A new insight into the impact of different proteases on SILAC quantitative proteome of the mouse liver

In this study, we examined the use of multiple proteases (trypsin, LysC, tandem LysC/trypsin) on both protein identification and quantification in the Lys‐labeled SILAC mouse liver. Our results show that trypsin and tandem LysC/trypsin digestion are superior to LysC in peptides and protein identification while LysC shows advantages in quantification of Lys‐labeled proteins. Combination of experimental results from different proteases (LysC and trypsin) enabled a significant increase in the number of identified protein and protein can be quantified. Thus, taking advantage of the complementation of different protease should be a good strategy to improve both qualitative and quantitative proteomics research.     

Electrochemical surface modification of carbon mesh anode to improve the performance of air-cathode microbial fuel cells

A convenient and promising alternative to surface modification of carbon mesh anode was fulfilled by electrochemical oxidation in the electrolyte of nitric acid or ammonium nitrate at ambient temperature. It was confirmed that such an anode modification method was low cost and effective not only in improving the efficiency of power generation in microbial fuel cells (MFCs) for synthetic wastewater treatment, but also helping to reduce the period for MFCs start-up. The MFCs with anode modification in electrolyte of nitric acid performed the best, achieving a Coulombic efficiency enhancement of 71 %. As characterized, the electrochemical modification resulted in the decrease of the anode potential and internal resistance but the increase of current response and nitrogen-containing and oxygen-containing functional groups on the carbon surface, which might contribute to the enhancement on the performances of MFCs.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Systematic research on the pretreatment of peptides for quantitative proteomics using a C18 microcolumn

Reversed phase microcolumns have been widely used for peptide pretreatment to desalt and remove interferences before tandem LC–MS in proteomics studies. However, few studies have characterized the effects of experimental parameters as well as column characteristics on the composition of identified peptides. In this study, several parameters including the concentration of ACN in washing buffer, the microcolumn's purification effect, the peptide recovery rate, and the dynamic‐binding capacity were characterized in detail, based upon stable isotope labeling by amino acids in a cell culture quantitative approach. The results showed that peptide losses can be reduced with low ACN concentration in washing buffers resulting in a recovery rate of approximately 82%. Furthermore, the effects of ACN concentration and loading amount on the properties of identified peptides were also evaluated. We found that the dynamic‐binding capacity of the column was approximately 26 μg. With increased loading amounts, more hydrophilic peptides were replaced by hydrophobic peptides.     

Negative Refraction and Focusing of Photonic Crystals with Graded Negative Index in Visible Regime

In this paper, we designed a hexagonal lattice photonic crystal (PC) which presents negative refraction behavior in the broadband visible region. By varying the PC parameters, a graded index PC was obtained for the purpose of focusing a plane wave with large transmission. Finite-difference and time-domain algorithm-based numerical calculation was adopted to demonstrate the negative refraction and analyze the focusing effect. Calculation results demonstrate that the designed PCs have good focusing property together with large transmission. The proposed structures provide an approach for designing the negative refraction-based imaging systems.     

Plasmonic Spectral Detection of Carcinoembryonic Antigen by Preventing the Direct Binding of Rhodamine 6G with Au Nanoparticles

Carcinoembryonic antigen (CEA) was used as a separator to prevent the Rhodamine 6G (R6G)-induced aggregation of colloidal gold nanoparticles. The destroyed aggregation has been monitored by measuring the absorption and resonance light scattering peaks corresponding to the longitudinal surface plasmon resonance (SPR) of the chain-like aggregated gold nanoparticles (AuNPs). It was found that the pre-adding of CEA with different concentrations to the gold colloids before mixing them with R6G could lead to the longitudinal SPR peak decrease and blue shift. By analysing the intensity changing and wavelength shifting of the absorption spectra, CEA could be detected in a linear range from 0.2 to 4 ng/mL, and the limit of detection reaches to 0.1 ng/mL. The sensitivity of the CEA concentration dependent shifting and quenching of the plasmonic absorption and scattering corresponding to the AuNPs aggregation presents a well potential application of biologic spectral sensing.     

Epigenetic regulation of DACH1, a novel Wnt signaling component in colorectal cancer

Colorectal cancer (CRC) is one of the common malignant tumors worldwide. Both genetic and epigenetic changes are regarded as important factors of colorectal carcinogenesis. Loss of DACH1 expression was found in breast, prostate, and endometrial cancer. To analyze the regulation and function of DACH1 in CRC, 5 colorectal cancer cell lines, 8 cases of normal mucosa, 15 cases of polyps and 100 cases of primary CRC were employed in this study. In CRC cell lines, loss of DACH1 expression was correlated with promoter region hypermethylation, and re-expression of DACH1 was induced by 5-Aza-2'-deoxyazacytidine treatment. We found that DACH1 was frequently methylated in primary CRC and this methylation was associated with reduction in DACH1 expression. These results suggest that DACH1 expression is regulated by promoter region hypermethylation in CRC. DACH1 methylation was associated with late tumor stage, poor differentiation, and lymph node metastasis. Re-expression of DACH1 reduced TCF/LEF luciferase reporter activity and inhibited the expression of Wnt signaling downstream targets (c-Myc and cyclinD1). In xenografts of HCT116 cells in which DACH1 was re-expressed, tumor size was smaller than in controls. In addition, restoration of DACH1 expression induced G2/M phase arrest and sensitized HCT116 cells to docetaxel. DACH1 suppresses CRC growth by inhibiting Wnt signaling both in vitro and in vivo. Silencing of DACH1 expression caused resistance of CRC cells to docetaxel. In conclusion, DACH1 is frequently methylated in human CRC and methylation of DACH1 may serve as detective and prognostic marker in CRC.     

Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.     

KSHV MicroRNAs Mediate Cellular Transformation and Tumorigenesis by Redundantly Targeting Cell Growth and Survival Pathways

Kaposi''s sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi''s sarcoma, primary effusion lymphoma and multicentric Castleman''s disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.