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

Phomopsis longanae Chi-Induced Change in ROS Metabolism and Its Relation to Pericarp Browning and Disease Development of Harvested Longan Fruit

Phomopsis longanae Chi is a major pathogenic fungus that infects harvested longan fruit. This study aimed to investigate the effects of P. longanae on reactive oxygen species (ROS) metabolism and its relation to the pericarp browning and disease development of harvested longan fruit during storage at 28°C and 90% relative humidity. Results showed that compared to the control longans, P. longanae-inoculated longans displayed higher indexes of pericarp browning and fruit disease, higher O2-. generation rate, higher accumulation of malondialdehyde (MDA), lower contents of glutathione (GSH) and ascorbic acid (AsA), lower 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability and reducing power in pericarp. In addition, P. longanae-infected longans exhibited higher activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in the first 2 days of storage, and lower activities of SOD, CAT, and APX during storage day 2–5 than those in the control longans. These findings indicated that pericarp browning and disease development of P. longanae-infected longan fruit might be the result of the reducing ROS scavenging ability and the increasing O2-. generation rate, which might lead to the peroxidation of membrane lipid, the loss of compartmentalization in longan pericarp cells, and subsequently cause polyphenol oxidase (PPO) and peroxidase (POD) to contact with phenolic substrates which result in enzymatic browning of longan pericarp, as well as cause the decrease of disease resistance to P. longanae and stimulate disease development of harvested longan fruit

&epsilon;-Poly-l-Lysine Enhances Fruit Disease Resistance in Postharvest Longans (Dimocarpus longan Lour.) by Modulating Energy Status and ATPase Activity

&epsilon;-poly-l-lysine (&epsilon;-PL) holds a strong antibacterial property and is widely used for food preservation. However, the application of &epsilon;-PL to enhance fruit disease resistance in postharvest longans (Dimocarpus longan Lour.) has not been explored. The objective of this study was to explore the impact of &epsilon;-PL treatment on disease occurrence and energy metabolism of longans infected with Phomopsis longanae Chi (P. longanae). It was found that, in comparison with P. longanae-inoculated longans, &epsilon;-PL could decrease the fruit disease index and adenosine monophosphate (AMP) content, increase the amounts of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and energy charge, and enhance the activities of adenosine triphosphatase (ATPase) (such as H+-, Mg2+-, and Ca2+-ATPase) in the mitochondria, protoplasm, and vacuole. The results suggest that the higher levels of ATPase activity and energy status played essential roles in disease resistance of postharvest longan fruit. Therefore, the &epsilon;-PL treatment can be used as a safe and efficient postharvest method to inhibit the disease occurrence of longan fruit during storage at room temperature

Analysis of Effect of Grouser Height on Tractive Performance of Tracked Vehicle under Different Moisture Contents in Paddy Soil

Grouser height and soil moisture content have a significant effect on the tractive performance of tracked vehicles. Paddy soil has the mechanical properties of both clay soil and sandy soil and can have a wide range of water content values, and it has a considerable influence on the tractive performance of tracked agricultural machinery. To study the influence of grouser height on the tractive performance of a single-track shoe under different soil moisture contents, a three-dimensional shearing model of the single-track shoe and the contact soil was established based on the ground vehicle mechanics theory, and an experimental platform with a soil bin, sensors, and a control system was established. Six preset levels of moisture contents (7%, 12%, 17%, 22%, 27%, and 32%) of paddy soil were prepared for the research experiment. The mechanical properties of the soil with different moisture contents were obtained through the use of a direct shear test, penetration test, and compaction test. The obtained physical parameters of the soil have special characteristics that are different from typical soil. Combined with the three-dimensional model and the obtained soil parameters, the parameters of the tractive performance, such as thrust, running resistance, and traction with different moisture contents were analyzed and calculated. The test results revealed that the thrust at different grouser heights shows a regular waveform growth trend with an increase in soil moisture content. The minimum value and the maximum value of thrust were obtained at moisture contents of 7% and 12%, respectively. The curve of different grouser heights of the running resistance shows similar changes with different moisture contents. The two peak points and inflection points of the fluctuation curve are for moisture contents of 17% and 27%. The change curve of the traction is highly similar to the curve of thrust. The maximum value of the traction was found at a moisture content of 12%, and the minimum value at 22% or 27%. Under different moisture conditions, tracked vehicles with higher grousers have better tractive performances