Moderate alternate wetting and drying irrigation enhances drought-resistance abilities by improving structural mesophyll conductance of water-saving and drought-resistant rice under severe drought.

Water-saving and drought-resistant rice (WDR) coupled with alternate wetting and drying irrigation (AWDI) possesses a high photosynthetic potential due to higher mesophyll conductance (gm) under drought conditions. However, the physiological and structural contributions to the gm of leaves and their mechanisms in WDR under AWDI are still unclear. In this study, WDR (Hanyou 73) and drought-sensitive rice (Huiliangyou 898) were selected as materials. Three irrigation patterns were established from transplanting to the heading stage, including conventional flooding irrigation (W1), moderate AWDI (W2), and severe AWDI (W3). A severe drought with a soil water potential of -50 kPa was applied for a week at the heading stage across all treatments and cultivars. The results revealed that severe drought reduced gas exchange parameters and gm but enhanced antioxidant enzyme activities and malondialdehyde content in the three treatments and both cultivars. The maximal photosynthetic rate (Amax) of HY73 in the W2 treatment was greater than that in the other combinations of cultivars and irrigation patterns. The contribution of leaf structure (54%) to gm (gm-S, structural gm) was higher than that of leaf physiology (46%) to gm (gm-P, physiological gm) in the W2 treatment of Hanyou 73. Additionally, gm-S was significantly and linearly positively correlated with gm under severe drought. Moreover, both the initial and apparent quantum efficiencies were significantly and positively with gm in rice plants (p < 0.05). These results suggest that the improvements in photosynthesis and yield in the WDR combined with moderate AWDI can mainly be attributed to the enhancement of gm-S under severe drought conditions. Quantum efficiency may be a potential factor in regulating photosynthesis by cooperating with the gm of rice plants under severe drought conditions.

Development and application of a low-priced duplex quantitative PCR assay based on SYBR Green I for the simultaneous detection of porcine deltacoronavirus and porcine sapelovirus.

Porcine deltacoronavirus (PDCoV) and porcine sapelovirus (PSV) are two viruses that can cause diarrhoea in pigs and bring great economic loss to the pig industry. In this research, a duplex real-time quantitative polymerase chain reaction (qPCR) assay based on SYBR Green I was developed to simultaneously detect PDCoV and PSV. No specific melting peaks were found in other porcine diarrhoea-associated viruses, indicating that the method developed in this study had good specificity. The detection limits of PDCoV and PSV were 1.0 × 101 copies µl-1 and 1.0 × 102 copies µl-1, respectively. The duplex real-time qPCR assay tested two hundred and three (203) intestinal and faecal samples collected from diarrhoeal and asymptomatic pigs. The positive rates of PDCoV and PSV were 20.2% and 23.2%, respectively. The co-infection rate of PDCoV and PSV was 13.8%. To evaluate the accuracy of the developed method, conventional PCR and singular TaqMan real-time qPCR assays for PDCoV/PSV were also used to detect the samples. The results showed that the duplex real-time qPCR assay was consistent with the singular assays, but its sensitivity was higher than conventional PCR methods. This duplex real-time qPCR assay provides a rapid, sensitive and reliable method in a clinic to simultaneously detect PDCoV and PSV.

Expression of porcine interferon-α and its bioactivity analysis in vitro and in vivo.

Interferon α (IFN-α) plays a crucial role in the host's immune response. In this study, the amino acid sequence of porcine interferon α (PoIFN-α) was analyzed. Seven substitutions, S38F, H40Q, F43L, N78D, Y86C, S151A, and R156T, were mutated and obtained by aligning the sequences of PoIFN-α subtypes. The PoIFN-α mutants were designed, expressed, and purified in E. coli. The antiviral activities of these PoIFN-αs were measured in Vero and swine testis cells against vesicular stomatitis virus (VSV). Their inhibitory abilities on pseudorabies virus (PRV) were also examined. Commercial PoIFN-α was used as a control. We found the ideal inducer concentration of isopropyl ß-D-thiogalactoside was 1 mM, and the best time-point for induction was 8 h. The PoIFN-α mutant named PoIFN-α-156s had the highest antiviral activity, which was about 200-fold more than that of PoIFN-α. PoIFN-α-156s could inhibit VSV and PRV replication in a dose-dependent manner in vitro. The half-life of PoIFN-α-156s was longer than that of PoIFN-α in mice, and the effective antiviral action was higher than PoIFN-α. Animal experiments showed that PoIFN-α-156s could decrease the viral load after infection with VSV. Overall, these results suggest that recombinant PoIFN-α-156s has the ability of antivirus, and is feasible for veterinary clinical applications and fundamental research.

High Throughput Screening of Transcription Factor LysG for Constructing a Better Lysine Biosensor.

The biosensors based on transcription factors (TFs) are widely used in high throughput screening of metabolic overproducers. The unsatisfactory performances (narrow detection and dynamic ranges) of biosensors limit their practical application and need more improvement. In this study, using the TF LysG (sensing lysine) as an example, a biosensor optimization method was constructed by growth-coupled screening of TF random mutant libraries. The better the performance of the biosensor, the faster the strain grows under screening pressure. A LysGE15D, A54D, and I164V-based biosensors were obtained, which were about 2-fold of the control in the detection and dynamic ranges. A lysine high-producer was screened effectively using the optimized biosensor with the production at 1.51 ± 0.30 g/L in flasks (2.22-fold of the original strain). This study provided a promising strategy for optimizing TF-based biosensors and was of high potential to be applied in the lysine high-producers screening process.

Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody.

The African swine fever virus (ASFV) is an ancient, structurally complex, double-stranded DNA virus that causes African swine fever. Since its discovery in Kenya and Africa in 1921, no effective vaccine or antiviral strategy has been developed. Therefore, the selection of more suitable vaccines or antiviral targets is the top priority to solve the African swine fever virus problem. B125R, one of the virulence genes of ASFV, encodes a non-structural protein (pB125R), which is important in ASFV infection. However, the epitope of pB125R is not well characterized at present. We observed that pB125R is specifically recognized by inactivated ASFV-positive sera, suggesting that it has the potential to act as a protective antigen against ASFV infection. Elucidation of the antigenic epitope within pB125R could facilitate the development of an epitope-based vaccine targeting ASFV. In this study, two strains of monoclonal antibodies (mAbs) against pB125R were produced by using the B cell hybridoma technique, named 9G11 and 15A9. The antigenic epitope recognized by mAb 9G11 was precisely located by using a series of truncated ASFV pB125R. The 52DPLASQRDIYY62 (epitope on ASFV pB125R) was the smallest epitope recognized by mAb 9G11 and this epitope was highly conserved among different strains. The key amino acid sites were identified as D52, Q57, R58, and Y62 by the single-point mutation of 11 amino acids of the epitope by alanine scanning. In addition, the immunological effects of the epitope (pB125R-DY) against 9G11 were evaluated in mice, and the results showed that both full-length pB125R and the epitope pB125R-DY could induce effective humoral and cellular immune responses in mice. The mAbs obtained in this study reacted with the eukaryotic-expressed antigen proteins and the PAM cell samples infected with ASFV, indicating that the mAb can be used as a good tool for the detection of ASFV antigen infection. The B cell epitopes identified in this study provide a fundamental basis for the research and development of epitope-based vaccines against ASFV.

Evaluation of the Potential Flight Ability of the Casuarina Moth, Lymantria xylina (Lepidoptera: Erebidae).

Lymantria xylina Swinhoe (Lepidoptera: Erebidae) is a potentially invasive pest, similar to Lymantria dispar asiatica Vnukovskij and Lymantria dispar japonica Motschulsky (Lepidoptera: Erebidae). To evaluate its potential for spread and flight distance related to egg deposition on vessels at ports, we employed a flight mill to assess the flight capabilities of its adults under varying conditions. Our findings revealed that females primarily flew short distances and ceased flying after 3:00 AM, whereas males covered much longer distances throughout the day. Sex, age, and flight duration significantly influenced flight ability. Females exhibited weaker flight capability than males, and their ability declined with increasing age or flight duration. Notably, 1-day-old moths displayed the strongest flight ability, with average flight distances of up to 3.975 km for females and 8.441 km for males. By the fifth day, females no longer flew, and males experienced reduced flight ability. After continuous hanging for 16 h, females lost most of their flight capacity, while males remained capable of flight even after 32 h. Additionally, female flight ability decreased significantly after mating, possibly due to factors such as egg-carrying capacity, weight, and load ratio. This study provides a foundation for assessing the risk of long-distance dispersal of L. xylina via ocean-going freighters, considering female moths' phototactic flight and oviposition.

Co-targeting CDK 4/6 and C-MYC/STAT3/CCND1 axis and inhibition of tumorigenesis and epithelial-mesenchymal-transition in triple negative breast cancer by Pt(II) complexes bearing NH3 as trans-co-ligand.

In search of potential anticancer agents, we synthesized SNO-donor salicylaldimine main ligand-based Pt(II) complexes bearing NH3 as co-ligand at trans-position (C1-C6). These complexes showed similarity in structure with transplatin as the two N donor atoms of the main ligand and NH3 co-ligand were coordinated to Pt in trans position to each other. Each complex with different substituents on the main ligand was characterized thoroughly by detailed spectroscopic and spectrophotometric methods. Four of these complexes were studied in solid state by single crystal X-ray analysis. The stability of reference complex C1 was measured in solution state in DMSO­d6 or its mixture with D2O using 1H NMR methods. These complexes were further investigated for their anticancer activity in triple-negative-breast (TNBC) cells including MDA-MB-231, MDA-MB-468 and MDA-MB-436 cells. All these complexes showed satisfactory cytotoxic effect as revealed by the MTT results. Importantly, the highly active complex C4 anticancer effect was compared to the standard chemotherapeutic agents including cisplatin, oxaliplatin and 5-fluorouracil (5-FU). Functionally, C4 suppressed invasion, spheroids formation ability and clonogenic potential of cancer cells. C4 showed synergistic anticancer effect when used in combination with palbociclib, JQ1 and paclitaxel in TNBC cells. Mechanistically, C4 inhibited cyclin-dependent kinase (CDK)4/6 pathway and targeted the expressions of MYC/STAT3/CCND1/CNNE1 axis. Furthermore, C4 suppressed the EMT signaling pathway that suggested a role of C4 in the inhibition of TNBC metastasis. Our findings may pave further in detailed mechanistic study on these complexes as potential chemotherapeutic agents in different types of human cancers.

Preparation and epitope mapping of monoclonal antibodies against African swine fever virus p22 protein.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is now widespread in many countries and severely affects the commercial rearing of swine. Rapid and early diagnosis is crucial for the prevention of ASF. ASFV mature virions comprise the inner envelope protein, p22, making it an excellent candidate for the serological diagnosis and surveillance of ASF. In this study, the prokaryotic-expressed p22 recombinant protein was prepared and purified for immunization in mice. Four monoclonal antibodies (mAbs) were identified using hybridoma cell fusion, clone purification, and immunological assays. The epitopes of mAbs 14G1 and 22D8 were further defined by alanine-scanning mutagenesis. Our results showed that amino acids C39, K40, V41, D42, C45, G48, E49, and C51 directly bound to 14G1, while the key amino acid epitope for 22D8 included K161, Y162, G163, D165, H166, I167, and I168. Homologous and structural analysis revealed that these sites were highly conserved across Asian and European ASFV strains, and the amino acids identified were located on the surface of p22. Thus, our study contributes to a better understanding of the antigenicity of the ASFV p22 protein, and the results could facilitate the prevention and control of ASF.

An improved faster R-CNN algorithm for assisted detection of lung nodules.

The morbidity and mortality of lung cancer are increasing rapidly in every country in the world, and pulmonary nodules are the main symptoms of lung cancer in the early stage. If we can diagnose pulmonary nodules in time at the early stage and follow up and treat suspicious patients, we can effectively reduce the incidence of lung cancer. CT (Computed Tomography) has been applied to the screening of many diseases because of its high resolution. Pulmonary nodules show white round shadows in CT images. With the popularity of CT equipment, doctors need to review a large number of imaging results every day. Doctors will misjudge and miss the lesions because of reviewing CT scanning results for a long time. At this time, the method of automatic detection of pulmonary nodules by computer can relieve the pressure of doctors in reviewing CT scan results. Traditional lung nodule detection methods, such as gray threshold method and region growing method, divide the detection process into two steps: extracting candidate regions and eliminating false regions. In addition, the traditional detection method can only operate on a single image, which leads to the inability of this method to detect the batch scanning results in real time. With the continuous development of computer equipment performance and artificial intelligence, the relationship between medical image processing and deep learning is getting closer and closer. In deep learning, object detection methods such as Faster R-CNN、YOLO can complete parallel detection of batch images, and deep structure can fully extract the features of input images. Compared with traditional lung nodule detection methods, it has the characteristics of high efficiency and high precision. Faster R-CNN is a classical and high-precision two-stage object detection method. In this paper, an improved Faster R-CNN model is proposed. On the basis of Faster R-CNN, multi-scale training strategy is used to fully mine the features of different scale spaces and perform path augmentation on lower-dimensional features, which improves the small object detection ability of the model. Through Online Hard Example Mining (OHEM), the loss value is used to quantify the difficulty of candidate region detection, and the training times of the region to be detected are adaptively adjusted. Make full use of prior information to customize the size and proportion of preset boundary anchor boxes. Using deformable convolution to improve the visual field to enhance the global features and enhance the ability to extract the feature information of pulmonary nodules in the same scale space. The new model was tested on LUNA16 (Lung Nodule Analysis 2016) dataset. The detection precision of the improved Faster R-CNN model for pulmonary nodules increased from 76.4% to 90.7%, and the recall rate increased from 40.1% to 56.8% Compared with the mainstream object detection algorithms YOLOv3 and Cascade R-CNN, the improved model is superior to the above models in every index.

Preparation of a Single-Chain Antibody against Nucleocapsid Protein of Porcine Deltacoronavirus by Phage Display Technology.

Porcine deltacoronavirus (PDCoV) mainly causes severe diarrhea and intestinal pathological damage in piglets and poses a serious threat to pig farms. Currently, no effective reagents or vaccines are available to control PDCoV infection. Single-chain fragment variable (scFv) antibodies can effectively inhibit virus infection and may be a potential therapeutic reagent for PDCoV treatment. In this study, a porcine phage display antibody library from the peripheral blood lymphocytes of piglets infected with PDCoV was constructed and used to select PDCoV-specific scFv. The library was screened with four rounds of biopanning using the PDCoV N protein, and the colony with the highest affinity to the PDCoV N protein was obtained (namely, N53). Then, the N53-scFv gene fragment was cloned into plasmid pFUSE-hIgG-Fc2 and expressed in HEK-293T cells. The scFv-Fc antibody N53 (namely, scFv N53) was purified using Protein A-sepharose. The reactive activity of the purified antibody with the PDCoV N protein was confirmed by indirect enzyme-linked immunosorbent assay (ELISA), western blot and indirect immunofluorescence assay (IFA). Finally, the antigenic epitopes that the scFv N53 recognized were identified by a series of truncated PDCoV N proteins. The amino acid residues 82GELPPNDTPATTRVT96 of the PDCoV N protein were verified as the minimal epitope that can be recognized by the scFv-Fc antibody N53. In addition, the interaction between the scFv-Fc antibody N53 and the PDCoV N protein was further analyzed by molecule docking. In conclusion, our research provides some references for the treatment and prevention of PDCoV.

Preparation of monoclonal antibody and identification of two novel B cell epitopes to VP1 protein of porcine sapelovirus.

Porcine sapelovirus (PSV) is an important emerging swine pathogen that causes diarrhoea, respiratory distress, severe reproductive system and neurological disorders in pigs, posing huge threat to swine industry. However, there are no effective serological diagnostic products and the epitope characterization of PSV VP1 protein is still largely unknown. In current study, we successfully expressed recombinant His-VP1 protein by prokaryotic expression system and the recombinant VP1 protein had good immunogenicity. BALB/C mice were then selected and immunized with purified recombinant VP1 protein, and two monoclonal antibodies (Mabs) 9F10 and 15E4 against VP1 were successfully prepared by hybrioma technology. The isotype of these two Mabs were identified and showed that Mab 9F10 with the heavy chain subtype was IgG1 and the light chain subtype was kappa. Mab 15E4 was identified as IgG2 for the heavy chain subtype and Kappa for the light chain subtype. The antigen epitopes of prepared two VP1 Mabs were clearly identified. The minimal unit of B cell specific epitope recognized by Mab 15E4 was 203YDGDG207 and conserved in different strain genotypes of PSV, indicating this epitope may be a good target for serological detection of PSV. However, the epitope recognized by Mab 9F10 was 8QAIVNRT14 and varied greatly among different PSV strains. Structural modeling analysis showed that the identified two novel B cell epitopes were located on the surface of VP1. Our study provides useful tool for the establishment the serological detection methods of PSV and may support the study of VP1 protein function.