Effective inhibition of foot-and-mouth disease virus (FMDV) replication in vitro by vector-delivered microRNAs targeting the 3D gene

<p>Abstract</p> <p>Background</p> <p>Foot-and-mouth disease virus (FMDV) causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined.</p> <p>Results</p> <p>Four pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV.</p> <p>Conclusion</p> <p>Our results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication <it>in vitro</it>. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.</p

Insulin-like peptide 8 (Ilp8) regulates female fecundity in flies

Introduction: Insulin-like peptides (Ilps) play crucial roles in nearly all life stages of insects. Ilp8 is involved in developmental stability, stress resistance and female fecundity in several insect species, but the underlying mechanisms are not fully understood. Here we report the functional characterization of Ilp8s in three fly species, including Bactrocera dorsalis, Drosophila mercatorum and Drosophila melanogaster.Methods: Phylogenetic analyses were performed to identify and characterize insect Ilp8s. The amino acid sequences of fly Ilp8s were aligned and the three-dimensional structures of fly Ilp8s were constructed and compared. The tissue specific expression pattern of fly Ilp8s were examined by qRT-PCR. In Bactrocera dorsalis and Drosophila mercatorum, dsRNAs were injected into virgin females to inhibit the expression of Ilp8 and the impacts on female fecundity were examined. In Drosophila melanogaster, the female fecundity of Ilp8 loss-of-function mutant was compared with wild type control flies. The mutant fruit fly strain was also used for sexual behavioral analysis and transcriptomic analysis.Results: Orthologs of Ilp8s are found in major groups of insects except for the lepidopterans and coleopterans, and Ilp8s are found to be well separated from other Ilps in three fly species. The key motif and the predicted three-dimensional structure of fly Ilp8s are well conserved. Ilp8 are specifically expressed in the ovary and are essential for female fecundity in three fly species. Behavior analysis demonstrates that Ilp8 mutation impairs female sexual attractiveness in fruit fly, which results in decreased mating success and is likely the cause of fecundity reduction. Further transcriptomic analysis indicates that Ilp8 might influence metabolism, immune activity, oocyte development as well as hormone homeostasis to collectively regulate female fecundity in the fruit fly.Discussion: Our findings support a universal role of insect Ilp8 in female fecundity, and also provide novel clues for understanding the modes of action of Ilp8

Lentviral-mediated RNAi to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor, and against FMDV infection in PK-15 cells

<p>Abstract</p> <p>Background</p> <p>shRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV) receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT™, which expressed siRNA targeting the FMDV receptor, the porcine integrin αv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF) and cell enzyme linked immunosorbent assays (cell ELISA), and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells.</p> <p>Results</p> <p>Our results indicated successful establishment of the iαv U6 RNAi entry vector and the iαv pLenti6/BLOCK -iT expression vector. The functional titer of obtained virus was 1.0 × 10⁶TU/mL. To compare with the control and mock group, the iαv-PK-15 group αv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold and there was a > 99% reduction in virus titer when cells were challenged with 10²TCID₅₀of FMDV.</p> <p>Conclusions</p> <p>Iαv-PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.</p

Optical and Near-Infrared Observations of the Highly Reddened, Rapidly Expanding Type Ia Supernova 2006X in M100

We present extensive optical (UBVRI), near-infrared (JK) light curves and optical spectroscopy of the Type Ia supernova (SN) 2006X in the nearby galaxy NGC 4321 (M100). Our observations suggest that either SN 2006X has an intrinsically peculiar color evolution, or it is highly reddened [E(B - V)_{host} = 1.42+/-0.04 mag] with R_V = 1.48+/-0.06, much lower than the canonical value of 3.1 for the average Galactic dust. SN 2006X also has one of the highest expansion velocities ever published for a SN Ia. Compared with the other SNe Ia we analyzed, SN 2006X has a broader light curve in the U band, a more prominent bump/shoulder feature in the V and R bands, a more pronounced secondary maximum in the I and near-infrared bands, and a remarkably smaller late-time decline rate in the B band. The B - V color evolution shows an obvious deviation from the Lira-Phillips relation at 1 to 3 months after maximum brightness. At early times, optical spectra of SN 2006X displayed strong, high-velocity features of both intermediate-mass elements (Si, Ca, and S) and iron-peak elements, while at late times they showed a relatively blue continuum, consistent with the blue U-B and B-V colors at similar epochs. A light echo and/or the interaction of the SN ejecta and its circumstellar material may provide a plausible explanation for its late-time photometric and spectroscopic behavior. Using the Cepheid distance of M100, we derive a Hubble constant of 72.7+/-8.2 km s^{-1} Mpc^{-1}(statistical) from the normalized dereddened luminosity of SN 2006X. We briefly discuss whether abnormal dust is a universal signature for all SNe Ia, and whether the most rapidly expanding objects form a subclass with distinct photometric and spectroscopic properties.Comment: 48 pages, 20 figures and 11 tables. Accepted Version (ApJ, 2008, March issue

Systematic analysis of the necroptosis index in pan-cancer and classification in discriminating the prognosis and immunotherapy responses of 1716 glioma patients

Necroptosis is a programmed form of necrotic cell death that serves as a host gatekeeper for defense against invasion by certain pathogens. Previous studies have uncovered the essential role of necroptosis in tumor progression and implied the potential for novel therapies targeting necroptosis. However, no comprehensive analysis of multi-omics data has been conducted to better understand the relationship between necroptosis and tumor. We developed the necroptosis index (NI) to uncover the effect of necroptosis in most cancers. NI not only correlated with clinical characteristics of multiple tumors, but also could influence drug sensitivity in glioma. Based on necroptosis-related differentially expressed genes, the consensus clustering was used to classify glioma patients into two NI subgroups. Then, we revealed NI subgroup I were more sensitive to immunotherapy, particularly anti-PD1 therapy. This new NI-based classification may have prospective predictive factors for prognosis and guide physicians in prioritizing immunotherapy for potential responders