This study aims to establish a high-performance size-exclusion chromatography (HPSEC) method for determining the content of the classical swine fever virus (CSFV) E2 protein and screen the optimal stabilizer to enhance the stability of this protein. The optimal detection conditions were determined by optimizing the composition of the mobile phase, and characteristic chromatographic peaks were identified by SDS-PAGE and Western blotting. The specificity, repeatability, precision, linearity, limit of detection (LOD), and limit of quantitation (LOQ) of the method were assessed. The method established was used to determine the content of CSFV E2 protein antigen and vaccine. Differential scanning fluorimetry (DSF) was employed to screen the buffer system, pH, and salt ion concentrations, and sugar, amino acid, and alcohol stabilizers were further screened. The results showed that using a 200 mmol/L phosphate buffer provided the best column efficiency. An antigen-specific chromatographic peak appeared at the retention time of 18 min, which was identified as the CSFV E2 protein by SDS-PAGE and Western blotting. The method exhibited high specificity for detecting the CSFV E2 protein, with no absorbance peak observed in the blank control. The relative standard deviation (RSD) of the peak area for six repeated injections of the CSFV E2 protein was 0.74%, indicating good repeatability of the method. The RSD for repeated detection of two different concentrations of CSFV E2 protein samples by different operators at different time points was less than 2%, suggesting good intermediate precision of the method. The peak area of the CSFV E2 protein was linearly related to its concentration, with the regression equation showing R² of 1.000. The LOD and LOQ of the method were 14.88 μg/mL and 29.75 μg/mL, respectively. Application of the developed method in the detection of three batches of CSFV E2 protein antigen and three batches of vaccine demonstrated results consistent with those from the bicinchoninic acid (BCA) assay, which meant that the method could accurately determine the content of CSFV E2 protein antigen and vaccine. The DSF method identified 50 mmol/L Tris-HCl at pH 8.0 as the optimal buffer, and the addition of sugar and alcohol stabilizers further improved the stability of the CSFV E2 protein. The HPSEC method established in this study is simple, fast, and exhibits good accuracy and repeatability, enabling precise measurement of the CSFV E2 protein content. It is expected to play a crucial role in the quality control of the CSFV E2 vaccine. Furthermore, the strategy for improving the CSFV E2 protein stability, identified through DSF screening, has significant implications for enhancing the stability of the CSFV E2 vaccine.
Classical Swine Fever Virus/chemistry* ; Chromatography, Gel/methods* ; Animals ; Swine ; Viral Envelope Proteins/immunology*

Objective To investigate the progression of breast cancer in mice induced by chronic binding stress and the regulatory mechanism of Xiaoyao SAN based on TGF-β1/CD147 signal pathway.Methods 40 BABL/c mice were randomly divided into tumor group,model group,Xiaoyaosan group and Mifepristone group,and then 4T1 cell line was inoculated into the armpits of each group of mice.After Tumor formation,mice in all groups except tumor group were subjected to chronic restraint stress for 21 days.Meanwhile,mice in Xiaoyaosan and Mifepristone groups were gavaged with the corresponding drugs,and mice in the other two groups were gavaged with normal saline.After the modeling,the mice were sacrificed after anaesthesia.The weight and volume of the tumors and visceral index of the mice were measured.The contents of serum tumor markers(CA199,CEA,VEGF),serum neurotransmitters(DA and CORT),and inflammatory mediators(TGF-β1 and IL-10)in tumor tissues were detected by Elisa.The expressions of iNOS and Arg-1,the polarization markers of macrophages,and the expressions of CD147 and its downstream signaling molecules MMP2,MMP9 and VEGF in tumor tissues were all detected by immunohistochemistry and Western blot.Results Compared with tumor group,in model group,tumor weight and volume,serum CA199,CEA,VEGF,CORT content,tumor TGF-β1 and IL-10 content were significantly increased;visceral index and serum DA content were significantly reduced;the expression of M2-type polarization marker Arg-1 in tumor macrophages was significantly increased,while the expression of M1-type polarization marker iNOS was significantly decreased;the expressions of CD147 and its downstream signaling molecules MMP2,MMP9 and VEGF were significantly increased.Both Xiaoyaosan and mifepristone could effectively reverse the above changes.Conclusion The mechanism of chronic restraint stress promoting breast cancer progression in mice is related to the increased release of TGF-β1 from M2-type polarization of tumor-associated macrophages,which activates CD147 and its downstream related signals.Xiaoyaosan could relieve the M2-type polarization of macrophages caused by increased corticosterone under stress conditions,reduce the production of TGF-β1,inhibit CD147 and its downstream signal,and thus inhibit the progression of breast cancer caused by chronic restraint stress in mice.

Objective To study the core behavioral symptoms,biological indicators,and pathological changes of a mouse model of breast cancer complicated with depression induced using 4T1 breast cancer cell inoculation combined with chronic restraint stress(CRS).Methods BABL/c mice were randomly divided into Control,Stress,Tumor,and stress combined with tumor(S+T)groups.Mice in the tumor and S+T groups were inoculated under the front legs with breast cancer 4T1 cells.After tumor formation,mice in the stress and S+T groups were subjected to CRS for 21 days.The body weight and food intake of each group were monitored during modeling.After the experiment,the occurrence of depression-like behavior of mice in each group was evaluated by sucrose preference test,open field test,elevated plus-maze test,and forced swimming test.After the mice were decapitated,the weights and volumes of the tumors were measured.Concentrations of serum tumor markers,including carbohydrate antigen(CA199),carcinoembryonic antigen(CEA),and vascular endothelial growth factor(VEGF),and related neurotransmitters,including 5-hydroxytryptamine(5-HT),norepinephrine(NE),and corticosterone(CORT),were determined using ELISA.HE staining was used to observe histopathological changes to the hippocampus and tumor.Results In S+T group mice,body weight and food intake were significantly decreased,tumor weight and volume were significantly increased,serum tumor marker(CA199,CEA,VEGF)levels were significantly increased,enthusiasm and desire to explore a new environment were reduced,stress and despair behaviors were significantly increased,and levels of the serum neurotransmitters 5-HT and NE and levels of CORT were significantly increased.In addition,the cell arrangement in the tumor tissue was loose,the amount of intercellular substance decreased,the pathological nuclear classification phase was increased,the arrangement and morphology of neurons in the CA3 region of the hippocampus were disordered,and there were obvious nuclear vacuolation-like changes.Conclusions A mouse model of breast cancer complicated with depression induced by 4T1 breast cancer cell inoculation combined with CRS showed the typical dual symptoms and biological indicators of breast cancer and depression and can be used as a good reference model for experimental studies of breast cancer complicated with depression.

Pathological mood changes,mainly depression,occurring during the diagnosis and treatment of breast cancer are referred to as breast cancer-related depression(BCRD).Numerous epidemiological and clinical studies have confirmed that BCRD is a complex condition that is difficult to treat and has a poor prognosis.Most existing clinical treatments involve the use of postoperative chemotherapy for breast cancer,and antidepressant drugs,which treat breast cancer and depression as two independent diseases and have various disadvantages such as low efficiency and strong adverse reactions.Traditional Chinese Medicine(TCM)has a unique value in the prevention and treatment of BCRD via its ability to regulate multiple pathways and targets using multiple components at the same time.In this paper,we review the mechanism of BRCD and the therapeutic mechanisms of TCM from the aspects of neurological disorders,inflammatory immune response,and intestinal flora disorders,with a view to providing references for the clinical application and research of TCM in the treatment of BCRD.

Objective:To explore the curative effects of ultrathin anterolateral femoral flap in one-stage split-finger repair of palmar combined with multiple finger wounds.Methods:A retrospective observational study was conducted. From October 2016 to June 2018, 20 patients with wounds on palms and multiple fingers who met the inclusion criteria were admitted to Zhengzhou First People's Hospital, including 15 males and 5 females, aged 18 to 77 years. After debridement, the wound area was 8 cm×4 cm-17 cm×12 cm. The wound was repaired by ultrathin anterolateral femoral flap with area of 9 cm×5 cm to 19 cm×13 cm. According to the wound condition of finger, the finger division was performed in one stage, and the length-to-width ratio of the split-finger flap was 2.0:1.0-2.5:1.0. During the surgery, the descending branches of lateral circumflex femoral artery and accompanying vein of flap were anastomosed end-to-end to the radial artery and vein in the recipient area, respectively, and the anterolateral femoral cutaneous nerve of flap was bridged with the superficial branch of radial nerve in the recipient area. The wounds in the donor area of flap in 14 patients were sutured directly, the wounds in the donor area of flap in 3 patients were repaired by relay superficial iliac circumflex artery perforator flap, and the wounds in the donor area of flap in 3 patients were covered by free trunk medium-thick skin graft. The survival of flap, occurrence of vascular crisis and other complications, and healing of wounds in the donor area of flap were recorded. The appearance of flap, scar hyperplasia in the donor and recipient areas and the patients' satisfaction with the shape and function of the donor and recipient areas were followed up. In 1 year after surgery, the two-point discrimination distance of the flap was measured, and the recovery of hand function was evaluated by the trial standard for the evaluation of the functions of the upper limbs of the Hand Surgery Society of the Chinese Medical Association.Results:The flaps of 17 patients survived without vascular crisis or other complications after surgery. The flap of 1 patient had poor blood circulation and partial necrosis, and the wound was healed 14 days after dressing change and grafting of split-thickness skin graft from head. Two patients had mild cyanosis at the margin of flap after surgery, which disappeared spontaneously 5 days later. Incisions at donor site, relay flaps, and skin grafts of all patients survived well. After surgery, the color and texture of flap were basically the same as that of the normal skin of hand, and linear scars were observed in the donor and recipient areas. The patients were satisfied with the recovery of appearance and function of donor and recipient areas. After 1 year of follow-up, the patients' hand sensory function recovered well, the two-point discrimination distance of flap was 4-6 mm, and the recovery of hand function was evaluated as excellent in 18 cases and good in 2 cases.Conclusions:The ultrathin anterolateral femoral flap in repairing the palmar combined with multiple finger wounds in one-stage split-finger can significantly reduce the number of surgeries and improve the function and beauty of the hand, so it is worthy of clinical promotion.

Objective:To establish the method for detecting lower respiratory infections (LRIs) bacterialpathogens using nanopore sequencing, and evaluate the feasibility of this method.Methods:Bronchoalveolar lavage fluid (BALF) samples from 33 patients with LRIs who visited the Department of Respiratory and Critical Care Medicine of Beijing Hospital from July 2019 to September 2020 were collected.Nanopore 16S amplicon sequencing were performed on these samples. In order to evaluate the clinical value of the nanopore sequencing, χ 2 test was used to analyze the pathogen differences between the detection rate and pathogen types results found with using the nanopore 16S sequencing and the results found with bacterial culture. Results:The process and method of nanopore sequencing used in the detection of the LRIs pathogens were established. The pathogen detection rate of the 16S sequencing was higher than that of the traditional bacterial culture (75.8% [25/33], 45.5% [15/33], χ2=5.140, P<0.05). From the 25 positive samples found with nanopore 16S sequencing, 16 pathogens were detected, including Haemophilus parainfluenzae, Haemophilus influenzae, Streptococcus pneumoniae, Streptomonas maltophilia, Acinetobacter baumannii, and Acinetobacter junii, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, Enterococcus gallinarum, Corynebacterium striatum, Mycobacterium paraintracellulare, Serratia marcescens, Achromobacter insuavis, Citrobacter murliniae and Mycoplasma pneumoniae. More than 6 pathogens were tested in clinical culture, including Haemophilus parainfluenzae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and Streptomonas maltophilia (χ2=7.949, P<0.05). 16S sequencing aligned to species level sequences accounted for 80.0 (60.0, 86.0)% of the genus level. The results obtained by using16S sequencing and bacterial culture were consistent in 11 (33.3%) samples. Conclusions:Nanopore 16S amplicon sequencing can quickly identify pathogenic bacteria from BALF in LRIs patients. Nanopore 16S amplicon sequencing has a high detection rate, it can detect more pathogens than traditional bacterial culture, and it can also identify most bacteria to the species level. This technology is a very promising platform with broad application prospects.

OBJECTIVE: To explore the genetic basis for a pedigree affected with Alport syndrome. METHODS: Next generation sequencing and Sanger sequencing was carried out to detect potential variant of the COL4A5 gene among members from the pedigree and 100 unrelated healthy controls. RESULTS: A novel missense c.3293G>T (p.Gly1098Val) variant was found in the COL4A5 gene among 6 affected members but not the unaffected members of the pedigree or the 100 healthy controls. According to the American College of Medical Genetics and Genomics standards and guidelines, the c.3293G>T variant was classified as pathogenic (PP1-strong+PM1+PM2+PP3+PP4). CONCLUSION By destructing the Gly-X-Y structure of its protein product, the c.3293G>T variant of the COL4A5 gene probably underlies the Alport syndrome in this pedigree. Above finding has enriched the spectrum of COL4A5 variants.

OBJECTIVE: To analyze variant of IDS gene in a pedigree affected with mucopolysaccharidosis type II (MPS II). METHODS: The proband was subjected to next generation sequencing and Sanger sequencing to identify potential variants. Suspected variant was analyzed by its co-segregation with the disease in the pedigree. Its impact on mRNA splicing was analyzed by using reverse transcription PCR (RT-PCR). RESULTS: A hemizygous IVS1-3T>G variant was found in the IDS gene in the proband. RT-PCR results revealed two abnormal cDNA fragments of 600 bp and 300 bp. The 600 bp fragment had inserted 216 nucleotides at the 3' end of intron 1, while the 300 bp fragment had lost 109 nucleotides at the 5' end of exon 2, which resulted in two truncated proteins comprising 38 and 92 amino acids, respectively, instead of the normal product (550 amino acids). The proband and his mother were respectively hemizygous and heterozygous for the variant. The same variant was not found among 100 normal controls. CONCLUSION The IVS1-3T>G variant of the IDS gene probably underlies the MPS II in this pedigree by causing reduction or elimination of the IDS protein.

Objective Toexploretheimagingcharacteristicsofileocecaltuberculosisbycontrast-enhancedCT.Methods TheCT findingsof16ileocecaltuberculosispatientswereretrospectivelyanalyzed.AllcasesunderwentplainandenhancedCTscan.Results 14casesshowedinhomogeneousthickeningoftheintestinalwallandnarrowingofthelumen,and2casesshowedextensiveanduniform thickeningoftheintestinalwallwithnoobviousnarrowingofthelumen.Theenhancedimagesshowedthat13caseswereobviously heterogeneousenhancement,2caseswerering-likeenhancement,and1casewashomogeneousenhancement.Blurredserosasurface wasseeninallpatients,withsurroundingexudationin14casesandperitoneuminvolvementin12cases.Inallcases,enlargedlymph nodeswerefoundneartheabdominalaortaandonmesentery,withhomogeneousenhancementin10cases,annularenhancementin5casesand calcificationin1case.Conclusion Ileocecaltuberculosisismainlycharacterizedbyinhomogeneousthickeningandheterogeneousenhancementofthe intestinalwall,aswellasnarrowingofthelumen.Thereisoftenobviousexudationaroundtheintestines.Ileocecaltuberculosisoften combineswithenlargedlymphnodesandintrapulmonarytuberculosis.TheenhancedCTscanisofgreatvaluefortheevaluationofileocecal tuberculosis.

Objective To evaluate the efficacy and safety of tip-flexible ureterorenoscope (tf-URS) with holmium laser for one-stage management of parapelvic cyst.Methods The clinical data of 26 patients treated with tf-URS incision and drainage using holmium laser from February 2016 to August 2018 were reviewed.The study were including 15 male and 11 female patients,ranging from 32 to 68 years old,with an average of 53.5 years old.There were 24 cases of unilateral single renal parapelvic cyst and 2 cases of bilateral single renal parapelvic cyst.There were 4 cases in 26 cases with unilateral single renal parapelvic cyst and contralateral single renal cyst,2 cases with unilateral single renal parapelvic cyst and ipsilateral kidney of stones.The diameter of parapelvic cyst was 3.4-5.6 cm,average 4.8 cm.All patients had undergone holmium laser endo-decortication of parapelvic cyst by tf-URS.With general anesthesia,tf-URS accessed pelvis retrogradely and decorticated parapelvic cyst with 200 μm Holium laser to drainage the cyst to pelvis.If the tf-URS was not placed successfully for the first time,Double-J tubes were retained for 1-2 weeks before treatment.The operative time,hospitalization time,blood loss,postoperative complications and clinical symptoms were collected and analyzed.Results The one-time access success rate of insertion of ff-URS was 88.5％ (23/26).All operations were successful without severe complications.The average time of operation was 17.2 min,ranging from 11 to 25 min.In 3-30 months follow-up,the cysts disappeared in 22 patients and reduced by more than one half in 1 patients.Flank pain relieved in 19 patients.Conclusions Holmium laser endo-decortication of parapelvic cyst by tf-URS could be a simple,minimally invasive,safe and effective method for parapelvic cyst,which is worthy of further promotion and application in clinical practice.