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 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: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 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.

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.

Oral and maxillofacial-head and neck soft tissue defects affect the appearance of patients, as well as pronunciation, swallowing and other functions. Introduction of the propeller flap in 1991 has improved reconstruction procedures for oral and maxillofacial-head and neck soft tissue defects. A propeller flap has several advantages over traditional local flaps. It improves mobility, colour and texture matching for maxillofacial defect, surgical procedure, and individual satisfaction. Therefore, it can be used as a complement to the traditional flap by providing surgeons with more options. This paper reviews the classification, surgical procedures, and recent clinical applicatiosn and indications of the propeller flap.

Objective:To analyze 12 antithrombins (AT) gene mutations that cause AT deficiency and discuss the relationship between the SERPINC1 gene. mutations and venous thrombotic events.Methods:This study belongs to case series of observational studies. Collected the clinical data of 12 AT deficiency cases in the First Affiliated Hospital of Wenzhou Medical University from April 2014 to April 2021 and collected the blood samples before treatment. The AT activity (AT: A) and AT antigen (AT: Ag) was detected by chromogenic substrate and immunoturbidimetry, respectively. The 7 exons and flanking sequences of the SERPINC1 gene were sequenced directly by PCR, the suspected mutations were validated by reverse sequencing. Analyzed the correlation between the SERPINC1 gene. mutations and venous thrombotic events and figured out the proportion.Results:The AT: A of the 12 patients all decreased significantly, ranging from 30% to 66%, and the AT: Ag of the 7 patients decreased accordingly, showing type Ⅰ AT deficiency, and the AT: Ag of the other 5 patients were normal, presented type Ⅱ AT deficiency. 12 mutations were found including 6 heterozygous mutations which were discovered for the first time: c.456_458delCTT(p.phe121del), c.318_319insT(p.Asn75stop), c.922G>T(p.Gly276Cys), c.938T>C (p.Met281Thr), c.1346T>A(p.Leu417Gln)and c.851T>C(p.Met252Thr). All 12 patients had venous thrombosis, and 3 cases including 2 compound heterozygotes and 1 single heterozygote all suffered from deep venous thrombosis (DVT) when they were younger without obvious triggers. The other 9 patients all combined with the other thrombotic factors including old age, hypertensive, smoking, pregnancy, and prolonged immobilization.Conclusion:Patients with AT deficiency caused by SERPINC1 gene defects are prone to venous thrombosis, especially combined with other thrombotic factors.

Objective:To investigate the efficacy of strategies for minimizing small bowel resection during surgery for pelvic radiation-induced terminal small intestinal stenosis in preventing postoperative complications such as anastomotic leakage and short bowel syndrome.Methods:This was a retrospective cohort study. There are two subtypes of chronic radiation enteritis (CRE) with combined intestinal stenosis and intestinal obstruction: (1) Type I: terminal ileal lesions with a normal ileal segment of 2–20 cm between the ileal lesion and ileocecal junction; and (2) Type II: the lesion is located in the small bowel at a distance from the ileocecal region, usually accompanied by extensive damage to the bowel segments outside the lesion. The indications for minimal bowel resection are as follows: (1) diagnosis of Type I small bowel CRE; (2) absence of radiological evidence of rectosigmoid damage; and (3) absence of colonic obstruction. The contraindications are: (1) stenotic, penetrating lesions of the distal cecum; (2) emergency surgery; (3) recurrence of malignant tumor or history of radiotherapy for recurrent malignant tumor; (4) interval between radiotherapy and surgery <6 months; and (5) history of preoperative small bowel resection or abdominal chemotherapy. Case data of 40 patients with Type I CRE who met the above criteria and had undergone minimal bowel resection between April 2017 and December 2019 were retrospectively analyzed (minimal bowel resection group; including 13 patients from Jinling Hospital, 16 from the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, and 11 from the Affiliated Hospital of Xuzhou Medical University). Forty patients with Type I CRE who had undergone resection of intestinal stenosis lesions and the ileocecal region between October 2015 and March 2017 were included as historical controls (conventional resection group; all from Jinling Hospital). The specific strategy for minimal bowel resection was one-stage partial ileal resection+ileo anastomosis+protective small bowel stoma. In contrast, conventional resection comprised ileocecal resection+ileocecal-ascending colon anastomosis. Postoperative complications, intraoperative and postoperative recovery, and changes in postoperative quality of life were analyzed in both groups. The severity of postoperative complications was assessed by Clavien-Dindo and the Comprehensive Complication Index (CCI). Karnofsky performance scores (KPS) were used to evaluate the quality of life of patients in the two groups preoperatively and postoperatively. The higher the KPS score, the better the quality of life.Results:Baseline patient characteristics did not differ significantly between the two groups ( P>0.05). Compared with the conventional resection group, the length of small bowel resected in the minimal bowel resection group (51 [20–200] cm vs. 91 [60–200] cm, Z=5.653, P<0.001), duration of postoperative total enteral nutrition [9 (3–18) days vs. 12 (4–50) days, Z=2.172, P=0.030], and duration of postoperative hospital stay [17 (9–24) days vs 29 (13–57) days, Z=6.424, P<0.001] were shorter; all of these differences are statistically significant. The overall incidence of postoperative complications was lower in the minimal bowel resection group than in the conventional resection group [20.0% (8/40) vs. 70.0% (28/40), χ 2=19.967, P<0.001], These comprised short bowel syndrome [5.0% (2/40) vs. 25.0% (10/40), χ 2=6.274, P=0.012], anastomotic leakage or fistula [2.5% (1/40) vs. 22.5% (9/40), χ 2=7.314, P=0.014], and pleural effusion [7.5% (3/40) vs. 25.0% (10/40), χ 2=4.500, P=0.034], all of which occurred less often in the minimal bowel resection than conventional resection group. The CCI index was also lower in the minimal bowel resection group than in the conventional resection group [CCI>40: 2.5% (1/40) vs. 12.5% (5/40), Z=18.451, P<0.001]. KPS scores were higher in the minimal bowel resection group 1 and 3 months postoperatively than they had been 1 day preoperatively (79.9±4.7 vs. 75.3±4.1, 86.2±4.8 vs. 75.3±4.1, both P<0.05). In the minimal bowel resection group, seven patients were satisfied with their current quality of life and refused to undergo stoma reduction at follow-up and one deferred stoma reduction because of rectal bleeding. The remaining 32 patients underwent stoma reduction 3 to 12 months after surgery, 26 of whom underwent ileo-cecal anastomosis. The remaining six underwent resection of the stoma and anastomosis of the ileum to the ascending colon. Conclusions:The strategy of minimal small bowel resection in patients with radiation-induced bowel injuries reduces the length of resected small bowel, decreases the risk and severity of postoperative complications, and is associated with a better prognosis and quality of life than conventional resection.

There are many validated surgical techniques for the repair and reconstruction of facial defects, such as skin grafting, free flap grafting and local flap grafting, all of which have achieved good results within their scope of application. As a local flap, the keystone design perforator island flap (KDPIF) has the advantages of easy harvest and design, abundant blood supply, and a wide range of applicability. However, this flap has not been frequently applied in facial defect reconstruction. In reviewing the literatures, the authors believe that the KDPIF is a worthy method for facial defect repair. The principles, types, surgical techniques and applications, and considerations are also summarized.