Detection of Cytomegalovirus Antibodies Using a Biosensor Based on Imaging Ellipsometry

Background

Cytomegalovirus (CMV) is the most common infectious cause of mental disability in newborns in developed countries. There is an urgent need to establish an early detection and high-throughput screening method for CMV infection using portable detection devices.

Methods

An antibody analysis method is reported for the detection and identification of CMV antibodies in serum using a biosensor based on high spatial resolution imaging ellipsometry (BIE). CMV antigen (CMV-3A) was immobilized on silicon wafers and used to capture CMV antibodies in serum. An antibody against human immunoglobulin G (anti-IgG) was used to confirm the IgG antibody against CMV captured by the CMV-3A.

Results

Our results show that this assay is rapid and specific for the identification of IgG antibody against CMV. Further, patient serum was quantitatively assessed using the standard curve method, and the quantitative results were in agreement with the enzyme-linked immunosorbent assay. The CMV antibody detection sensitivity of BIE reached 0.01 IU/mL.

Conclusions

This novel biosensor may be a valuable diagnostic tool for analysis of IgG antibody against CMV during CMV infection screening.     

Synthetic viability genomic screening defines Sae2 function in DNA repair

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2Δ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.     

气候假说对内蒙古草原群落物种多样性格局的解释

物种丰富度的地理格局是宏观生态学和生物地理学的中心议题之一。本文基于内蒙古草原192个野外样地的调查数据, 结合各样地年平均气温、年降水量等9个气候因子, 探讨内蒙古草原物种丰富度格局及其主导因素, 以确定气候假说在该区的适用性。结果表明: (1)内蒙古草原物种丰富度经度格局显著, 呈现沿经度升高而增加的趋势, 同时由于经纬度的共线性, 也呈现出沿纬度升高而增加的趋势。(2)方差分解显示, 能量单独解释率为2.7%, 水分单独解释率为11.4%, 水分和能量共同解释率为46.3%, 未解释部分为39.6%, 可见能量与水分的共同作用在物种丰富度格局形成中占主导地位, 支持水热动态假说。这说明水热动态假说适用于解释内蒙古草原物种丰富度 格局。     

KNOCKDOWN OF THE IONOTROPIC γ‐AMINOBUTYRIC ACID RECEPTOR (GABAR) RDL GENE DECREASES FIPRONIL SUSCEPTIBILITY OF THE SMALL BROWN PLANTHOPPER,Laodelphax striatellus (HEMIPTERA: DELPHACIDAE)

Insect γ‐aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil‐susceptible and fipronil‐resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance‐conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27–82%, and greatly decreased fipronil‐induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth‐instar nymphs, and female adults than in third‐instar and fourth‐instar nymphs. Lsrdl was predominantly expressed in the heads of 2‐day‐old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects.     

Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1–8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species.     

Oxidative stress causes a decline in lysosomal integrity during hypothermic incubation of rat hepatocytes

Oxidative stress during cold preservation has been identified as a significant cause of cell injury but the process by which injury occurs is poorly understood. We examined loss of lysosomal integrity as a possible cause of cell injury during extended cold storage of isolated rat hepatocytes. After 21 h of hypothermia, there was a marked decline in lysosomal integrity, which was correlated with an increase in lipid peroxidation. When lipid peroxidation was prevented with the antioxidant Trolox (a vitamin E analog) or the iron chelator desferrioxamine, lysosomal integrity was preserved. In contrast, increasing lysosomal iron with ferric chloride caused an increase in lipid peroxidation and decreased lysosomal integrity. Loss of lysosomal integrity during cold preservation in this experimental model was consistent with iron-initiated oxidative stress. The progressive loss of lysosomal integrity during hypothermic incubation has the potential to affect liver function after transplantation.     

Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal α-Hemolysin

α-Hemolysin (α-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, α-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to α-HL. Due to the binding of those compounds, the conformational transition of α-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of α-HL. However, these results have not indicated how the binding of the α-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of α-HL, could bind to the “stem” region of α-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of α-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the “stem” region of α-HL, the conformational transition of α-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of α-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.     

Cell origins and diagnostic accuracy of interleukin 27 in pleural effusions

The objective of the present study was to investigate the presence of interleukin (IL)-27 in pleural effusions and to evaluate the diagnostic significance of pleural IL-27. The concentrations of IL-27 were determined in pleural fluids and sera from 68 patients with tuberculous pleural effusion, 63 malignant pleural effusion, 22 infectious pleural effusion, and 21 transudative pleural effusion. Flow cytometry was used to identify which pleural cell types expressed IL-27. It was found that the concentrations of pleural IL-27 in tuberculous group were significantly higher than those in malignant, infectious, and transudative groups, respectively. Pleural CD4(+) T cells, CD8(+) T cells, NK cells, NKT cells, B cells, monocytes, macrophages, and mesothelial cells might be the cell sources for IL-27. IL-27 levels could be used for diagnostic purpose for tuberculous pleural effusion, with the cut off value of 1,007 ng/L, IL-27 had a sensitivity of 92.7% and specificity of 99.1% for differential diagnosing tuberculous pleural effusion from non-tuberculous pleural effusions. Therefore, compared to non-tuberculous pleural effusions, IL-27 appeared to be increased in tuberculous pleural effusion. IL-27 in pleural fluid is a sensitive and specific biomarker for the differential diagnosing tuberculous pleural effusion from pleural effusions with the other causes.     

Overexpression of Aurora-A enhances invasion and matrix metalloproteinase-2 expression in esophageal squamous cell carcinoma cells

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers, and metastasis is the principal cause of death in ESCC patients. It has been shown that amplification and overexpression of mitotic serine/threonine kinase Aurora-A occur in several types of human tumors, including ESCC. Moreover, increase in expression levels of Aurora-A has been predicted to correlate with the grades of tumor differentiation and invasive capability. However, the mechanisms by which Aurora-A mediates its invasive effects still remain elusive. In this article, we showed that Aurora-A overexpression significantly increased cell migration and invasion as well as secretion and expression of matrix metalloproteinase-2 (MMP-2). Conversely, siRNA-mediated knockdown of Aurora-A expression in human ESCC cells led to inhibition of cell invasiveness as well as secretion and expression of MMP-2. In addition, Aurora-A overexpression increased phosphorylation levels of p38 mitogen-activated protein kinase (MAPK) and Akt, and the knockdown of Aurora-A by siRNA decreased the activity of p38 MAPK and Akt. Moreover, the blocking of the activity of above kinases using chemical inhibitors suppressed the ability of Aurora-A to induce MMP-2 secretion and expression as well as cell invasion. These data show that overexpression of Aurora-A contributes to the malignancy development of ESCC by enhancing tumor cell invasion as well as MMP-2 activity and expression, which can occur through signaling pathways involving p38 MAPK and Akt protein kinases. Taken together, these studies provide a molecular basis for promoting the role of Aurora-A in malignancy development of ESCC.     

Embryological Studies of Codonopsis pilosula Nannf.

This paper reports the studies of megasporogenesis and microsporogenesis, development of female and male gametophytes, fertilization, and development of embryo and endosperm, The anther wall consists of four layers, i.e. epidermis, endothecium, middle layer and tapetum. Part of the tapetum cells originates from the primary parietal cells, and the other part comes from the basic tissue of the anther partition. Tapeta? cells are uninucleate or binucleate, and belong to the secretory type. Microsporocyte originates directly from the primary sporogenous cell, Cytokinesis is of the simultaneous type. Arrangement of microspores in tetrad is isobilateral. Mature pollen grain is of the 2-celled type. The ovary is tricarpellum, trilocular with many ovules. The ovule is mono-integinous, tenui-nucellar and anatropous. The embryo sac originates from the single-archesporial cell. The one chalazal megaspore in linear tetrad is the functional megaspore. The development of embryo sac is of the Polygonum type. Before fertilization, two polar nuclei fuse in to a secondary nucleus and the antipodal cells degenerate. Fertilization is porogamy, fusion of one sperm with secondary nucleus is faster than that of one sperm with egg nucleus. The development of endosperm is of the cellular type. The first three divisions of endosperm ceils are regular. Two endosperm cells near the ends of chalaza and the micropyle develop into haustorium without division. The haustoria gradually degenerate at the late stage of globular embryo. The mature seeds contain abundant endosperm. The development of embryo is of the Solanad type. The suspensor consists of 12–20 cells. The optimum development of the suspensor is at the early stage of the globular embryo. It begins to degenerate after late globular stage. The embryo develops from proembryo, heartshaped embryo, dicotyledenous- to mature embryo.