Tunable Surface-Enhanced Raman Spectroscopy via Plasmonic Coupling Between Nanodot-Arrayed Ag Film and Ag Nanocube

Herein, a flexible surface-enhanced Raman spectroscopy (SERS) substrate composed of nanodot-arrayed Ag film and Ag nanocubes was fabricated through a simple method. The large-area nanodot-arrayed Ag film was produced at low cost and high reproducibility. The experimental results show that the coupled structure produces a much stronger SERS signal than the Ag nanocube alone and the isolated nanodot-arrayed Ag film. Furthermore, the coupling effect is sensitive to geometrical parameter of the period of the dot-array. Numerical simulations are performed to verify the electric field enhancement of the composite SERS substrate and support the experimental results.     

Atomic Model of Rabbit Hemorrhagic Disease Virus by Cryo-Electron Microscopy and Crystallography

Rabbit hemorrhagic disease, first described in China in 1984, causes hemorrhagic necrosis of the liver. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. The detailed molecular structure of any lagovirus capsid has yet to be determined. Here, we report a cryo-electron microscopic (cryoEM) reconstruction of wild-type RHDV at 6.5 Å resolution and the crystal structures of the shell (S) and protruding (P) domains of its major capsid protein, VP60, each at 2.0 Å resolution. From these data we built a complete atomic model of the RHDV capsid. VP60 has a conserved S domain and a specific P2 sub-domain that differs from those found in other caliciviruses. As seen in the shell portion of the RHDV cryoEM map, which was resolved to ∼5.5 Å, the N-terminal arm domain of VP60 folds back onto its cognate S domain. Sequence alignments of VP60 from six groups of RHDV isolates revealed seven regions of high variation that could be mapped onto the surface of the P2 sub-domain and suggested three putative pockets might be responsible for binding to histo-blood group antigens. A flexible loop in one of these regions was shown to interact with rabbit tissue cells and contains an important epitope for anti-RHDV antibody production. Our study provides a reliable, pseudo-atomic model of a Lagovirus and suggests a new candidate for an efficient vaccine that can be used to protect rabbits from RHDV infection.     

Potent Antitumor Activity Generated by a Novel Tumor Specific Cytotoxic T Cell

Hepatocellular carcinoma is one of the most common malignant neoplasms in the world and is the main cause of death in patients with liver cirrhosis. Surgical intervention is not suitable for majority of hepatocellular carcinoma. Investigation of the effective targeting to the tumor cells is essential for both primary tumors and metastases. Tumor specific cytotoxic T lymphocytes (CTL) have been considered to be the attractive vehicles for delivering therapeutic agents toward various tumor diseases. This study was to explore the distribution pattern of CTL carrying the lentiviral vectors with the characteristic of adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand promoter (Lenti/hCD40L/E1AB). Following transduction with adenoviral vectors containing chimeric type 5 and type 35 fiber proteins (Ad5/35-TRAIL), these CTLs produced infectious virus when exposed to HepG2 cells. We assessed the therapeutic ability of CTLs using MTT, Western blot and colony formation assay. The novel CTL harboring Lenti/hCD40L/E1AB and Ad5/35-TRAIL caused proliferation inhibition and significant apoptosis in hepatocellular carcinoma cell lines. Thus, the novel CTL may be useful for the development of gene therapy approaches to hepatocellular carcinoma.     

miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha ^loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.     

Low Divergence of Clonorchis sinensis in China Based on Multilocus Analysis

Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb]) was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of C. sinensis.     

A Role for Innexin2 and Innexin3 Proteins from Spodoptera litura in Apoptosis

Gap junctions formed by two hemichannels from two neighboring cells are cell-to-cell communication channels; hemichannels are communication channels between intracellular and extracellular environments. Hemichannels are hexameric proteins formed by connexins, pannexins, innexins and vinnexins. Innexin-hemichannels (innexons) exist in the lepidopteran cell surface, but their component innexins and functions have not been reported. Recent studies by others have demonstrated that hemichannels, connexons and pannexons from vertebrates serve as regulators of apoptosis via inactivating the PI3K/Akt signaling pathway. Here, the apoptogenic properties of innexons are demonstrated using two innexin cDNAs, Spli-inx2 and Spli-inx3, which were isolated from hemocytes of lepidopteran Spodoptera litura. Alignment analysis revealed that these two genes belong to a conserved innexin family, as they contain the insect signature YYQWV motif at the beginning of the second transmembrane domain. Immunofluorescence showed that two fusion proteins, Inx2-V5 and Inx3-V5, were localized predominantly in the cell membrane, cytoplasm and also nuclei. Ectopic expression in Sf9 cells and over-expression of Inx2 and Inx3 in Spli221 cells promoted apoptosis. In the Spli221 cells, apoptotic cells presented remarkable membrane blebbing. This study also showed that Sf9 and Spli221 cells undergo low level apoptosis under normal culture conditions, but not Hi5 cells. In Hi5 stable cell lines, biotinylation was used to isolate surface proteins and confirm Inx2 and Inx3 localization in the cell membrane and also further data showed that Hi5 cells may activate the PI3K signaling pathway via phosphorylating molecular Akt downstream. This result suggests that innexon-promoted apoptosis may be involving the PI3K/Akt signaling pathway. These findings will facilitate further examinations of the apoptotic regulation by the PI3K/Akt signaling pathway and comparative studies of innexons, connexons, pannexons, and vinnexons.     

P16 and P53 Play Distinct Roles in Different Subtypes of Breast Cancer

Breast cancers are heterogeneous and complex diseases, and subtypes of breast cancers may involve unique molecular mechanisms. The p16^INK4a and p53 pathways are two of the major pathways involved in control of the cell cycle. They also play key roles in tumorigenesis. However, whether the roles of these pathways differ in the subtypes of breast cancer is unclear. Therefore, p16 and p53 expression were investigated in different breast cancer subtypes to ascertain their contributions to these cancers. A total of 400 cases of non-invasive ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC), including the major molecular subtypes luminal-A, luminal-B, Her-2, and triple-negative subtypes, and 50 cases of normal controls were compared. Luminal-A cancers expressed the lowest level of p16 among the subtypes in DCIS, and the level of p16 expression was up-regulated in the luminal-A of IDC (P<0.008). Triple-negative breast cancers were characterized by a correlation of p53 overexpression with a high level of p16 expression. Luminal lesion types with high p16 expression in DCIS were found to be more likely to develop into aggressive breast cancers, possibly promoted by p53 dysfunction. Taken together, the present study suggest that p16 expression in luminal-A breast cancers is associated with their progression from DCIS to IDC, and both p53 and p16 expressions are important for the development of triple-negative breast cancers in DCIS and IDC.     

Anti-Arrhythmic Effect of Verapamil Is Accompanied by Preservation of Cx43 Protein in Rat Heart

The present study was to test the hypothesis that anti-arrhythmic properties of verapamil may be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of verapamil. Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein.