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Jiyun Jeong, Yeolin Lee, Yeongeun Yoo, Myung Kyu Lee
Agarose gel can be used for three dimensional (3D) cell culture because it prevents cell attachment. The dried agarose film coated on a culture plate also protected cell attachment and allowed 3D growth of cancer cells. We developed an efficient method for agarose film coating on an oxygen-plasma treated micropost polystyrene chip prepared by an injection molding process. The agarose film was modified to maleimide or Ni-NTA groups for covalent or cleavable attachment of photoactivatable Fc-specific antibody binding proteins (PFcBPs) via their N-terminal cysteine residues or 6xHis tag, respectively...
December 5, 2017: Colloids and Surfaces. B, Biointerfaces
Linpeng Wang, Jing Yan, Jingyun Yan, Huanhuan Xu, Dengyang Zhang, Xuanjun Wang, Jun Sheng
In the fields of drug discovery and protein science, small quantities of proteins are always needed to investigate or validate protein-protein (or protein-small molecule) interactions. Traditional transient or stable expression method to obtain recombinant proteins in eukaryotic systems can be laborious and time-consuming, especially when multiple protein variants are required. Here, we present a fast and convenient method for obtaining small quantities of recombinant human epidermal growth factor receptor (rhEGFR) ectodomain protein, which could be efficiently extended to the expression of other eukaryotic proteins...
December 4, 2017: Protein Expression and Purification
James M Rice, Jeromey B Temple, Peter F McDonald
OBJECTIVE: To investigate intergenerational equity in consumption using the Australian National Transfer Accounts (NTA). METHODS: Australian NTA estimates of consumption were used to investigate disparities in consumption between people of different ages and generations in Australia between 1981-1982 and 2009-2010. RESULTS: There is a clear patterning of consumption by age, with the distribution by age of consumption funded by the private sector being very different to that of consumption funded by the public sector...
December 2017: Australasian Journal on Ageing
Jeromey B Temple, James M Rice, Peter F McDonald
OBJECTIVE: To illustrate the use of National Transfer Accounts (NTA) for understanding ageing and the economic life cycle in Australia. METHODS: The NTA methodology is applied utilising a range of unit record, demographic and administrative data sets from 1981 to 2010. RESULTS: During early and later life, total consumption (public and private) is greater than labour income. On a time series and cohort basis, we show that each successive generation has improved their level of well-being (as measured by consumption) relative to the previous years or previous cohorts from 1981 to 1982 onwards...
December 2017: Australasian Journal on Ageing
Yaofang Zhang, Li Du, Yanling Xu, Lin Tian, Peipei Tu, Minggang Li
BACKGROUND: Glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) play a similar but complementary role in the regulation of glucose levels in islet β-cells. This study was aimed to develop a fusion peptide, which combines 4 tandem repeated GLP-1 and 4 tandem repeated GIP (4rolGG), and to investigate its therapeutic effect on type 2 diabetes using a diabetic mice model. METHOD: A 4rolGG expression plasmid was constructed and expressed in BL21 (DE3)...
December 3, 2017: Current Pharmaceutical Biotechnology
Qianni Liu, Meijuan Xu, Rongzhen Zhang, Meizhou Wang, Xian Zhang, Taowei Yang, Zhiming Rao
Arginine deiminase (ADI) was first high-efficient expressed in Corynebacterium crenatum SYPA 5-5. The ADI was purified by Ni-NTA affinity chromatography and SDS-PAGE analysis showed the molecular weight (MW) was 46.8 kDa. The optimal temperature and pH of ADI were 37 ℃ and 6.5 respectively. The Michaelis constant was 12.18 mmol/L and the maximum velocity was 0.36 μmol/(min·mL). Under optimal conditions, 300 g/L of arginine was transformed and the productivity reach 8 g/(L·h). The recombinant strain was cultivated in a 5-L fermentor and used for whole-cell transformation of 300 g/L arginine, under repeated-batch bioconversion, the cumulative production reached 1 900 g/L...
November 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Huawei He, Yejing Wang, Kai Song, Jiao Wang, Shuguang Wei, Peng Zhao, Ping Zhao
Protein tyrosine phosphatase (PTP, EC specifically catalyzes the removal of phosphate groups from phosphorylated tyrosine residues, resulting in protein dephosphorylation, thus regulates life activities such as cell growth, proliferation, differentiation and immunization. Protein tyrosine phosphatase h of Bombyx mori (BmPTP-h) is involved in the replication of nucleopolyhedrovirus (NPV) in Bombyx mori, but the structure and properties of BmPTP-h are little known so far. In this study, the coding sequence of BmPTP-h gene was cloned from the midgut of Bombyx mori, and its genomic structure, mRNA structure, sequence signature, secondary structure and the state in solution were analyzed...
November 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Arsham Banisadr, Yaghoub Safdari, Anvarsadat Kianmehr, Mahdieh Pourafshar
The aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody)were grafted on framework regions derived from type 3 (VH3 and VL3 kappa)human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly4Ser)3]to form a single chain antibody (scFv). Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E...
November 29, 2017: Human Vaccines & Immunotherapeutics
Xiufeng Liu, Xintong Liu, Suwen Sunchen, Meixia Liu, Chen Shen, Juanjuan Wu, Wanli Zhao, Boyang Yu, Jihua Liu
OBJECTIVE: The aim of this research was to develop a novel ALA fusion protein for target to the malignant cells surface with high uPAR expression and locally release of the scorpion toxin AGAP in an uPA-cleavable manner. It will provide an effective approach for controlled release of the peptide toxins to treat cancerous cells. METHODS: The ALA fusion proteins were expressed in pichia pastoris, and the recombinant proteins were purified by Ni-NTA affinity chromatography...
November 2017: Drug Delivery
Jane Marie Alsweiler, Caroline A Crowther, Jane E Harding
INTRODUCTION: Neonatal hypoglycaemia is a common condition that can cause developmental delay. Treatment of neonatal hypoglycaemia with oral dextrose gel has been shown to reverse hypoglycaemia and reduce admissions to neonatal intensive care for hypoglycaemia. An evidence-based clinical practice guideline was written to guide the use of dextrose gel to treat neonatal hypoglycaemia in New Zealand. However, it is unclear what clinical discipline might most effectively lead the implementation of the guideline recommendations...
November 22, 2017: BMJ Open
Pascal Röthlisberger, Fabienne Levi-Acobas, Ivo Sarac, Bruno Baron, Patrick England, Philippe Marlière, Piet Herdewijn, Marcel Hollenstein
Methods for immobilization of DNA on solid supports are in high demand. Herein, we present a generally applicable enzymatic method for the immobilization of DNA without any prior chemical derivatization. This strategy relies on the homopolymerization of the modified triphosphate dImTP by the TdT. The resulting enzymatic his-tag mimic ensures binding of DNA on Ni-NTA agarose. The usefulness of this method is highlighted by the immobilization of functional nucleic acids without impairing their specific activities...
November 22, 2017: Chemical Communications: Chem Comm
Martin E M Parsons, Damien McParland, Paulina B Szklanna, Matthew Ho Zhi Guang, Karen O'Connell, Hugh D O'Connor, Christopher McGuigan, Fionnuala Ní Áinle, Amanda McCann, Patricia B Maguire
Nanoparticle tracking analysis (NTA) can be used to quantitate extracellular vesicles (EVs) in biological samples and is widely considered a useful diagnostic tool to detect disease. However, accurately profiling EVs can be challenging due to their small size and heterogeneity. Here, we aimed to provide a protocol to facilitate high-precision particle quantitation by NTA in plasma, the supernatant of activated purified platelets [the platelet releasate (PR)] and in serum, to increase confidence in NTA particle enumeration...
2017: Frontiers in Cardiovascular Medicine
Mara F Cuebas-Irizarry, Ricardo A Irizarry-Caro, Carol López-Morales, Keyla M Badillo-Rivera, Carlos M Rodríguez-Minguela, Rafael Montalvo-Rodríguez
We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6...
November 21, 2017: Life
Thomas C Davis, Daniel S Jones, Arianna J Dino, Nicholas I Cejda, Jing Yuan, Andrew C Willoughby, Sharon A Kessler
MLOs in Plant Reproduction. The MILDEW RESISTANCE LOCUS-O (MLO) protein family, comprised of 15 members, plays roles in diverse cell-cell communication processes such as powdery mildew susceptibility, root thigmomorphogenesis, and pollen tube reception. The NORTIA (NTA, AtMLO7) gene is expressed in the synergid cells of the female gametophyte where it functions in intercellular communication with the pollen tube. Discrepancies between previously published promoter::GUS and promoter::gene-GUS constructs expression patterns led us to explore the regulation of NTA expression...
November 20, 2017: Plant Reproduction
Saurabh Lal, Richard M Hall, Joanne L Tipper
This article refers to the paper "A novel method for isolation and recovery of ceramic nanoparticles and metal wear debris from serum lubricants at ultra-low wear rates" (Lal et al., 2016) [1] and describes the concentration and size distribution data of silicon nitride nanoparticles measured using nanoparticle tracking analysis (NTA). A NanoSight LM10 instrument was used to capture the video data of silicon nitride nanoparticles moving under Brownian motion in the water. The video data was then analyzed using the NanoSight NTA software...
December 2017: Data in Brief
Yang Zhou, Dandan Yan, Shaofei Yuan, Yawen Chen, Emmanuella E Fletcher, Haifeng Shi, Bangxing Han
In previous studies, we synthesized the magnetic core-shell structured Fe3O4/PMG/IDA-Ni(2+) nanoparticles. The Ni(2+) on the surface of nanoparticles provides abundant docking sites for histidine, and the composite nanoparticles showed potential applications in the separation and purification of histidine-tagged (His-tagged) proteins. Meanwhile, the presence of the superparamagnetic core (Fe3O4) in the nanoparticles allows them to be quickly separated and purified by an external magnetic field. Herein, the ability of magnetic nanoparticles to purify His-tagged human superoxide dismutase 1 (hSOD1) was verified...
November 15, 2017: Protein Expression and Purification
Lara A B C Carneiro, Richard J Ward
A paramagnetic nanocomposite coated with chitosan and N-(5-Amino-1-carboxy-pentyl) iminodiacetic acid (NTA) that is suitable for protein immobilization applications has been prepared and characterized. The nanoparticle core was synthesized by controlled aggregation of Fe3O4 under alkaline conditions, and Transmission Electron Microscopy revealed a size distribution of 10-50 nm. The nanoparticle core was coated with chitosan and derivatized with glutaraldehyde and NTA, as confirmed by Fourier Transform Infrared Spectroscopy...
November 18, 2017: Analytical Biochemistry
Suveen Kumar, Ashish, Saurabh Kumar, Shine Augustine, Santosh Yadav, Birendra Kumar Yadav, Rishi Pal Chauhan, Ajay Kumar Dewan, Bansi Dhar Malhotra
We report results of the studies relating to fabrication of nanostructured metal oxide (NMO) based cancer biosensor. With the help of 2D electroactive reduced graphene oxide (RGO), we successfully inhibited the Brownian motion of NMO that led to reduced agglomeration of NMO. The nanostructured hafnium oxide (nHfO2) was used as a model NMO. The reduced agglomeration of nHfO2 was achieved through controlled hydrothermal synthesis and investigated via nanoparticles tracking analysis (NTA). X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscope (TEM) techniques were used for phase identification as well as morphological analysis of the synthesized nanohybrid (nHfO2@RGO) material...
November 10, 2017: Biosensors & Bioelectronics
Alexander Jacobs, Haley Hoover, Edward Smith, David E Clemmer, Chul-Hyun Kim, C Cheng Kao
In the brome mosaic virus (BMV) virion, the coat protein (CP) selectively contacts the RNA motifs that regulate translation and RNA replication (Hoover et al., 2016. J. Virol. 90, 7748). We hypothesize that the unstructured N-terminal arm (NTA) of the BMV CP can specifically recognize RNA motifs. Using ion mobility spectrometry-mass spectrometry, we demonstrate that peptides containing the NTA of the CP were found to preferentially bind to an RNA hairpin motif that directs the initiation of BMV RNA synthesis...
November 11, 2017: Nucleic Acids Research
Md Reyazul Islam, Nan-Sun Kim, Jae-Wan Jung, Hyo-Boon Kim, So-Chon Han, Moon-Sik Yang
A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis...
January 2018: Enzyme and Microbial Technology
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