Pseudorabies virus(PRV)is the etiological agent of pseudorabies in pigs,which is char-acterized by dyspnea,reproductive disorders,and neurological diseases,and it spreads widely a-round the world.Since 2011,the newly emerged PRV variants have resulted in poor immunity pro-tection of traditional vaccine strains,and the original method of vaccine strain preparation is time-consuming and labor-intensive.Therefore,it is urgently needed to develop an efficient screening method of the vaccine strain at present.Using CRISPR/Cas9 gene editing technology in this study,two single guide RNAs(sgRNA)were designed targeting the virulence gene TK of PRV variant strain CH/JX/2016,and then the enhanced green fluorescent protein the reporter(EGFP)gene was inserted at the TK locus by a homologous repair plasmid.After multiple rounds of plaque puri-fication,the rPRV-ΔTK/EGFP strain was obtained.The results showed the cleavage efficiency of the two sgRNAs was extremely high.The preparation of rPRV-ΔTK/EGFP strain was succeed af-ter only three rounds of purification,and the EGFP expressed normally.The CRISPR/Cas9 system can edit the PRV gene simply,rapidly,and efficiently,and exhibits great potential in the construction of vaccine candidate strains.Meanwhile,the rescued rPRV-ΔTK/EGFP strain not only could be used as a tracer strain in PRV variant infection progresses,but also for subsequent antivi-ral drug screening.

Objective:To explore the adjustment ranges of auto-segmentation contours for organs at risk (OAR) in patients with nasopharyngeal carcinoma and assess the dosimetric impacts of the contours from varying sources on radiotherapy plans.Methods:Twenty-five patients with early-stage nasopharyngeal carcinoma were investigated. Through expert delineation, deep learning-based automatic delineation, and atlas-based automatic delineation of their spinal cord, brainstem, optic nerves, optic chiasm, parotid glands, oral cavity, hypopharynx, and mandible, as well as expert correction of these automatic delineations, five structure sets were formed. Moreover, the contours delineated by experts (also referred to as the expert contours) of the target volumes and other OARs were copied into the images for subsequent research. The Dice similarity coefficients (DSCs) of the structure sets were calculated. Using the radiotherapy plans optimized based on expert contours as templates, the radiotherapy plans and dose distributions of all the structure sets were established. The expert contours and contours determined using automatic delineation and corrected by experts (also referred to as the corrected contours) were defined as clinical contours. Then, three research objectives were set: the dosimetric effects of inter-observer clinical contour variations, the impacts of contour variations on plan optimization, and the impacts of contour variations on plan evaluation.Results:The average DSC of the visual pathway was 0.62±0.10, lower than that of other OARs (0.86±0.04). After expert correction, the DSCs of contours obtained using deep learning- and atlas-based automatic delineation increased by 7.61% and 10.69%, respectively. For the dosimetric effects of inner-observer contour variations, the Dmax of the optic chiasm was the maximum (3.96±6.02)Gy, while the Dmean of the hypopharynx was the minimum (0.81±0.55 Gy). When the impacts of contour variations on plan optimization were assessed based on expert contours, the dose differences (Δ D) exceeding ±3 Gy accounted for 22%, 14%, 46%, and 42%, respectively for the spinal cord, brainstem, optic nerve, and optic chiasm and accounted for only 2% for other OARs. After expert correction, the Δ D between automatic and expert contours decreased, with Δ D exceeding ±3 Gy decreased by 16% and 14%, respectively for the optic nerves and optic chiasm. When the average distance of the overlap volume histogram (OVH) exceeded 3.5 cm, all Δ Dmax fell within ±3 Gy. When the average distance of OVH was greater than 1.5 cm, all Δ Dmean fell within ±2 Gy. For contours obtained using deep learning and atlas-based automatic delineation, the doses of 50.0%±17.3% and 52.6%±19.3% of patients fell within the dose ranges of clinical contours, respectively. The numbers of patients for whom the Dmax of the spinal cord, optic nerve, optic chiasm and the D1 cm 3 of the mandible in the two types of automatic contours fell within the dose ranges of clinical contours were statistically different ( t = -4.24, -3.99, -3.16, 3.51, P < 0.05). Conclusions:After expert correction, the automatic delineation results from different sources exhibited certain geometric differences. The expert correction reduced the impacts of automatic contours on plan optimization. The average distance of OVH is identified as an important feature used to determine dose differences. For small-volume serial organs close to the target volumes, meticulous corrections are required before applying auto-segmentation to clinical practice.

OBJECTIVE: To investigate the effect of miR-4443 expression on migration and invasion of breast cancer. METHODS: We examined the expression of miR-4443 in breast carcinoma in situ and paired adjacent tissues from 3 breast cancer patients with high-throughput sequencing and verified the results using TCGA database. We also detected miR-4443 expressions using real-time quantitative PCR (RT-qPCR) in low invasive and highly invasive breast cancer cells (MCF-7 and MDA-MB-231 cells, respectively). The changes in apoptosis, migration and invasion of MCF-7 and MDA-MB-231 cells after transfection with miR-4443 mimics, mimics-NC, miR-4443 inhibitor or inhibitor-NC were analyzed using flow cytometry, wound healing assay and Transwell invasion assay. The target gene of miR-4443 was predicted by bioinformatics software and validated by a dual luciferase reporter gene system. RT-qPCR and Western blotting were performed to detect the expression of recombinant human phosphatidyl ethanolamine binding protein 1 (PEBP1) in the transfected cells. RESULTS: The expression of miR-4443 was significantly higher in the breast cancer tissues than in the adjacent tissues ( CONCLUSIONS MiR-4443 promotes the migration and invasion of breast cancer cells by inhibiting the expression of PEBP1, suggesting the possibility of suppressing miR-4443 expression as a potential therapeutic strategy for breast cancer.
Breast Neoplasms/genetics* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; MicroRNAs/genetics* ; Neoplasm Invasiveness/genetics* ; Phosphatidylethanolamine Binding Protein

Objective:To observe the clinical effect of drospirone ethinylestradiol (Yasmin) in the treatment of vaginal bleeding and irregular menstruation after induced abortion.Methods:From January 2018 to June 2018, 540 patients with vaginal bleeding and irregular menstruation after induced abortion treated in Pinghu Hospital of Traditional Chinese Medicine were selected and divided into two groups according to the random digital table method, with 270 cases in each group.The control group was treated with routine treatment, while the observation group was treated with Yasmin on the basis of routine treatment.The clinical effects, vaginal bleeding time, menstrual rehydration time, menstrual status and adverse reactions of the two groups were observed.Results:The total effective rate (97.04%, 262/270), normal menstruation rate (90.74%, 245/270) in the observation group were significantly higher than those in the control group (81.48%, 220/270; 74.07%, 200/270), the differences were statistically significant (χ 2=34.074, 25.866, all P<0.05). The time of vaginal bleeding and menstrual reactivation in the observation group were (3.79±1.11)d and (24.07±4.23)d, respectively, which were significantly shorter than those in the control group [(5.88±1.40)d and (31.36±5.38)d] ( t=19.222, 17.503, all P<0.05). There was no statistically significant difference in the incidence of adverse reactions between the two groups ( P>0.05). Conclusion:Yasmin is effective in treating vaginal bleeding and irregular menstruation after induced abortion.It can significantly improve the condition of vaginal bleeding, shorten the time of menstruation and has high safety.

A novel protein encoded by the open reading frame 4 (ORF4) was recently discovered in porcine circovirus type 2 (PCV2). However, little is known about the interaction proteins of ORF4 which hindered better understanding the biological functions of ORF4 in the life cycle of PCV2. In the present study, the ORF4 was inserted into the multiple cloning site of pCMV-N-Flag-GST, yielding recombinant plasmid pCMV-N-Flag-GST-ORF4. The recombinant plasmid was transfected into 293T cells and the intracellular interaction complex of ORF4 were enriched and separated by GST pull-down and SDS-PAGE, sequentially. The potential interacting proteins of PCV2 ORF4 were stained with silver and identified by mass spectrometry (MS). Finally, five candidate ORF4-interacting proteins, including Serine/threonine-protein phosphatase 6 catalytic subunit, alpha cardiac muscle 1, actin, SEC14-like protein 5 and myosin 9 were identified. These results would benefit a better understanding of the biological function of ORF4 in PCV2 infected cells.
Animals ; Circoviridae Infections ; Circovirus ; HEK293 Cells ; Humans ; Mass Spectrometry ; Open Reading Frames ; Swine ; Viral Proteins

Porcine deltacoronavirus (PDCoV) is a newly emerging enteropathogenic swine coronavirus causing acute diarrhea and vomiting in pigs. The apoptosis of ST cells induced by PDCoV infection was studied in this research. In ST cells, caspase activity assay showed that the activity of caspase 3, caspase 8 and caspase 9 increased significantly with the infection of PDCoV, but not observed in UV irradiated PDCoV-infected cells, indicating that PDCoV infection activated both endogenous and exogenous apoptotic pathways in ST cells, and the induction of apoptosis depended on viral replication. To further investigate the endogenous apoptosis induced by PDCoV, cytochrome C and apoptosis-inducing factors in cytoplasm and mitochondria were detected. Compared with normal cells, the amount of cytochrome C released from mitochondria to cytoplasm increased significantly in PDCoV-infected cells, and the release increased with the prolongation of infection, while the apoptosis-inducing factor was always localized to mitochondria, suggesting that PDCoV induced apoptosis was initiated through caspase-dependent mitochondrial apoptosis pathway by promoting cytochrome C in the mitochondrial membrane gap into the cytosol. In conclusion, this study reveals the mechanism of PDCoV inducing apoptosis.
Animals ; Apoptosis ; Coronavirus ; Coronavirus Infections ; Mitochondria ; Swine ; Swine Diseases

Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the rep gene promoter, but also enhance the transcription/translation activity of the rep/cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interactions, and provided a theoretical basis for developing highly effective PCV2 vaccines.
Animals ; Cell Line ; Chromatography, Liquid ; Circoviridae Infections ; Circovirus ; DNA Helicases ; Diabetes Mellitus, Type 2 ; Promoter Regions, Genetic ; Swine ; Tandem Mass Spectrometry ; Transcription Factor AP-2 ; Virus Replication

Objective To explore the clinical effect of psychological nursing pathway for patients with hepatocellular carcinoma (HCC) who are receiving 125I particle implantation therapy.Methods A total of 130 HCC patients,who were scheduled to receive 125I particle implantation therapy,were randomly and equally divided into the study group and the control group with 65 patients in each group.Traditional routine psychological nursing method was conducted for patients of the control group,while routine psychological nursing that was based on psychological nursing pathway was carried out for patients of the study group.The patient's conditions of both groups were evaluated by using self-rating anxiety scale (SAS),trait coping style questionnaire (TCSQ) and the quality of life assessment scale (SF-36).The results were statistically analyzed with t test.Results After nursing intervention,SAS scores of the study group and the control group were (43.76±6.25) and (46.16±7.19) points respectively,the difference between the two groups was statistically significant (t=-3.92,P＜0.001).The PC and NC values of the TCSQ score in the study group were (41.61±4.82) points and (20.53±5.88) points respectively,while those in the control group were (37.32±4.62) and (22.80±6.93) respectively,the differences between the two groups were statistically significant (t=5.944 and t=-3.316 respectively,P＜0.01).One month after discharge,the mental health scores of the study group and the control group were (74.58±8.68) points and (71.53±8.16) points respectively,the emotion function scores were (79.46±10.05) points and (75.13±15.09) points respectively,and the differences in both mental health score and emotion function score between the two groups were statistically significant (P＜0.05).Conclusion Standardized psychological nursing pathway can make nurses more clear about the content of psychological nursing work and enable nurses to actively plan and complete psychological nursing care measures,so that patients can get prospective,individual and comprehensive psychological care to alleviate his or her negative emotion,and to improve the quality of life.

Immune-checkpoint blockers(ICBs)have been well received in a variety of tumors,and the quality of patient life has improved significantly.However,the reasons why not all patients treated with ICBs benefit from lesion control,symptom improvement,and survival time.Many patients are resistant to the first time when they have been using ICBs for a period of time.This is a clinical challenge.This review lists possible causes of primary drug resistance and acquired resistance to ICBs.The primary resistance is associated with several mechanisms,including tumor microenvironment,cancer cells themselves and other related factors.The acquired resistance includes nonclassical immunoprecipitation molecules secondary overexpression,abnormalities of antigen presenting signal pathway and dysfunction of T cell activation killer.Finally,we have described a variety of possible new combination of treatment,including combined radiotherapy and chemotherapy,and combined targeted therapy with other measures.

Porcine circovirus type 2 (PCV2) can cause immunosuppression on herds. PCV2, as an essential pathogen of PCV2-systemic disease (PCV2-SD), has caused considerable economic losses in pig industry worldwide. Here we review and address the evolution, viral protein and immunolesion of PCV2 and preventive techniques of PCV2-SD.
Animals ; Circoviridae Infections ; veterinary ; Circovirus ; genetics ; Phylogeny ; Swine ; Swine Diseases ; virology ; Viral Proteins ; genetics