Objective We aimed to investigate the effects of different non-surgical embryo transfer devices,number of transferred embryos,embryo stage,and embryos obtained from different mouse strains on the efficiency of non-surgical embryo transfer in mice,and to compare the efficiencies of surgical and non-surgical embryo transfer,in order to establish a stable non-surgical embryo transfer technology system.Methods Mouse embryo transfer was carried out using non-surgical method.Results The pregnancy rates using two different non-surgical transfer devices were(75.00±0.00)％and(66.67±14.43)％,and the birth rates were(46.11±6.31)％and(18.89±0.96)％,respectively.Transfer of 10,15,and 20 embryos resulted in pregnancy rates of(66.67±11.55)％,(80.00±0.00)％,and(66.67±23.09)％,and birth rates of(29.33±4.16)％,(38.67±4.81)％,and(17.00±3.46)％,respectively.When blastocysts and morulae were transferred non-surgically,the resulting pregnancy rates were(80.00±0.00)％and(46.67±11.55)％and the birth rates were(38.67±4.81)％and(10.22±2.77)％,respectively.Four strains(C57BL/6J,ICR,genetically modified mice A,genetically modified mice B)were used as donors for non-surgical embryo transfer,with resulting pregnancy rates of(66.67±11.55)％,(80.00±0.00)％,(73.33±11.55)％,and(80.00±0.00)％,and birthrates of(26.67±2.67)％,(38.67±4.81)％,(32.00±3.53)％,and(29.34±2.31)％,respectively.Fifteen pseudo-pregnant mice were transplanted surgically and 15 were transplanted non-surgically,with pregnancy rates of(80.00±0.00)％and(86.67±11.55)％,and birth rates of(38.67±4.81)％and(36.00±5.82)％,respectively.Conclusions Transfer device A resulted in a higher birth rate in this study.The embryo transfer efficiency was higher when 15 embryos were transferred into unilateral uterine horns of pseudo-pregnant 2.5-day recipients.Blastocyst-stage embryo transfer was more efficient than morula-stage transfer.There was no significant difference in efficiency between surgical and non-surgical embryo transfer procedures.

This study aims to establish an antibody detection method for porcine deltacoronavirus (PDCoV). The recombinant proteins PDCoV-N1 and PDCoV-N2 were expressed via the prokaryotic plasmid pColdII harboring the N gene sequence of the PDCoV strain CH/XJYN/2016. The reactivity and specificity of PDCoV-N1 and PDCoV-N2 with anti-PEDV sera were analyzed after the recombinant proteins were analyzed by SDS-PAGE and purified by the Ni-NTA Superflow Cartridge. Meanwhile, Western blotting and indirect immunofluorescence assay were carried out separately to validate the recombinant proteins PDCoV-N1 and PDCoV-N2. Finally, we established an indirect ELISA method based on the recombinant protein PDCoV-N2 after optimizing the conditions and tested the sensitivity, specificity, and reproducibility of the method. Then, the established method was employed to examine 102 clinical serum samples. The recombinant protein PDCoV-N2 showed low cross-reactivity with anti-PEDV sera. The optimal conditions of the indirect ELISA method based on PDCoV-N2 were as follows: the antigen coating concentration of 1.25 μg/mL and coating at 37 ℃ for 1 h; blocking by BSA overnight at 4 ℃; serum sample dilution at 1:50 and incubation at 37 ℃ for 1 h; secondary antibody dilution at 1:80 000 and incubation at 37 ℃ for 1 h; color development with TMB chromogenic solution at 37 ℃ for 10 min. The S/P value ≥ 0.45, ≤0.38, and between 0.45 and 0.38 indicated that the test sample was positive, negative, and suspicious, respectively. The testing results of the antisera against porcine epidemic diarrhea virus (PEDV), porcine circovirus 2 (PCV2), transmissible gastroenteritis virus (TGEV), foot-and-mouth disease virus (FMDV), and African swine fever virus (ASFV) showed that the S/P values were all less than 0.38. The testing results of the 800-fold diluted anti-PDCoV sera were still positive. The results of the inter- and intra-batch tests showed that the coefficients of variation of this method were less than 10%. Clinical serum sample test results showed the coincidence rate between this method and neutralization test was 94.12%. In this study, an ELISA method for the detection of anti-PDCoV antibodies was successfully established based on the truncated N protein of PDCoV. This method is sensitive, specific, stable, and reproducible, serving as a new method for the clinical diagnosis of PDCoV.
Animals ; Enzyme-Linked Immunosorbent Assay/methods* ; Swine ; Antibodies, Viral/blood* ; Recombinant Proteins/genetics* ; Deltacoronavirus/isolation & purification* ; Coronavirus Infections/virology* ; Swine Diseases/diagnosis* ; Coronavirus Nucleocapsid Proteins ; Sensitivity and Specificity

Objective:To investigate the inhibitory effect of Xiaojianzhong Granule(XJZG)on food allergy(FA)and related mecha-nisms in terms of gut microbiota,zonula occluden-1(ZO-1),and Occludin.Methods:A total of 24 specific pathogen-free female BALB/c mice were randomly divided into normal group,model group,prevention group,and treatment group,with 6 mice in each group.The mice in the prevention group were given XJZG by gavage at a standard dose of 5.85 g/kg/day from 3 days before the first challenge till 4 hours before the last challenge;the mice in the treatment group were given XJZG at the double dose for 3 days based on the allergy score;the mice in the other groups were given an equal volume of distilled water by gavage.At the end of the experiment,al-lergy score and anal temperature were measured;flow cytometry was used to measure eosinophils and mast cells in mesenteric lymph nodes(MLNs);toluidine blue staining was performed for mast cells in jejunal tissue;immunohistochemistry was used to measure the expression of ZO-1 and Occludin;16S rRNA sequencing was per-formed to analyze the microbiota in the intestinal content;high-performance liquid chromatography-mass spectrometry was used to measure the content of short-chain fatty acids(SCFAs)in jejunal lavage fluid.Results:Compared with the model group,the prevention group and the treatment group had significant reductions in al-lergy score(P=0.000,P=0.000),anal temperature(P=0.002,P=0.000),the proportion of eosinophils and mast cells in MLNs(P<0.05),and mast cell infiltration in jejunal tissue(P=0.000,P=0.000).Compared with the normal group,the model group had signifi-cant increases in the relative abundances of Erysipelaceae and Turicibacter,while the prevention group and the treatment group had disappearance of Erysipelaceae and Turicibacter and an increase in the relative abundance of Porphyromonadaceae.Compared with the normal group,the model group had a significant reduction in the content of propionate in jejunal lavage fluid(P=0.014),and compared with the model group,the prevention group had a significant increase in the content of propionate in jejunal lavage fluid(P=0.024),as well as a significant increase in the treatment group(P=0.008).In the model group,the expression of ZO-1 was downregulated(P=0.010),and the expression of Occludin was significantly downregulated(P=0.002),while the expression of ZO-1 and Occludin re-turned to normal levels in the prevention group and the treatment group(P=0.001,P=0.013;P=0.025,P=0.015).Conclusion:XJZG can change the composition and abundance of gut microbiota,increase the concentration of SCFAs,upregulate the expression of ZO-1 and Occludin,promote the repair of intestinal barrier,and inhibit food allergy.

Objective We aimed to investigate the effects of different non-surgical embryo transfer devices,number of transferred embryos,embryo stage,and embryos obtained from different mouse strains on the efficiency of non-surgical embryo transfer in mice,and to compare the efficiencies of surgical and non-surgical embryo transfer,in order to establish a stable non-surgical embryo transfer technology system.Methods Mouse embryo transfer was carried out using non-surgical method.Results The pregnancy rates using two different non-surgical transfer devices were(75.00±0.00)％and(66.67±14.43)％,and the birth rates were(46.11±6.31)％and(18.89±0.96)％,respectively.Transfer of 10,15,and 20 embryos resulted in pregnancy rates of(66.67±11.55)％,(80.00±0.00)％,and(66.67±23.09)％,and birth rates of(29.33±4.16)％,(38.67±4.81)％,and(17.00±3.46)％,respectively.When blastocysts and morulae were transferred non-surgically,the resulting pregnancy rates were(80.00±0.00)％and(46.67±11.55)％and the birth rates were(38.67±4.81)％and(10.22±2.77)％,respectively.Four strains(C57BL/6J,ICR,genetically modified mice A,genetically modified mice B)were used as donors for non-surgical embryo transfer,with resulting pregnancy rates of(66.67±11.55)％,(80.00±0.00)％,(73.33±11.55)％,and(80.00±0.00)％,and birthrates of(26.67±2.67)％,(38.67±4.81)％,(32.00±3.53)％,and(29.34±2.31)％,respectively.Fifteen pseudo-pregnant mice were transplanted surgically and 15 were transplanted non-surgically,with pregnancy rates of(80.00±0.00)％and(86.67±11.55)％,and birth rates of(38.67±4.81)％and(36.00±5.82)％,respectively.Conclusions Transfer device A resulted in a higher birth rate in this study.The embryo transfer efficiency was higher when 15 embryos were transferred into unilateral uterine horns of pseudo-pregnant 2.5-day recipients.Blastocyst-stage embryo transfer was more efficient than morula-stage transfer.There was no significant difference in efficiency between surgical and non-surgical embryo transfer procedures.

Feces and intestinal contents of pigs suspected with porcine epidemic diarrhea virus were collected from a farm in Minqin County,Gansu Province,China.After the suspected positive sam-ples were detected by RT-PCR,Vero cells were used to isolate and culture them in vitro.The suc-cessfully isolated virus was identified in the laboratory,and its whole genome sequence was ana-lyzed for genetic evolution.The pathogenicity was evaluated by animal regression test.The results showed that typical syncytial lesions could be observed when the PEDV-positive treatment solu-tion was inoculated with Vero cells in the 4th generation,and the virus titer in the 6th generation reached 10-4 75TCID50/mL.PEDV-like virions with a diameter of about 100 nm and a round shape with obvious capsular membranes and spikes were observed by electron microscopy.Whole genome sequencing analysis showed that the total length of this strain was 28 085 bp,which was far from the G1 subtype represented by the classical strain CV777(96.6％),and had a high homology with the G2b strains BC-2011-1,IA1,USA/Colorado/2013 and WELL(98.6％).This indicated that the strain belonged to the G2b epidemic strain.The animal regression test showed that the 5-day-old piglets developed vomiting,acute watery diarrhea,emaciation and mental depression within 12 h after the attack,and the symptoms worsened and died within 24 h.After autopsy,the infected piglets could be observed with stomach swelling,high intestinal heave,thin and transparent intesti-nal wall,and undigested milk clots inside.In summary,a PEDV G2b epidemic strain was success-fully isolated and identified in this study,and its whole genome sequence and pathogenicity were analyzed,providing research materials for future studies on PEDV gene function,pathogenic mech-anism and vaccine development.

To screen and identify the key host proteins interacting with the non-structural protein 15 (Nsp15) of porcine epidemic diarrhea virus (PEDV). The IP/pull-down assay and mass spectrometry were employed to screen and identify the host proteins interacting with Nsp15. The interaction between the host protein and Nsp15 was studied by co-immunoprecipitation and laser scanning confocal microscopy. Finally, Western blotting and RT-qPCR were employed to examine the interaction between SLC25a3 and PEDV. The recombinant eukaryotic expression vector pcDNA3.1(+)-Flag-Nsp15 was successfully constructed, and the host protein SLC25a3 interacting with PEDV Nsp15 was screened out. An interaction existed between SLC25a3 and Nsp15, and SLC25a3 significantly inhibited PEDV replication in a dose-dependent manner. SLC25a3 inhibits PEDV replication. The results of this study provide a basis for deciphering the role and mechanism of SLC25a3 in the host immune response to PEDV infection.
Porcine epidemic diarrhea virus/genetics* ; Viral Nonstructural Proteins/metabolism* ; Animals ; Swine ; Virus Replication ; Coronavirus Infections/veterinary* ; Swine Diseases/metabolism*

Many people affected by fragile X syndrome (FXS) and autism spectrum disorders have sensory processing deficits, such as hypersensitivity to auditory, tactile, and visual stimuli. Like FXS in humans, loss of Fmr1 in rodents also cause sensory, behavioral, and cognitive deficits. However, the neural mechanisms underlying sensory impairment, especially vision impairment, remain unclear. It remains elusive whether the visual processing deficits originate from corrupted inputs, impaired perception in the primary sensory cortex, or altered integration in the higher cortex, and there is no effective treatment. In this study, we used a genetic knockout mouse model (Fmr1^KO), in vivo imaging, and behavioral measurements to show that the loss of Fmr1 impaired signal processing in the primary visual cortex (V1). Specifically, Fmr1^KO mice showed enhanced responses to low-intensity stimuli but normal responses to high-intensity stimuli. This abnormality was accompanied by enhancements in local network connectivity in V1 microcircuits and increased dendritic complexity of V1 neurons. These effects were ameliorated by the acute application of GABA_A receptor activators, which enhanced the activity of inhibitory neurons, or by reintroducing Fmr1 gene expression in knockout V1 neurons in both juvenile and young-adult mice. Overall, V1 plays an important role in the visual abnormalities of Fmr1^KO mice and it could be possible to rescue the sensory disturbances in developed FXS and autism patients.
Animals ; Disease Models, Animal ; Fragile X Mental Retardation Protein/metabolism* ; Fragile X Syndrome/metabolism* ; Humans ; Mice ; Mice, Knockout ; Neurons/metabolism*

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×10^9.1 TCID₅₀/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.
Adenoviridae/genetics* ; Adenoviruses, Human/genetics* ; Animals ; Antibodies, Viral ; Capsid/metabolism* ; Capsid Proteins ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins/genetics* ; Serogroup ; Swine ; Viral Proteins ; Viral Vaccines/genetics*

Objective: To evaluate the role of morphine preconditioning on necroptosis during myocardial ischemia-reperfusion (I/R) injury in the rats with heart failure. Methods: Clean-grade adult male Sprague-Dawley rats, weighing 200-230 g, were injected with 2 mg/kg doxorubicin via the tail vein once a week for 6 consecutive weeks to establish the chronic heart failure model.Thirty rats with chronic heart failure at the end of 8th week were divided into 3 groups (n=10 each) using a random number table method: sham operation group (group S), I/R group and morphine preconditioning group (group MPC). Myocardial I/R was induced by occlusion of anterior descending branch of left coronary artery for 30 min followed by 120 min reperfusion in each group except group S. In group MPC, the rats were subjected to 3 cycles of 5-min infusion of 0.1 mg/kg morphine via the femoral vein at 5 min intervals before ischemia.The animals were sacrificed at the end of reperfusion, and the myocardial specimens were obtained for determination of the area at risk (AAR), infarct size (IS), expression of Fas mRNA (by quantitative real-time polymerase chain reaction) and expression of Fas, receptor-interacting protein 1 (RIP1) and RIP3 (by Western blot). The IS/AAR ratio was calculated. Results: Compared with group S, the IS and IS/AAR ratio were significantly increased at the end of reperfusion, and the expression of Fas protein and mRNA, RIP1 and RIP3 was up-regulated in group I/R (P<0.05). Compared with group I/R, the IS and IS/AAR ratio were significantly decreased at the end of reperfusion, and the expression of Fas protein and mRNA, RIP1 and RIP3 was down-regulated in group MPC (P<0.05). Conclusion The mechanism by which morphine preconditioning reduces myocardial I/R injury is related to inhibiting necroptosis in the rats with heart failure.

Objective To investigate the prevalence and drug resistance of clinical Klebsiella vari-icola ( K. variicola ) isolates and to illuminate the mechanism of drug resistance in carbapenem-resistant strains. Methods Clinical K. variicola isolates were identified with matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry ( MALDI-TOF MS ) . The antimicrobial susceptibility profile of these strains was determined using broth microdilution. Resistance genes carried by carbapenem-resistant K. vari-icola strains were detected by PCR with specific primers. Multilocus sequence typing ( MLST) was used for molecular typing. A pan-drug resistant strain which was isolated from cerebrospinal fluid sample was ana-lyzed with whole genome sequencing ( WGS) . Results Twenty-six isolates were identified as K. variicola by MALDI-TOF MS. Results of the antimicrobial susceptibility test showed that there were 15. 4％ (4/26) re-sistant to carbapenem and 11. 5％ (3/26) unsusceptible to tigecycline. These strains were highly suscepti-ble to amikacin and gentamicin, which accounted for 96. 2％ (25/26). As for the third-and fourth-genera-tion cephalosporins, the resistance rate was 23. 1％ (6/26). All of the four carbapenem-resistant isolates carried the resistance genes of blaIMP-4 , qnrA/B and blaTEM , and one of them was also positive for blaNDM-1 gene. The fosfomycin resistance gene, fosA, was detected in three of them. Molecular typing analysis indica-ted these isolates belonged to two sequence types ( ST) of ST357 ( three strains) and ST1737 ( one strain) . Two plasmids were obtained from the pan-drug resistant strain by WGS, including IncFⅡ/FIB( k) type plas-mid (160 kb) that was highly homologous to LMG 23571 plasmid (GenBank: CP013986. 1) and IncHⅠ1B/FIB type plasmid (260 kb) sharing high homology with pIMP4 LL34 (GenBank: CP025964. 1). Be-sides the resistance genes mentioned above, the two plasmids also carried a variety of other genes that media-ted the resistance to aminoglycosides (strB, strA, armA, aac3-Ⅱd, aadA2), macrolides (msrE, mphE), chloramphenicol (catA2), sulfonamides (sulⅠ) tigecycline (tetA variant) and trimethoprim (dfrA16). However, no virulence genes were detected. Conclusions In general, the resistance profile of K. variicola was similar to that of Klebsiella pneumoniae, but the differences were that carbapenem-resistant K. variicola strains mainly belonged to ST357 and the leading causes of resistance were carrying the genes encoding IMP-4 and NDM-1 metalβ-lactamases. WGC analysis revealed that the pan-drug resistant K. variicola strain carried multiple drug resistance genes without virulence determinants, which might be resulted from the evo-lution of drug resistance.