthesis and thermal degradation mechanism of polyorganocarboranesiloxanes containing m-carboranylmethyl unit

This paper reports a high-yielding, efficient and facile method in preparing monomers, namely 1,7-bis(hydroxyl(dimethyl)silylmethyl)-m-carborane (M1), 1,7-bis(diethylamino(dimethyl)silylmethyl)-m-carborane (M2), 1,3-dimethyl-1,3-diphenyl-siloxanediol (M3) and diethylaminomethylphenylsilane (M4). The reaction of M2 and M3 to get polymer P1, and the reaction of M1 and M4 to get polymer P2 which has a promising lead in searching for materials with high-temperature properties. The structure of monomers and linear polymers were characterized by NMR and FTIR. Thermal properties of the polyorganocarboranesiloxane elastomers were characterized by DSC and TGA. The results showed that elastomers E1 and E2 have better thermal stability and thermal oxidative stability with 5% weight loss temperatures above 570 °C, 650 °C in nitrogen and 536 °C, 730 °C in air, respectively than that of linear polymers P1 and P2. The highlight was to study the thermal degradation mechanism of polyorganocarboranesiloxane elastomers using XPS and Solid-state 29Si NMR. Carborane and organosiloxane outside of samples were gradually oxidized to B2O3 and SiO2 respectively, the inside only occurred the cleavage of the Si-Ph bond caused by terminal hydroxyl groups which resulted in chain branching and illustrated there was no access for oxygen easy to internal oxidation.

High-Yielding Syntheses of Crown Ether-Based Pyridyl Cryptands

Pyridinium bis(trifluoromethylsulfonyl)imide (PyTFSI)-templated syntheses of 2,6-pyridyl cryptands of cis(4,4')-dibenzo-30-crown-10 (3a), the p-bromobenzyloxy derivative 3b, bis(m-phenylene)-32-crown-10 (5), cis(4,4')-dibenzo-27S-crown-9 (7), cis(4,4')-dibenzo-27L-crown-9 (9), and cis(4,4')-dibenzo-24-crown-8 (11) are reported. Here we provide a fast (12 h), high-yielding (89%, 74%, 80%, and 62% for 3a, 3b, 5, and 9, respectively) templation method without the use of a syringe pump. The yields for 7 (19%) and 11 (26%) were lower than with the previous pseudo-high-dilution method, indicating ineffective templation in these cases. Coupled with our previously developed templated syntheses of dibenzo crown ethers, this protocol makes powerful cryptand hosts readily available in gram quantities in good yields from methyl 4(or 3)-hydroxy-3(or 4)-benzyloxybenzoate.

Investigation of Mechanical Properties of Graphene and Reduced Graphene Oxide Reinforced Epoxy Matrix Composites

Abstract Graphene has generated great excitement in the scientific community due its unique mechanical and electronic properties, and also the availability of bulk quantities of graphene as both colloidal dispersion and powder. With the development of relatively high yield and defect-free synthesis methods, this exciting material is ready for practical application in the preparation of polymer nanocomposites. Here, we reported on the mechanical performances of epoxy nanocomposites which have been reinforced with exfoliated graphene nano platelet (GNP) and reduced graphene oxide (RGO) at a loading of 0 to 0.5 wt. %. A soft molding method was used for the preparation of epoxy nanocomposites. The reinforcing effects of GNP and RGO on epoxy resin were examined by tensile testing and dynamic mechanical analysis (DMA). The morphology of the epoxy/GO and epoxy/RGO nanocomposites were investigated using a scanning electron microscope (SEM). A significant improvement on mechanical properties of epoxy/GO and epoxy/RGO nanocomposites was observed at low GNP and RGO loading. Contrary to the literature, the ultimate tensile strength values have mainly decreased, although the Young’s modulus has improved. Dynamic mechanical analysis has shown that with the addition of both GNP and RGO, storage modulus was significantly enhanced at 40 °C. The loss factor was almost not affected by both RGO and GNP loading. SEM investigation of the fractured surface indicates that GNP and RGO fillers are dispersed uniformly in the epoxy matrix.

ゆうだい21の高品質・多収安定栽培法の検討 ―リン酸追肥に着目した追肥技術と異なる移植時期が収量・品質に及ぼす影響―

ゆうだい21の高品質・多収安定栽培法の検討 ―リン酸追肥に着目した追肥技術と異なる移植時期が収量・品質に及ぼす影響―

Alumina nanoparticle-assisted enzyme refolding: A versatile methodology for proteins renaturation

We present a high-yield method for the renaturation of negatively charged enzymes. The approach is based on the use of alumina nanoparticles, which after electrostatic interaction with denatured protein molecules, prevent their aggregation and make the process of refolding controllable. The method, demonstrated by the renaturation of several enzymes, is efficient, rapid, employs a minimal amount of reagents and even can be applied to renature mixture of the denatured enzymes.

Search for arc-produced heterofullerenes

Replacing one or more of the carbon atoms in a fullerene cage with one or more non-carbon atoms is expected to result in a structure (heterofullerene) with significantly altered and potentially useful properties. While (C59N)- and (C69N)-based compounds can be synthesized via solution-phase modification of C60 and C70, respectively, a general, high-yield method for producing heterofullerenes has not been identified. We report the results of a search for heterofullerenes using atmospheric pressure photo-ionization mass spectrometry on samples of fullerene soot produced in nitrogen and carbon monoxide via the Krätschmer-Huffman arc technique. Contrary to earlier reports, we found no evidence of the presence of heterofullerenes in any of the samples we examined. X-ray photoelectron spectroscopy of fullerene soot produced in nitrogen indicates that while nitrogen is incorporated into the carbon matrix, the observed nitrogen-carbon bonding configurations do not permit the formation of fullerenes. We also report the discovery of production conditions under which argon can be used as a buffer gas and the yield of soluble fullerenes is comparable to the best yield achievable using helium. These findings may shed light on the general problem of the fullerene formation process.

High-yield production of pure tagatose from fructose by a three-step enzymatic cascade reaction.

To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. Fructose at 1M (180gl-1) was converted to 0.8M (144gl-1) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16h with a productivity of 9gl-1 h-1. No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%. This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

Low Temperature Synthesis and Properties of BiFeO3

Extensive efforts have been made to synthesize single phase and stoichiometric BiFeO3. Some modified techniques have been tried in synthesizing BiFeO3 as compared with the conventionally used co-precipitation method. Thermogravimetric Analysis/Differential Scanning Calorimetry and x-ray diffraction experiments were used exclusively to explore the effects of heat treatment temperature and time on crystallographic behavior of the prepared BiFeO3 powder. Field emission scanning electron microscopy was used to explore the microstructure of the synthesized BiFeO3. The appearance of different magnetic phases in 57Fe Mossbauer spectra and field dependent magnetization was confirmed on the basis of particle size distribution. In this research, an easy, low cost and high-yield method for the low temperature single phase and stoichiometric synthesis of BiFeO3 has been suggested.

Salt-Templated Synthesis of 2D Metallic MoN and Other Nitrides.

Two-dimensional (2D) transition-metal nitrides just recently entered the research arena, but already offer a potential for high-rate energy storage, which is needed for portable/wearable electronics and many other applications. However, a lack of efficient and high-yield synthesis methods for 2D metal nitrides has been a major bottleneck for the manufacturing of those potentially very important materials, and only MoN, Ti4N3, and GaN have been reported so far. Here we report a scalable method that uses reduction of 2D hexagonal oxides in ammonia to produce 2D nitrides, such as MoN. MoN nanosheets with subnanometer thickness have been studied in depth. Both theoretical calculation and experiments demonstrate the metallic nature of 2D MoN. The hydrophilic restacked 2D MoN film exhibits a very high volumetric capacitance of 928 F cm-3 in sulfuric acid electrolyte with an excellent rate performance. We expect that the synthesis of metallic 2D MoN and two other nitrides (W2N and V2N) demonstrated here will provide an efficient way to expand the family of 2D materials and add many members with attractive properties.

Nickel-Salen supported paramagnetic nanoparticles for 6-His-target recombinant protein affinity purification

In this research, a simple, efficient, inexpensive, rapid and high yield method for the purification of 6×histidine-tagged recombinant protein was developed. For this purpose, manganese ferrite magnetic nanoparticles (MNPs) were synthesized through a co-precipitation method and then they were conveniently surface-modified with tetraethyl orthosilicate (TEOS) in order to prevent oxidation and form high density of hydroxyl groups. Next, the salen ligand was prepared from condensation reaction of salicylaldehyde and 3-aminopropyl (trimethoxy) silane (APTMS) in 1:1 molar ratio; followed by complexation with Ni(OAc)2.4H2O. Finally, the prepared Ni(II)-salen complex conjugated to silica coated MNPs and MnFe2O4@SiO2@Ni-Salen complex nanoparticles were obtained. The functionalized nanoparticles were spherical with an average diameter around 70nm. The obtained MNPs had a saturation magnetization about 54 emu/g and had super paramagnetic character. These MNPs were used efficiently to enrich recombinant histidine-tagged (His-tagged) protein-A from bacterial cell lysate. In about 45min, highly pure His-tagged recombinant protein was obtained, as judged by SDS-PAGE analysis and silver staining. The amount of target protein in flow through and washing fractions was minimal denoting the high efficiency of purification process. The average capacity of the matrix was found to be high and about 180±15mgg−1 (protein/MnFe2O4@SiO2@Ni-Salen complex). Collectively, purification process with MnFe2O4@SiO2@Ni-Salen complex nanoparticles is rapid, efficient, selective and whole purification can be carried out in only a single tube without the need for expensive systems.

In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines.

RNA plays a fundamental, ubiquitous role as either substrate or functional component of many large cellular complexes-"molecular machines"-used to maintain and control the readout of genetic information, a functional landscape that we are only beginning to understand. The cellular mechanisms for the spatiotemporal organization of the plethora of RNAs involved in gene expression are particularly poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent mechanistic parameters that govern cellular RNA functions, including those of protein coding messenger RNAs (mRNAs). Progress has been hampered, however, by the scarcity of efficient high-yield methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ T7 RNA polymerase to body label an RNA with a cyanine dye, as well as yeast poly(A) polymerase to strategically place multiple 2'-azido-modifications for subsequent fluorophore labeling either between the body and tail or randomly throughout the tail. Using a combination of biochemical and single-molecule fluorescence microscopy approaches, we demonstrate that both yeast poly(A) polymerase labeling strategies result in fully functional mRNA, whereas protein coding is severely diminished in the case of body labeling.

Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System

The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 1014 vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 1015 VG in crude cell harvests and an average of 3.5 × 1014 total VG of purified rAAV9 material, resulting in greater than 1 × 105 VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.

Preparation of Photoluminescent Porous Silicon Nanoparticles by High‐Pressure Microfluidization

The use of high-shear microfluidization as a rapid, reproducible, and high-yield method to prepare nanoparticles of porous silicon (pSi) with a narrow size distribution is described. Porous films prepared by electrochemical etch of a single-crystal silicon wafer are removed from the substrate, fragmented, dispersed in an aqueous solution, and then processed with a microfluidizer, which generates high yields (57%) of pSi nanoparticles of narrow size distribution (PDI = 0.263) without a filtration step. Preparation of pSi nanoparticles via microfluidization improves yields (by 2.4-fold) and particle size uniformity (by 1.8-fold), and it lowers the total processing time (by 36-fold) over standard ultrasonication or ball milling methods. The average diameter of the nanoparticles can be adjusted over the range 150–350 nm by appropriate adjustment of processing steps. If the fluid carrier in the microfluidizer contains an oxidant for Si, the resulting pSi particles are prepared with a core–shell structure, in which an elemental Si core is encased in a silicon oxide shell. When an aqueous sodium tetraborate processing solution is used, microfluidization generates photoluminescent core–shell pSi particles with a quantum yield of 19% in a single step in less than 20 min.

High photoluminescent TPA and the ratiometric sensor for free chlorine

A facile and high yield method is introduced to synthesize 3,5-dihydro-5-oxo-2H-thiazolo[3,2-a]pyridine-3,7-dicarboxylic acid (TPA) through the “one-step” condensation reaction of citric acid and l-cysteine. For the first time, the produced TPA molecules are purified by the simple crystallization, and the photoluminescence (PL) properties of the TPA are investigated. The obtained purified TPA shows bright blue PL, with a PL quantum yield as high as 90.5%. The sulfur atom of TPA molecule can be selectively oxidized by free chlorine into sulfoxide, which is a strong electron withdrawing group. As a result, the conjugation is decreased, leading to a great PL quenching. On the basis, a sensitive and selective PL sensor is developed for the detection of free chlorine. Furthermore, tris(2,2-bipyridine)ruthenium(II) chloride hexahydrate, whose red PL signal is inert to the free chlorine, can be used to provide a background PL signal. Thus, a visual PL ratiometric sensor is developed, which is convenient for the on-site and on-line detection of free chlorine.

A high-yield and versatile method for the synthesis of carbon dots for bioimaging applications.

A facile and versatile molten-salt method was developed to prepare hydrosoluble carbon dots (CDs) from various precursors, in high yields and on a large scale. Citric acid-based CDs (CA-CDs) were obtained in a maximum yield of 39.6% and exhibited a high fluorescence quantum yield of 20.8% without any passivation. The CA-CDs showed little cytotoxicity even at a concentration as high as 800 μg mL-1. In addition, CA-CDs could be used as multicolour fluorescence imaging agents in vitro with blue, green, and red fluorescence emissions at excitation wavelengths of 405, 488, and 543 nm, respectively. Moreover, the CA-CDs could be chelated with gadolinium ions (Gd3+) to construct Gd-CA-CDs for dual-mode magnetic resonance and fluorescence imaging. The Gd-CA-CDs showed good water dispersibility, excellent biocompatibility, a strong fluorescence quantum yield of 13.1%, and a high magnetic resonance relaxivity of 22.45 mM-1 s-1. The molten-salt method was demonstrated to be applicable to other precursors, such as sodium lignosulphonate, sucrose, glucose, and p-phenylenediamine, and the maximum yield of the four as-prepared CDs was as high as 66.7%, which is much higher than the value reported in previous studies. This study proves that the molten-salt synthesis is a versatile method to obtain CDs in high yields, which will promote the application of CDs in the field of bioimaging.

An atlas of endohedral Sc2S cluster fullerenes.

Structural identification is a difficult task in the study of metallofullerenes, but understanding of the mechanism of formation of these structures is a pre-requisite for new high-yield synthetic methods. Here, systematic density functional theory calculations demonstrate that metal sulfide fullerenes Sc2S@Cn have similar cage geometries from C70 to C84 and form a close-knit family of structures related by Endo-Kroto insertion/extrusion of C2 units and Stone-Wales isomerization transformations. The stabilities predicted for favoured isomers by DFT calculations are in good agreement with available experimental observations, have implications for the formation of metallofullerenes, and will aid structural identification from within the combinatorially vast pool of conceivable isomers.

Synthesis and Growth Stimulant Properties of 2-Acetyl-3,7-dimethyl-5H-thiazolo[3,2-a]pyrimidin-5-one Derivatives

A convenient, accessible, and high yield method for preparing of 6-methyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (1) by treatment of acetoacetic acid ethyl ester with thiourea in sodium methylate was developed. The alkylation of the latter with 3-chloro-pentane-2,4-dione and further regioselective cyclization of intermediate compound (2) in high yield afforded 2-acetyl-3,7-dimethyl-5H-thiazolo[3,2-a]pyrimidin-5-one (3). The halogenation and some transformations of synthesized thiazolo[3,2-a]pyrimidine (3) due to its ketone group were carried out to obtain the corresponding carboxamide, carbothioamide, sulfonohydrazide, and oxime and its alkylated derivatives (5). At preliminary biological studies the synthesized compounds have shown growth stimulant properties. The activity of four of them was higher than 70%, compared with heteroauxin.