Indirect ultrasonication for protein quantification and peptide mass mapping through mass spectrometry-based techniques

We report in this work a fast protocol for protein quantification and for peptide mass mapping that rely on ¹⁸O isotopic labeling through the decoupling procedure. It is demonstrated that the purity and source of trypsin do not compromise the labeling degree and efficiency of the decoupled labeling reaction, and that the pH of the labeling reaction is a critical factor to obtain a significant ¹⁸O double labeling. We also show that the same calibration curve can be used for MALDI protein quantification during several days maintaining a reasonable accuracy, thus simplifying the handling of the quantification process. In addition we demonstrate that ¹⁸O isotopic labeling through the decoupling procedure can be successfully used to elaborate peptide mass maps. BSA was successfully quantified using the same calibration curve in different days and plasma from a freshwater fish, Cyprinus carpio, was used to elaborate the peptide mass maps.     

Structures of the reduced niobium oxides Nb12O29 and Nb22O54

The crystal structure of Nb₂₂O₅₄ is reported for the first time, and the structure of orthorhombic Nb₁₂O₂₉ is reexamined, resolving previous ambiguities. Single crystal X-ray and electron diffraction were employed. These compounds were found to crystallize in the space groups P2/m (, , , β=102.029(3)^°) and Cmcm (, , ), respectively and share a common structural unit, a 4×3 block of corner sharing NbO₆ octahedra. Despite different constraints imposed by symmetry these blocks are very similar in both compounds. Within a block, it is found that the niobium atoms are not located in the centers of the oxygen octahedra, but rather are displaced inward toward the center of the block forming an apparent antiferroelectric state. Bond valence sums and bond lengths do not show the presence of charge ordering, suggesting that all 4d electrons are delocalized in these compounds at the temperature studied, T=200 K.     

A combination of modified particle swarm optimization algorithm and support vector machine for gene selection and tumor classification

In the analysis of gene expression profiles, the number of tissue samples with genes expression levels available is usually small compared with the number of genes. This can lead either to possible overfitting or even to a complete failure in analysis of microarray data. The selection of genes that are really indicative of the tissue classification concerned is becoming one of the key steps in microarray studies. In the present paper, we have combined the modified discrete particle swarm optimization (PSO) and support vector machines (SVM) for tumor classification. The modified discrete PSO is applied to select genes, while SVM is used as the classifier or the evaluator. The proposed approach is used to the microarray data of 22 normal and 40 colon tumor tissues and showed good prediction performance. It has been demonstrated that the modified PSO is a useful tool for gene selection and mining high dimension data.     

Cross‐Platform DNA Encoding for Single‐Cell Imaging of Gene Expression

Integration of imaging data across different molecular target types can provide in‐depth insight into cell physiology and pathology, but remains challenging owing to poor compatibility between target‐type‐specific labeling methods. We show that cross‐platform imaging analysis can be readily achieved through DNA encoding of molecular targets, which translates the molecular identity of various target types into a uniform in situ array of ssDNA tags for subsequent labeling with complementary imaging probes. The concept was demonstrated through multiplexed imaging of mRNAs and their corresponding proteins with multicolor quantum dots. The results reveal heterogeneity of cell transfection with siRNA and outline disparity in RNA interference (RNAi) kinetics at the level of both the mRNA and the encoded protein.     

Differential display identifies genes in chinese hamster ovary cells sensitive to elevated ammonium

Ammonium is a toxic waste product that has been reported to negatively inhibit cell growth and recombinant glycosylation in Chinese hamster ovary (CHO) cells; however, the effect of this toxicity on intracellular gene expression has received only limited investigation. We used a differential display method to identify genes in CHO cells that were affected by ammonium stress. Eight genes whose mRNA levels significantly changed in response to elevated ammonium were isolated and identified. Five of the genes were identified as having lower expression under the ammonium stress, whereas three genes were identified as having higher expression. Sequence homology with other mammalian organisms was used to attribute function to these newly identified genes. The identified ammonium-sensitive genes were grouped into three broad functional groups: cellular processes, energy metabolism, and genetic-information processing. The three cellular process-related genes had lower expression (anaphase-promoting complex subunit 5, eukaryotic initiation factor 5A II, KIAA1091 protein). The two energy-related genes had higher expression under ammonium stress (adenosine triphosphate synthase subunit C and mitofusin 1). Both of the genetic information-processing genes (endoplasmic reticulum [ER]-resident protein ERdj5 and structure-specific recognition protein 1) had lower expression under the ammonium stress, whereas the 26S proteasome subunit adenosine triphosphatase 3 gene had higher expression. These preliminary results indicate that ammonium stress lowers expression of genes controlling cell cycle, protein folding, and quality and raises genes that control energy metabolism and degradation. Our findings demonstrate the usefulness of mRNA differential-display techniques for the detection of CHO cell genes affected by ammonium stress.     

Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

Phosphatase of regenerating liver 3(PRL3),which belongs to the superfamily of protein tyrosine phosphatases(PTPs),represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes.Presented here are the results of cloning,prokaryotic expression,purification,and polyclonal antibody preparation of PRL3.To obtain a specific polyclonal antibody against PRL3,the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns.Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein.The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.     

A genetically encoded metallocene containing amino acid

Redox active amino acids, cofactors, and metal ions are involved in a large number of catalytic, electron transfer, and regulatory processes in biology. Consequently, the ability to engineer redox active centers at defined sites in proteins would facilitate both the study and manipulation of a wide range of biological processes. Recently, we demonstrated that the redox active amino acid 3,4-dihydroxyphenylalanine could be efficiently and selectively incorporated into proteins in Escherichia coli using a nonsense codon and a corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. We now report that ferrocene derivative 1 can be genetically encoded in Saccharomyces cerevisiae (S. cerevisiae) in good yield in response to the amber codon, TAG.     

Ionic liquid-assisted electrochemical determination of pyrimethanil using reduced graphene oxide conjugated to flower-like NiCo2O4

The novel hierarchical flower-like superstructure NiCo₂O₄/reduced graphene oxide (rGO) hybrids have been successfully synthesized with a facile one-step hydrothermal process for the determination of fungicide pyrimethanil (PMT). For comparison, various structures of NiCo₂O₄/rGO including hexagonal nanoplates and nanorods were also synthesized. Among them, three-dimensional (3D) flower-like NiCo₂O₄/rGO exhibited the highest electrocatalytic activity for the oxidation of PMT. With the synergistic effect of [OMIM]PF₆ ionic liquid (IL), the electrochemical sensor film (NiCo₂O₄/rGO/IL) further facilitated interfacial electron transfer and enhanced electrocatalytic activity for the oxidation of PMT. Under the optimum conditions, the electrochemical sensor exhibited two linear ranges of 0.1–10.0 μmol/L and 20.0–140 μmol/L for PMT with a low detection concentration of 11.0 nmol/L. Besides, the interference, repeatability, reproducibility and stability measurements were also evaluated. The proposed method was successfully applied to the detection of PMT in water, seawater, fruits and vegetables with good recovery ranging from 93% to 105%, and possessed potential applications in the analysis of real samples.