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Koji Kosaka, Keiko Ohkubo, Michihiro Akiba
The occurrence of six haloacetamides (HAcAms), which are a group of emerging nitrogenous disinfection byproducts, was investigated in drinking water across Japan in September 2015 and February 2016. At least one of the six HAcAms were found in all of the drinking water samples and their total concentrations ranged from 0.3 to 3.8 μg/L. The detection frequencies and concentrations of 2,2-dichloroacetamide (DCAcAm) and 2-bromo-2-chloroacetamide (BCAcAm) were the largest among the targeted HAcAm species. The total HAcAm concentrations in the raw water after chlorination ranged from 0...
October 13, 2016: Water Research
Masayuki Tsuzuki, Yuichiro Watanabe
Small RNAs are key molecules in RNA silencing pathways that exert the sequence-specific regulation of gene expression and chromatin modifications in many eukaryotes. In plants, endogenous small RNAs, including microRNAs (miRNAs), trans-acting short interfering RNAs (tasiRNAs), and heterochromatic siRNAs (hc-siRNAs), play an important role in switching or orchestrating biological processes during the development and at the onset of stress responses. These endogenous and exogenous small RNAs are mainly 20-24 nucleotides in length...
2017: Methods in Molecular Biology
Zhuqing Leslie Li, Yonghui Shi, Yinyi Ding, Yumei Ran, Guowei Le
Oxidized tyrosine (O-Tyr) products have been detected in commercial food and have been demonstrated to induce liver injury in our previous study, but the precise mechanisms of the impact induced by dietary O-Tyr are still unclear. Kidney plays an important role in the metabolism of protein. Accumulation of O-Tyr products, especially the dityrosine (Dityr) and advanced oxidation protein products (AOPPs), in vivo was shown to be associated with many kidney diseases. Therefore, this study determined whether chronic exposure to dietary O-Tyr impaired renal function in rats...
October 21, 2016: Amino Acids
Changliang Zhu, Jingliang Zhang, Jing Zhang, Yanhui Jiang, Zhaopeng Shen, Huashi Guan, Xiaolu Jiang
An extracellular chondroitinase ABC (ChSase ABC, EC produced by cultivating Acinetobacter sp. C26, was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q-Sepharose Fast Flow and Sephadex G-100 chromatography. The 76kDa enzyme was purified 48.09-fold to homogeneity with specific activity of 348.64U/mg, Using the chondroitin sulfate A (CS-A) as substrate, the maximal reaction rate (Vmax) and Michaelis-Menten constant (Km) of ChSase ABC were found to be 10.471μmol/min/ml and 0...
October 18, 2016: International Journal of Biological Macromolecules
Alexandra McCarron, Martin Donnelley, Chantelle McIntyre, David Parsons
Lentiviruses are becoming an increasingly popular choice of gene transfer vehicle for use in the treatment of a variety of genetic and acquired human diseases. As research progresses from basic studies into pre-clinical and clinical phases, there is a growing demand for large volumes of high purity, concentrated vector, and accordingly, the means to produce such quantities. Unlike other viral vectors, lentiviruses are difficult to produce using stable cell lines, therefore transient transfection of adherent cell lines is conventionally used, and this method has proven challenging to up-scale...
October 18, 2016: Journal of Biotechnology
Rushd Khalaf, Bertrand Coquebert de Neuville, Massimo Morbidelli
Ion exchange chromatography materials functionalized with polyelectrolyte brushes (PEB) are becoming an integral part of many protein purification steps. Adsorption onto these materials is different than that onto traditional materials, due to the 3D partitioning of proteins into the polyelectrolyte brushes. Despite this mechanistic difference, many works have described the chromatographic behavior of proteins on polyelectrolyte brush type ion exchangers with much of the same methods as used for traditional materials...
October 13, 2016: Journal of Chromatography. A
Donatella Nardiello, Clelia Prattichizzo, Maria Teresa Rocchetti, Loreto Gesualdo, Diego Centonze
A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis...
October 12, 2016: Journal of Pharmaceutical and Biomedical Analysis
Jialin Zhang, Wenxing Liu, Weiye Chen, Cuicui Li, Meimei Xie, Zhigao Bu
From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations...
2016: PloS One
Syed T Ahmed, Fabio Parmeggiani, Nicholas J Weise, Sabine L Flitsch, Nicholas J Turner
Current routes to nitrogen-containing heteroarylalanines involve complex multistep synthesis and are often reliant on protection/deprotection steps and wasteful chromatographic purifications. In order to complement existing methodologies, a convenient telescopic strategy was developed for the synthesis of l-pyridylalanine analogues (12 examples) and other l-heteroarylalanines (5 examples) starting from the corresponding aldehydes. A phenylalanine ammonia lyase (PAL) from Anabaena variabilis was used as the biocatalyst to give conversions ranging between 88 and 95%, isolated yields of 32-60%, and perfect enantiopurity (>99% ee) by employing an additional deracemization cascade where necessary...
October 21, 2016: Organic Letters
Jean-Rene Arseneau, Royce Steeves, Mark Laflamme
The increasing use of high-throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that co-precipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often co-precipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low-salt CTAB (MoLSC) extraction protocol to accommodate contaminant rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle...
October 21, 2016: Molecular Ecology Resources
Purnandhu Bose, Pradip K Sukul, Omar M Yaghi, Kentaro Tashiro
We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications...
October 8, 2016: Journal of Visualized Experiments: JoVE
Gunaseelan Narayanan, Yuan Hong Yu, Muly Tham, Hui Theng Gan, Srinivas Ramasamy, Shvetha Sankaran, Srivats Hariharan, Sohail Ahmed
Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages - astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres...
October 4, 2016: Journal of Visualized Experiments: JoVE
Nitzan Samra, Yoav Arava
RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo...
September 30, 2016: Journal of Visualized Experiments: JoVE
Jongho Jeon, Ha Eun Shim, Sajid Mushtaq, Mi Hee Choi, Sang Hyun Park, Dae Seong Choi, Beom-Su Jang
Here, we demonstrate a detailed protocol for the radiosynthesis of a (125)I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [(125)I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified (125)I-labeled azide (1) was obtained with high radiochemical yield (75 ± 10%, n = 8) and excellent radiochemical purity (>99%)...
October 10, 2016: Journal of Visualized Experiments: JoVE
Erwann Le Gendre, Erwan Martin, Benoit Villemant, Pierre Cartigny, Nelly Assayag
RATIONALE: The O- and S- isotope compositions of sulfates can be used as key tracers of the fate and sink of sulfate in both terrestrial and extra-terrestrial environments. However, their application remains limited in those geological systems where sulfate occurs in low concentrations. Here we present a simple and reliable method to extract, purify and concentrate sulfate from natural samples. The method allows us to take into account the separation of nitrate, which is known to be an issue in O-isotope analysis...
October 21, 2016: Rapid Communications in Mass Spectrometry: RCM
Neha Kumari, Rajesh Kumar, Vandana Mishra, Savita Yadav
CC-NBS-LRR (CNL) plant proteins are related with highly conserved family of disease resistance protein distinguished by a coiled-coil domain, which plays an important role in innate immunity. The present study reports the purification and identification of CNL like protein fragment (CNL-LPF) by two step chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/MS), respectively. Furthermore, current study also illustrated the development of polyclonal antibody against purified CNL-LPF, which was used for immunolocalization of CNL-LPF in cytoplasm of cotyledon, using Fluorescence microscopy and Transmission electron microscopy...
October 20, 2016: Protein Journal
Yi Ma, Jieying Yu, Jinglian Lin, Shaomin Wu, Shan Li, Jufang Wang
Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10...
2016: BioMed Research International
Parvin Akbarzadehlaleh, Mona Mirzaei, Mahdiyeh Mashahdi-Keshtiban, Karim Shamsasenjan, Hamidreza Heydari
Human serum albumin (HSA) is a non-glycosylated, negatively charged protein (Mw: about 65-kDa) that has one free cystein residue (Cys 34), and 17 disulfide bridges that these bridges have main role in its stability and longer biological life-time (15 to 19 days). As HSA is a multifunctional protein, it can also bind to other molecules and ions in addition to its role in maintaining colloidal osmotic pressure (COP) in various diseases. In critical illnesses changes in the level of albumin between the intravascular and extravascular compartments and the decrease in its serum concentration need to be compensated using exogenous albumin; but as the size of HSA is an important parameter in retention within the circulation, therefore increasing its molecular size and hydrodynamic radius of HSA by covalent attachment of poly ethylene glycol (PEG), that is known as PEGylation, provides HSA as a superior volume expander that not only can prevent the interstitial edema but also can reduce the infusion frequency...
September 2016: Advanced Pharmaceutical Bulletin
Ravely Casarotti Orlandelli, Maria de Lourdes Corradi da Silva, Ana Flora Dalberto Vasconcelos, Igor Vivian de Almeida, Veronica Elisa Pimenta Vicentini, Alicia Prieto, Maria Dolores Diaz Hernandez, João Lúcio de Azevedo, João Alencar Pamphile
This study reports the characterization and antiproliferative activity of exopolysaccharides (EPS) produced by submerged cultures of the endophytes Diaporthe sp. JF766998 and Diaporthe sp. JF767007 isolated from the medicinal plant Piper hispidum Sw. Both strains secreted a crude EPS that, upon size exclusion chromatography, showed to contain a heteropolysaccharide (galactose, glucose and mannose) and a high-molecular weight glucan. Data from methylation analysis, FTIR and NMR spectroscopy ((1)H, COSY, TOCSY and HSQC-DEPT) indicated that the purified glucan consisted of a main chain of glucopyranosyl β-(1→3) linkages substituted at O-6 by glucosyl residues...
October 17, 2016: International Journal of Biological Macromolecules
Folasade M Olajuyigbe, Cornelius O Fatokun
Functionality of enzymes within narrow pH range and temperature is a major challenge which limits their industrial applications, hence, there is need to search for thermostable pH-versatile enzymes. Here, a novel thermostable pH-versatile laccase from Sporothrix carnis CPF-05 was purified by ion-exchange and gel filtration chromatography. Single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed homogeneity of the enzyme with molecular weight of 56kDa. Enzyme yield was 3...
October 17, 2016: International Journal of Biological Macromolecules
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