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

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

The Advantage of Low-Delta Electroencephalogram Phase Feature for Reconstructing the Center-Out Reaching Hand Movements

It is an emerging frontier of research on the use of neural signals for prosthesis control, in order to restore lost function to amputees and patients after spinal cord injury. Compared to the invasive neural signal based brain-machine interface (BMI), a non-invasive alternative, i.e., the electroencephalogram (EEG)-based BMI would be more widely accepted by the patients above. Ideally, a real-time continuous neuroprosthestic control is required for practical applications. However, conventional EEG-based BMIs mainly deal with the discrete brain activity classification. Until recently, the literature has reported several attempts for achieving the real-time continuous control by reconstructing the continuous movement parameters (e.g., speed, position, etc.) from the EEG recordings, and the low-frequency band EEG is consistently reported to encode the continuous motor control information. Previous studies with executed movement tasks have extensively relied on the amplitude representation of such slow oscillations of EEG signals for building models to decode kinematic parameters. Inspired by the recent successes of instantaneous phase of low-frequency invasive brain signals in the motor control and sensory processing domains, this study examines the extension of such a slow-oscillation phase representation to the reconstructing two-dimensional hand movements, with the non-invasive EEG signals for the first time. The data for analysis are collected on five healthy subjects performing 2D hand center-out reaching along four directions in two sessions. On representative channels over the cortices encoding the execution information of reaching movements, we show that the low-delta EEG phase representation is characterized by higher signal-to-noise ratio and stronger modulation by the movement tasks, compared to the low-delta EEG amplitude representation. Furthermore, we have tested the low-delta EEG phase representation with two commonly used linear decoding models. The results demonstrate that the low-delta EEG phase based decoders lead to superior performance for 2D executed movement reconstruction to its amplitude based counterparts, as well as the other-frequency band amplitude and power based features. Thus, our study contributes to improve the movement reconstruction from EEG by introducing a new feature set based on the low-delta EEG phase patterns, and demonstrates its potential for continuous fine motion control of neuroprostheses

Additional file 1: Table S1. of Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus

Comparison of CaPV real-time RPA assay and CaPV RPA LFD assay with real-time qPCR assay on spiked samples. (DOCX 18 kb

Additional file 2: Figure S1. of Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus

The detection limit of CaPV RPA LFD assay. This assay was performed using a dilution series of the SPPV/Gulang 2009 genomic DNA, and NC represents negative control. (PPTX 393 kb

P21-activated protein kinase (PAK2)-mediated c-Jun phosphorylation at 5 threonine sites promotes cell transformation

The oncoprotein c-Jun is one of the components of the activator protein-1 (AP-1) transcription factor complex. AP-1 regulates the expression of many genes and is involved in a variety of biological functions such as cell transformation, proliferation, differentiation and apoptosis. AP-1 activates a variety of tumor-related genes and therefore promotes tumorigenesis and malignant transformation. Here, we found that epidermal growth factor (EGF) induces phosphorylation of c-Jun by P21-activated kinase (PAK) 2. Our data showed that PAK2 binds and phosphorylates c-Jun at five threonine sites (Thr2, Thr8, Thr89, Thr93 and Thr286) in vitro and ex vivo. Knockdown of PAK2 in JB6 Cl41 (P+) cells had no effect on c-Jun phosphorylation at Ser63 or Ser73 but resulted in decreases in EGF-induced anchorage-independent cell transformation, proliferation and AP-1 activity. Mutation at all five c-Jun threonine sites phosphorylated by PAK2 decreased the transforming ability of JB6 cells. Knockdown of PAK2 in SK-MEL-5 melanoma cells also decreased colony formation, proliferation and AP-1 activity. These results indicated that PAK2/c-Jun signaling plays an important role in EGF-induced cell proliferation and transformation