Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.     

Dynamic response of HPV16/anti-HPV16 pairs with unbinding events studied by atomic force microscopy

This paper proposes an effective approach to distinguish whether samples include Human Papilloma virus type-16 (HPV16) by Atomic force microscopy (AFM). AFM is an important instrument in nanobiotechnology field. At first we identified the HPV16 by Polymerase chain reaction (PCR) analysis and Western blotting from specimen of the HPV patient (E12) and the normal (C2), and then we used an AFM to observe the surface ultrastructure by tapping mode and to measure the unbinding force between HPV16 coupled to an AFM tip and anti-HPV16 L1 coated on the substrate surface by contact mode. The experimental results by tapping mode show that the size of a single HPV viron was similar to its SEM image from the previous literatures; moreover, based on the purposed methods and the analysis, two obvious findings that we can determine whether or not the subject is a HPV patient can be derived from the results; one is based on the distribution of unbinding forces, and the other is based on the distribution of the stiffness. Furthermore, the proposed method could be a useful technique for further investigating the potential role among subtypes of HPVs in the oncogenesis of human cervical cancer.     

Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis

K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15–20% of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1–3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy.     

Graphene oxide supported rhombic dodecahedral Cu2O nanocrystals for the detection of carcinoembryonic antigen

In this work, a simple electrochemical immunosensor was developed for the detection of carcinoembryonic antigen (CEA) based on rhombic dodecahedral Cu₂O nanocrystals–graphene oxide–gold nanoparticles (rCu₂O–GO–AuNPs). GO as the template and surfactant resulting in rCu₂O exhibit improved rhombic dodecahedral structure uniformity and excellent electrochemical performance. Moreover, GO was found to be able to effectively improve the long stability of rCu₂O on the electrode response. Under optimal conditions, the immunosensor showed a low limit of detection (0.004 ng ml⁻¹) and a large linear range (0.01–120 ng ml⁻¹). This work presents a potential alternative for the diagnostic applications of GO-supported special morphology materials in biomedicine and biosensors.     

Graphene quantum dots as additives in capillary electrophoresis for separation cinnamic acid and its derivatives

A facile capillary electrophoresis (CE) method for the separation of cinnamic acid and its derivatives (3,4-dimethoxycinnamic acid, 4-methoxycinnamic acid, isoferulic acid, sinapic acid, cinnamic acid, ferulic acid, and trans-4-hydroxycinnamic acid) using graphene quantum dots (GQDs) as additives with direct ultraviolet (UV) detection is reported. GQDs were synthesized by chemical oxidization and further purified by a macroporous resin column to remove salts (Na₂SO₄ and NaNO₃) and other impurities. Transmission electron microscopy (TEM) indicated that GQDs have a relatively uniform particle size (2.3 nm). Taking into account the structural features of GQDs, cinnamic acid and its derivatives were adopted as model compounds to investigate whether GQDs can be used to improve CE separations. The separation performance of GQDs used as additives in CE was studied through variations of pH, concentration of the background electrolyte (BGE), and contents of GQDs. The results indicated that excellent separation can be achieved in less than 18 min, which is mainly attributed to the interaction between the analytes and GQDs, especially isoferulic acid, sinapic acid, and cinnamic acid.     

Genomic constitution and taxonomy of the Chinese hexaploids Elymus cylindricus and E. breviaristatus (Poaceae: Triticeae)

Elymus cylindricus (2n = 6x = 42) and E. breviaristatus (2n = 6x = 42) are distributed in grasslands and deserts of northern and north‐western China. Their genomic constitution and taxonomic status are unclear. Elymus cylindricus was crossed with E. wawawaiensis J.R.Carlson & Barkworth ( StH ), Roegneria grandis Keng ( StY ) and Campeiostachys dahurica (Turcz. ex Griseb.) B.R.Baum, J.L. Y ang & C. Y en var. dahurica ( StYH ). Meiotic pairing in the hybrids E. cylindricus × E. wawawaiensis ( StH ), E. cylindricus × R. grandis ( StY ) and E. cylindricus × C. dahurica var. dahurica ( StYH ) showed on average 10.00, 11.30 and 20.92 bivalents per cell, respectively. Elymus breviaristatus was crossed with C. dahurica var. dahurica ( StYH ) and E. cylindricus. Chromosome pairing in the hybrids of E. breviaristatus × C. dahurica var. dahurica and E. breviaristatus × E. cylindricus showed on average 19.60 and 19.27 bivalents, respectively. Genomic in situ hybridization (GI SH ) revealed the presence of St , Y and H genomes in E. cylindricus and E. breviaristatus. An intergenomic rearrangement was observed in E. cylindricus using GI SH . Meiotic pairing data and GI SH indicated that both E. cylindricus and E. breviaristatus are allohexaploids containing the StYH genomes. Elymus cylindricus and E. breviaristatus should be treated as Campeiostachys dahurica var. cylindrica and Campeiostachys breviaristata, respectively.