In order to explore the role of BspE protein in Brucella infection,yeast two-hybrid tech-nique was used to screen host cell proteins that interact with BspE protein.The constructed BspE recombinant plasmid pGBKT7-BspE was used as bait plasmid to hybridize with the RAW264.7-cD-NA library of mouse mononuclear macrophages by yeast two-hybridization technique.The positive clones were extracted by plasmid,sequenced and co-immunoprecipitation to determine the host cell proteins that could interact with BspE.The subcellular localization of BspE proteins was analyzed by confocal laser microscopy.The physical and chemical properties,protein structure and function of BspE interacting proteins were analyzed by bioinformatics.The siRNA for one of the BspE inter-acting proteins was synthesized,the expression of its gene was silenced in HEK293T cells,and the silenced cells was infected with Brucella M5-90 and the number of intracellular bacteria was coun-ted.The results showed that the decoy plasmid pGBKT7-BspE was successfully constructed,and the plasmid could express BspE protein in yeast.Eight positive clones were obtained from the host cell genome library by yeast two-hybridization.The positive clones were identified as RBM27 and PCBP1 by sequencing,backcross and co-immunoprecipitation.Bioinformatics was used to predict the cell location,protein structure and amino acid composition of RBM27 and PCBP1.After siRNA interference,the expression level of PCBP1 was significantly decreased and the amount of M5-90 in the cell was increased.Brucellosis secreted protein BspE interacts with host proteins RBM27 and PCBPl,and PCBP1 negatively regulates the proliferation of Brucellosis.

Objective To investigate the predictive value of serum lipoprotein-associated phospho-lipase A2(Lp-PLA2)for long-term prognosis of elderly patients with stable coronary heart dis-ease(CHD).Methods A retrospective trial was conducted on 198 patients with stable CHD ad-mitted to our hospital from January 2016 to December 2018.All of them were followed up for 5 years,and divided into adverse cardiovascular event group(n=42)and control group(n=156)according to whether adverse cardiovascular events occurred during follow-up.Clinical features and Lp-PLA2 level were compared between the two groups.The predictive value of Lp-PLA2 for adverse cardiovascular events was analyzed in elderly patients with stable CHD within 5 years.Re-sults The adverse cardiovascular event group had significantly older age(74.95±7.02 vs 70.17±6.30 years,P=0.000),larger proportions of diabetes(54.76％vs 27.56％,P=0.001),of coronary artery stenosis ≥75％(69.05％vs 47.44％,P=0.013)and of left ventricular ejection fraction(LVEF)＜50％(50.00％vs 28.21％,P=0.008),and higher Lp-PLA2 level(478.38±187.54 U/L vs 308.17±126.73 U/L,P=0.000)when compared with the control group.The AUC value of age and Lp-PLA2 was 0.683(95％CI:0.590--0.776,P＜0.001)and 0.763(95％CI:0.677--0.848,P=0.763),respectively,in predicting the long-term prognosis in elderly patients with stable CHD.Multivariate logistic regression analysis showed that age,diabetes,coronary artery stenosis ≥75％,LVEF＜50％and Lp-PLA2 were independent influencing factors for adverse cardiovascular events within 5 years in elderly patients with stable CHD(P＜0.05,P＜0.01).Con-clusion Increased Lp-PLA2 level is associated with adverse cardiovascular events within 5 years in patients with stable CHD.

Objective:To investigate the relationship between inherited protein S deficiency (PSD) and cerebral venous sinus thrombosis (CVST) by phenotype and gene mutation analysis of 2 inherited PSD pedigrees with nonsense mutations.Methods:Retrospective analysis of clinical and imaging data of 2 patients diagnosed with CVST who were treated in the Department of Neurology, the First Affiliated Hospital of Wenzhou Medical University in July and October 2023 was carried out. The peripheral blood samples were collected from proband 1 and her family members (3 subjects, 2 generations in total), and proband 2 and his family members (8 subjects of 3 generations in total). Their protein S (PS) activity (PS:A), the content of total PS antigen (TPS:Ag) and free PS antigen (FPS:Ag) were measured for definite diagnosis. Polymerase chain reaction was done in all 15 exons of the active PROS1 gene and its 5′ and 3′ untranslated regions and the amplification products were analyzed by direct sequencing. The results were compared with human PROS1 reference sequences, using Chromas software to find the mutation sites. Results:The proband 1 is a female, and the proband 2 is a male. Both of them had young onset and the clinical presentation of CVST. The PS:A level was reduced to 29% in the proband 1 and reduced to about 35% in her mother; PS:A was reduced to 21%-27% in the proband 2 and his 6 family members; a decline in the same proportion of TPS:Ag and FPS:Ag was found in the 2 probands and their family members, therefore they were primarily diagnosed as typeⅠPSD. Gene analysis showed that the proband 1 and her mother had a nonsense mutation of c.1680T>A in exon 14 (p.Tyr560 *) of the PROS1 gene; the proband 2 and his 6 family members had a nonsense mutation of c.1687C>T in exon 14 (p.Gln563 *) of the PROS1 gene. Conclusion:The reduced protein S levels in PSD patients and their family members may be associated with the p.Tyr560 * and p.Gln563 * nonsense mutations of the PROS1 gene, and the clinical manifestations of CVST in PSD patients may be related to these 2 nonsense mutations.

Objective:To investigate the influence of the interaction between gastric cancer (GC) cell-derived exosomes and hepatic Kupffer cells on GC with liver metastasis and analyze the potential mechanism.Methods:Cells with high hepatic metastatic potential (MKN 45-HL) were constructed from a parental GC cell line (MKN 45) using a nude mouse model and methods of viral transfection and flow sorting. Exosomes were collected using ultra-centrifugation and characterized by electron microscopy, nanoparticle tracking system and Western blot. A nude mouse model of liver metastasis induced by GC cell-derived exosomes was constructed, and the development of liver metastases was monitored by live imaging. The regulatory effects of GC cell-derived exosomes on macrophage polarization were assessed by cell culture, qRT-PCR, and immunofluorescence staining. Using the omics analysis of exosomal miRNA and qRT-PCR, the molecular targets by which exosomes specifically promoting macrophage M2 polarization were screened and validated.Results:GC cell-derived exosomes were mainly concentrated in the liver, most of which were ingested by intrahepatic macrophages, and could promote macrophages to M2 polarization in both in vitro culture and nude mice. Both groups of mice trained with MKN 45 and MKN 45-HL exosomes showed obvious liver metastases after mouse forestomach carcinoma (MFC) cells injection through the spleen, and MKN 45-HL exosomes showed a much stronger ability to promote hepatic macrophage M2 polarization and liver metastasis of MFC cells. Moreover, the miRNA omics analysis revealed a lot of differentially expressed miRNAs between MKN 45-derived and MKN 45-HL-derived exosomes. The expression of miR-519a-3p increased significantly in the exosomes derived from MKN 45-HL cell line and the clinical serum of GC patients with liver metastasis. It was found that miR-519a-3p could be internalized by macrophages through exosomes delivery. Furthermore, the miR-519a-3p in exosomes from patient′s serum had a predictive value for GC with liver metastasis and was closely associated with the prognosis of GC patients with liver metastasis. Conclusions:GC cell-derived exosomes trigger M2-like polarization of hepatic Kupffer cells via miR-519a-3p, thus promoting the progression of liver metastasis in GC and playing a critical role in shaping the pre-metastatic liver niche in gastric cancer. This study provides a new perspective on the mechanism of GC with liver metastasis and reveal potential targets for future therapeutic strategies.

[Objective]To investigate primary and secondary transfer of exfoliated cells from human hands on non-porous substrates such as plastic steering wheel or computer mouse.[Methods]DNA detection sensitivity and detection limit for mixed DNA profiling were examined to understand our laboratory's ability to test for trace DNA.Forensic swabs were used to collect samples from volunteers'one-hour-long unwashed hands,substrates touched by volunteers'immediately or 30 min following shaking hands,and individual A's daily-use substrates touched by individual B and then by individual A again.Simulations were conducted to assess the potential for introduction of another person's exfoliated cells from hands into routine casework samples.[Results]Our laboratory can obtain a full DNA profile from as little as 0.020 ng of DNA and detect minor components in a 1:9 mixed DNA sample.85%of samples from unwashed hands yielded a full DNA profile.Primary transfer of a full DNA profile was found in 77%of substrates touched by volunteers'dominant hand 30 min after hand washing,allowing differentiation between good and poor shedders,with no significant difference in genders and substrate types.75%of substrates touched 30 min after hand washing and then immediately following handshaking yielded the other individual's DNA profile(secondary transfer),with the number of short tandem repeat(STR)loci detected ranging from 0 to 23;the percentage and number decreased substantially when the substrates were touched 30 minutes later.No foreign DNA was detected in routine casework samples with introduced exfoliated cells from hands.When two individuals took turns touching items with their hands,the major contributor to the DNA profile was not always the individual who made the last contact.[Conclusions]Primary and secondary DNA transfer can be detected on non-porous substrates,and based on the deposit of hand exfoliated cells,individuals can be categorized as good or poor shedders,which is an important factor affecting detection of DNA transfer.Besides considering the laboratory's DNA detection sensitivity,if DNA is detected on substrates by hand contact,we need to take into account the potential for secondary transfer at different levels of activity when interpreting the results.

Objective:Molecular mechanisms underlying compound heterozygous mutations in a patient with inherited antithrombin (AT) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in November 2018 with a one-day history of sudden syncope and limb twitching. Peripheral venous blood was collected from the proband and members of his lineages, totaling nine persons across three generations, and a family lineage survey was conducted. AT activity (AT:A) was measured using a chromogenic substrate assay, while AT antigen (AT:Ag) was detected through an immunoturbidimetric assay. Mutation sites were identified by means of Sanger sequencing of the SERPINC1 gene, and silico tools were applied to predict the mutational conservation and hydrophobicity changes. Recombinant plasmid expression vectors were constructed and transfected into HEK293T cells for in vitro overexpression studies. The recombinant AT protein was characterized using Western Blotting, ELISA, and cellular immunofluorescence assays.Results:The proband was a 21-year-old man with type Ⅰ AT deficiency. His AT:A was 33%, along with a corresponding reduction in AT:Ag. The genetic analysis revealed there was a heterozygous insertion mutation at c.318_319insT (p.Asn107*) and a heterozygous missense mutation at c.922G>T (p.Gly308Cys) in exons 2 and 5, respectively. These mutation sites were entirely conserved among the homologous species. Additionally, hydrophobicity studies showed that the p.Gly308Cys mutation will decrease the hydrophilicity of amino acid residues 307-313. The in vitro expression studies indicated a reduction of approximately 46.98%±2.94% and 41.35%±1.48% in the amount of recombinant protein AT-G308C in transfected cell lysates and culture supernatants, respectively. Treatment with the proteasome inhibitor (MG132) restored the cytoplasmic levels of AT-G308C protein to a level similar to that of wild-type protein. However, neither cell lysate nor culture supernatant demonstrated the presence of the recombinant protein AT-N107*. Conclusions:The heterozygous insertion mutation of p.Asn107* and the heterozygous missense mutation of p.Gly308Cys have been associated with reduced AT levels in proband. The p.Asn107* heterozygous insertion mutation may initiate the degradation of mRNA via nonsense mutation-mediated mechanisms, which would remove the defective transcripts, as well as the p.The Gly308Cys heterozygous missense mutation may cause the AT protein to undergo proteasome-dependent degradation by modifying the hydrophobicity of nearby residues in the cytoplasm.

Objective To discuss the clinical application of intravoxel incoherent motion-diffusion weighted imaging(IVIM-DWI)in evaluating the efficacy and prognosis of transcatheter arterial chemoembolization(TACE)using different embolization materials for the treatment of hepatocellular carcinoma(HCC).Methods The clinical data of a total of 84 patients with inoperable HCC,who received TACE treatment at the Second Affiliated Hospital of Shandong First Medical University of China and the First Affiliated Hospital of Xinjiang Medical University of China between June 30,2019 and December 30,2022,were collected.According to the patient's condition,different embolization materials were used during TACE.IVIM-DWI check-up was performed before treatment as well as at one,6,12 months after treatment.Based on the fixed b-value set by IVIM-DWI sequence,the ADC value of the order index model for different embolization materials and the pure diffusion coefficient of double exponential model(D value),the pseudo-diffusion coefficient(D*value)and perfusion fraction(f value)were analyzed.According to modified Response Evaluation Criteria in Solid Tumors(mRECIST)and the embolization material used,the patients were divided into the stable group and progression group,and the changes in the ADC value,D value,D*value and f value were compared between the two groups.Multivariate Cox regression analysis was used to analyze the four clinical parameters(including age,Child-Pugh grade,AFP level and tumor size)and the eight functional quantitative indexes(including preoperative and postoperative ADC value,D value,D* value and f value)so as to determine the IVIM parameters with prognostic predictive value.Receiver operating characteristic(ROC)was adopted to analyze the diagnostic value and cut-off value of IVIM parameters with predictive value.Results After treatment,the ADC value of drug-loaded microspheres group(n=36)was significantly higher than that of iodized oil group(n=27),the D*value of drug-loaded microspheres group and iodized oil group was remarkably lower than that of PVA particle group(n=21),and the f value of drug-loaded microspheres group was strikingly lower than that of iodized oil group,the differences were statistically significant(all P＜0.01).In the stable group,the efficacy of drug loaded microspheres group was obviously better than that of the iodized oil group and the PVA particle group.In the progression group,the iodized oil group was more likely to develop disease progression than the drug-loaded microspheres group and the PVA particle group.The preoperative f value in the stable group was prominently higher than that in the progression group(P=0.005),and the postoperative ADC value in the stable group was obviously higher than that in the progression group(P=0.029).ROC analysis showed that the median follow-up time in the drug-loaded microspheres group,iodized oil group,and PVA particle group was 30,19,and 26 months respectively,the overall average survival time was 25 months,and the difference was statistically significant(P＜0.01).Multivariate Cox regression analysis showed that the preoperative D value(AUC=0.878),D*value(AUC=0.554)and postoperative D value(AUC=0.791),D*value(AUC=0.552),f value(AUC=0.467)were the independent factors affecting the short-term efficacy of TACE(all P＜0.05).The preoperative and postoperative D value had higher diagnostic efficacy,while a preoperative D value of＜0.505×10-3 mm2/s and a postoperative D value of＜0.785×10-3 mm2/s predicted a poor prognosis.Conclusion The preoperative and postoperative D value is the optimal parameter for predicting the curative efficacy of TACE using different embolization materials for the treatment of HCC.

Objective:To investigate the clinical efficacy of da Vinci Xi surgical system assisted programmed six-hole method anterior resection of rectal cancer.Methods:The retrospec-tive cohort study was conducted. The clinicopathological data of 102 patients with middle and low rectal cancer who were admitted to the Affiliated Hospital of Xuzhou Medical University from August 2020 to June 2021 were collected. There were 62 males and 40 females, aged (53±12)years. Of the 102 patients, 51 cases undergoing da Vinci Xi surgical system assisted programmed six-hole method anterior resection of rectal cancer were divided into the robotic group and 51 cases undergoing laparoscopic anterior resection of rectal cancer were divided into the laparoscopic group. Observa-tion indicators: (1) treatment; (2) postoperative pathological examination; (3) follow-up. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the independent sample t test. Measurement data with skewed distribution were represented as M(range), and comparison between groups was conducted using the Mann-Whitney U test. Count data were described as absolute numbers, and comparison between groups was conducted using the chi-square test or Fisher exact probability. Repeated measurement data were analyzed using the repeated ANOVA. Results:(1) Treatment. All patients of the two groups under-went radical resection of rectal cancer successfully, and none of patient with intraoperative blood transfusion, conversion to open surgery, and death within 30 days after surgery. The operation time, volume of intraoperative blood loss, number of lymph nodes dissected, time to postoperative first flatus, time to postoperative first liquid food intake, time to postoperative catheter removal, cases with postoperative pain grading as grade 1, grade 2, grade 3, grade 4, cost of treatment were (170±12)minutes, (73±50)mL, 23±6, (35.1±9.4)hours, (2.1±0.8)days, (2.9±2.7)days, 13, 15, 17, 6, (7.1±4.5) ten thousand yuan in patients of the robotic group, versus (153±22)minutes, (119±66) mL, 15±4, (40.7±1.9)hours, (2.9±0.4)days, (5.3±2.1)days, 6, 7, 26, 12, (6.7±1.6) ten thousand yuan in patients of the laparoscopic group, showing significant differences in the above indicators between the two groups ( t=6.79, -4.46,20.09, -3.01, -5.54, -16.69, Z=-2.87, t=4.22, P<0.05). (2) Postoperative patho-logical examination. The tumor diameter, length of specimen resected, distance of upper resection margin to tumor, distance of lower resection margin to tumor, cases with mesorectal specimens as integrity and mostly integrity, cases with tumor differentiation as high differentiation, moderate differentiation, low differentiation, cases with postoperative TNM staging as stage Ⅰ, stage Ⅱ, stage Ⅲ were (3.8±1.1)cm, (18.7±3.2)cm, (11.8±3.6)cm, (2.7±0.8)cm, 48, 3, 4, 41, 6, 6, 17, 28 in patients of the robotic group, versus (3.7±1.0)cm, (18.3±2.8)cm, (10.2±2.7)cm, (2.5±0.6)cm, 46, 5, 6, 39, 6, 5,20, 26 in patients of the laparoscopic group, showing no significant difference in the above indicators between the two groups ( t=1.72, 1.29, 1.64, 1.11, χ2=0.14, Z=-0.42, -0.26, P>0.05). Cases with positive circumferential margin and cases with destruction of mesentery was 0 and 0 in patients of the robotic group, versus 1 and 1 in patients of the laparoscopic group, showing no significant difference in the above indicators between the two groups ( P>0.05). (3) Follow-up. All patients in the two groups were followed up for 12 months after surgery and none of patient had postoperative local recurrence and distant metastasis of tumors. The anal incontinence score, low anterior resection syndrome score, international prostate symptom score, night urination score, international index of erectile score, female sexual function index score in patients of the robotic group were 0, 12.25±1.08, 4.43±0.33, 0.49±0.09, 24.07±2.75, 65.84±1.79 before surgery and 1.34±0.11, 18.11±3.54, 4.03±0.26, 1.08±0.28, 22.63±2.03, 38.57±6.13 at postoperative 12 months, respectively. The above indicators in patients of the laparoscopic group were 0, 12.60±1.11, 4.56±0.36, 0.46±0.07, 23.11±2.77, 66.31±1.73 before surgery and 1.99±1.33,20.85±6.19, 6.43±1.78, 2.27±0.23, 21.00±2.73, 27.62±8.20 at postoperative 12 months, respectively. There were significant differences in the above indicators between the two groups ( P<0.05). Conclusions:The oncological effects of da Vinci Xi surgical system assisted programmed six-hole method anterior resection of rectal cancer and lapa-roscopic anterior resection of rectal cancer are comparable. However, robotic surgery is superior to laparoscopic surgery in terms of intraoperative bleeding, lymph node dissection, gastrointestinal function recovery, and pelvic autonomic nerve protection.

Patients with deep vein thrombosis require long-term anticoagulant therapy to prevent the spread or recurrence of the thrombus. Self-management can enhance patients' awareness of the disease and adherence to anticoagulant therapy, reducing the recurrence rate. This study reviews the current status, evaluation tools, intervention methods, and influencing factors of self-management in patients with deep vein thrombosis, providing reference for improving the home self-management ability of patients with deep vein thrombosis.

Objective:TP53-abnormal MDS/acute myeloid leukemia (AML) patients’ allogeneic hematopoietic stem cell transplantation (allo-HSCT) treatment’s effectiveness and influencing factors should be studied.Methods:42 patients with TP53 gene status change MDS/AML who underwent allo-HSCT from 2014.8.1 to 2021.7.31 at the Hematology Hospital of the Chinese Academy of Medical Sciences were the subject of a retrospective analysis. The 42 patients were divided into three groups: the TP53 deletion group (group A) , TP53 mono-alle mutation group (group B) , and TP53 multi-hit group (group C) . The differences in clinical features and prognostic factors after transplantation were analyzed.Results:There were 42 MDS/AML patients, including 21 patients with MDS, and 21 patients with AML. The median follow-up period was 34.0 (7.5-75.0) months and the median patient age at the time of transplantation was 41.5 (18-63) years old. The total OS was 66.3% (95% CI 53.4%-82.4%) in 3 years after transplantation, and EFS was 61.0% (95% CI 47.7%-78.0%) in 3 years. For 3 years after receiving hematopoietic stem cell transplantation, there were no statistically significant differences in 3-year OS and EFS in groups A, B, and C ( P≥0.05) . The 3 years OS was 82.5% (95% CI 63.1%-100.0%) in group A, 60.6% (95% CI 43.5%-84.4%) in group B, and 57.1% (95% CI 30.1%-100.0%) in group C. Univariate analysis revealed that the number of co-mutant genes, pre-HSCT treatment, and disease type did not affect prognosis, while age, karyotype, co-mutation, positive blast cell before transplantation, and positive blast cell after transplantation were common prognostic factors for OS and EFS ( P<0.1) . MRD levels before transplantation were found to be independent risk factors for OS ( P=0.037, HR=33.40, 95% CI 1.24-901.17) in a multivariate analysis. Conclusion:Patients with MDS/AML who have TP53 mutations can benefit from allo-HSCT, but patients with complex karyotypes have a worse prognosis. Meanwhile, the final flow cytometry (FCM) monitoring blast cell test before HSCT has a certain guiding significance for prognostic assessment.