Objective To observe the expression of Toll-interacting protein ( Tollip) in appendix and analyze its significance during acute inflammation. Methods Thirty-three patients with acute appendicitis were included in the present study and 6 subjects with non-inflammatory appendixes were taken as controls ( without inflammatory changes). The pulse rate,body temperature ( BT) ,white blood cell ( WBC) count and neutrophil ( NEUT) count were observed one hour before appendectomy. Based on H-E staining and pathological examination,the appendix samples were divided into four groups:non-inflammatory appendix ( A) ,simple appendicitis ( B) ,suppurative appendicitis ( C) and gangrenous appendicitis ( D). The expression of Tollip protein ( the localization,qualitative and semiquantitative analysis) was analyzed using immunohistochemistry and digital image analysis. The correlation of Tollip with pusle rate,BT,WBC and NEUT was also analyzed. Results Significant differences in BT,WBC and NEUT were found between different pathological groups ( P

To design and construct an expression vector,PGEX-4T-1/MD-2, and to express human myeloid differentiation protein-2(hMD-2) and glutathione-S-transferase (GST) fusion protein in E. coli, the EcoRI/SalI sites and stop code were incorporated into the hMD-2 encoding fragment by PCR. After digesting with EcoRI/SalI, the hMD-2 encoding fragment was cloned into the expression vector PGEX-4T-1 at the corresponding sites. The positive clones selected with PCR and restriction endonuclease digestion were sequenced and the expression of GST/hMD-2 fusion protein in E. coli BL21 was analyzed with SDS-PAGE after induced by 0.4mmol/L IPTG for 3 to 5 hours. The hMD-2 encoding fragment containing stop code was correctly inserted into the expression vector PGEX-4T-1 and this was confirmed by PCR, double-enzyme identification and sequencing. The SDS-PAGE analysis indicated that the GST/hMD-2 fusion protein was successfully expressed in E. coli and the yield of the fusion protein was 30 percent of bacterial total protein. The construction of the expression vector PGEX-4T-1/hMD-2 and the expression of fusion protein GST/hMD-2 in E. coli would be useful for further investigation of MD-2.

Objective To explore the feasibility and safety of breast conserving surgery ( BCS) in patients with early breast cancer after local lumpectomy . Methods Clinical data of 26 patients who previously had received local lumpectomy from January 2009 to December 2011 were retrospectively analyzed .All the patients were diagnosed as solitary invasive ductal carcinoma with preoperative staging of T1N0M0.The interval from lumpectomy to BCS was 6-24 days (mean, 10 days) and the maximum diameter of tumors before first operation was 1-2 cm (mean, 1.6 cm).The shortest distance between nipple and operative incision was 2-6 cm (mean, 4.5 cm). Results The operations were successfully completed in all 26 patients, 15 of which received sentinel lymph node biopsy and 11 of which received axillary lymph node dissection .The hospital stay was 3-5 days with stage-Ⅰhealing of incision .All patients received whole breast radiation therapy after BCS .There was no local recurrence or distant metastasis during 2 -5 year ’ s follow-up (median, 32 months).All the patients were satisfied with the shape of breast and the quality of life . Conclusion For those patients with early breast cancer who have received local lumpectomy , meet the requirement of CBS , and have the willing of breast conserving , BCS is an ideal choice after rigid application of surgical indications .

Objective To observe the changes in proliferative activity of and autophagosome formation in human HaCaT keratinocytes and primary keratinocytes after different doses of ultraviolet B (UVB) radiation,and to assess the potential relationship between proliferation impairment and autophagosome formation.Methods Both cultured HaCaT cells and primary keratinocytes from human foreskin were irradiated with different doses (5,10,20and 40 mJ/cm2) of UVB.Those receiving no irradiation served as the control.After additional 12-hour culture,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of cells,monodansylcadaverin (MDC) staining to detect autophagosomes in cells.The number of autophagosome-positive or negative cells was counted using inverted fluorescence microscopy.Results UVB radiation induced a significant decrease in the proliferation of keratinocytes,especially in that of HaCaT cells.The proliferative activity expressed as the absorbance value at 490 nm was significantly lower in HaCaT cells (1.367 ± 0.035,1.173 ± 0.034 and 0.873 ±0.025 vs.1.519 ± 0.022,all P＜ 0.01) and primary keratinocytes (0.782 ± 0.012,0.773 ± 0.021 and 0.725 ± 0.031 vs.0.887 ± 0.035,all P ＜ 0.05) irradiated with UVB of 10,20 and 40 mJ/cm2 than in the unirradiated control cells.Significant differences were also observed in the proliferative activity among HaCaT cells irradiated with UVB of 10,20 and 40 mJ/cm2.The proportion of autophagosome-positive cells was increased after irradiation with UVB of 5,10 and 20 mJ/cm2,but decreased after irradiation with UVB of 40 mJ/cm2 in keratinocytes,especially in the primary keratinocytes.In detail,the proportion of autophagosome-positive cells was 22.69％ ± 2.15％,28.10％ ± 2.92％ and 22.92％ ± 2.61％ in HaCaT cells irradiated with UVB of 10,20 and 40 mJ/cm2 respectively,significantly higher than that in the unirradiated cells (10.18％ ± 1.50％,chi-square test for trends:x2 =27.48,P ＜ 0.01).No significant changes were observed in the proportion of autophagosome-positive cells in primary keratinocytes after irradiation with UVB of 5,10 and 20 mJ/cm2,but a marked decrease was found after irradiation with UVB of 40 mJ/cm2 compared with unirradiated keratinocytes (chi-square test for trends:x2 =6.86,P ＜ 0.01).Conclusions UVB radiation (10-40 mJ/cm2) decelerates the proliferation of HaCaT cells and primary keratinocytes in a dosedependent manner,and primary keratinocytes seem to be more resistant to UVB damage than HaCaT cells.Low to moderate doses (5-20 mJ/cm2) of UVB promote autophagosome formation in HaCaT cells in a dose-dependent manner,and exert no significant influence on that in primary keratinocytes; however,UVB of 40 mJ/cm2 suppresses autophagosome formation in keratinocytes,especially in primary keratinocytes.

Objective:This study aims to analyze the occurrence rate, positive rate, and other related factors influencing interpec-toral lymph nodes (IPNs) in breast cancer patients. This work further aims to explore the significance and indications of the surgical dis-section of IPNs. Methods:Clinical and pathological data from 1673 breast cancer patients were retrospectively analyzed. All patients were subjected to modified radical mastectomy, and IPNs were pathologically examined. The occurrence rate and metastasis of IPNs were recorded, and the relationship between the IPN positive rate and tumor size, axillary nodes, clinical stages, neo-adjuvant chemo-therapy, hormone receptors, Her-2 expression, and molecular subtypes of breast carcinoma was determined. Results:The occurrence rate, overall metastasis rate, and the positive rate of IPNs in patients with axillary lymph node metastasis were 13.39%, 4.30%, and 10.01%, respectively. IPN metastasis was significantly correlated with axillary node metastasis and the tumor, node and metastasis (TNM) stage of tumors (P<0.05). However, IPN metastasis was not significantly related with hormone receptor and Her-2 expressions. IPN metastasis rate may be unaffected by neo-adjuvant chemotherapy. Patients with IPNs metastasis were characterized by larger tu-mors, more positive axillary lymph nodes, and later TNM stages. Conclusion:IPN metastasis usually occurs in patients with larger tu-mors, more positive axillary lymph nodes, later TNM stages, as well as those with locally advanced cancer that meet the standard of neo-adjuvant chemotherapy. These indications suggest that the surgical dissection and pathological examination of IPNs should be rou-tinely performed.

[Objective] To observe the effects of autophagy induced by different doses of ultraviolet B (UVB) irradiation on the apeptosis in human skin fibroblasts.[Methods] Skin fibroblasts were isolated from the circumcision specimen of a 23-year-old healthy male,and subjected to a primary culture.After 3 to 10 passages,the cells were collected and applied in the following experiment.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of some fibroblasts treated with different concentrations (0,0.5,2.0,5.0and 10.0 mmol/L) of 3-methyladenine (3-MA).To qualitatively and quantitatively detect the autophagy in fibroblasts treated with different concentrations of 3-MA and in fibroblasts treated with 3-MA of 0.5 mmol/Lfollowing UVB irradiation,monodansylcadaverine (MDC) staining was carried out,and immunofluorescence was used to detect the expression of microtubule-associated protein 1 light chain (LC3).Some fibroblasts were classified into 8 groups to remain untreated,be irradiated with UVB of 30,50 and 100 mJ/cm2 alone,treated with 3-MA of 0.5 mmol/L alone,or treated with 0.5 mmol/L 3-MA following irradiation with UVB of 30,50 and 100 mJ/cm2,respectively,then,cell apoptosis was qualitatively detected by Hoechst and propidium iodide (PI)staining,and quantitatively detected by flow cytometry with annexin V and PI.[Results] The percentage of autophagic cells was (63.037 ± 5.876) ％ in fibroblasts treated with starvation condition,significantly decreased to (34.425 ± 5.183) ％ in fibroblasts treated with 3-MA of 0.5 mmol/L.The expression of LC3 showed a gradually increasing trend from untreated fibroblasts,to fibroblasts irradiated with UVB of 30,50 and 100 mJ/cm2,while the increase was attenuated by the 4-hour treatment with 3-MA immediately after the irradiation.Compared with the other concentrations,the 3-MA of 0.5 mmol/L showed the least influence on the viability of fibroblasts.The addition of 3-MA of 0.5 mmol/L increased the percentage of cells both positive for Hoechst and PI staining in fibroblasts irradiated with UVB of 50 mJ/cm2,but decreased that in fibroblasts irradiated with UVB of 100 mJ/cm2.Similarly,the percentage of middle and late apoptotic cells was significantly higher in fibroblasts irradiated with UVB of 50 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 50 mJ/cm2alone ((10.933 ± 0.839) ％ vs.(7.267 ± 0.473) ％,t =5.20,P＜ 0.05),but lower in fibroblasts irradiated with UVB of 100 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 100 mJ/cm2alone ( (7.100 ± 0.781 ) ％ vs.( 1 0.133 ± 0.681 ) ％,t =6.29,P ＜ 0.05 ).[Conclusion]s The irradiation with UVB of 50 mJ/cm2 may protect fibroblasts by inducing autophagy and suppressing apoptosis,while the high level of autophagy induced by UVB of 100 mJ/cm2 may lead to autophagic cell death in fibroblasts.

Objective To investigate the immunogenicity of immunodominant cytotoxic T lymphocyte epitope E749-57 of human papilloma virus (HPV) 16 oncoprotein E7 chaperoned by heat shock protein (HSP)110. Methods Mouse HSP110 gene was cloned into prokaryotic expression vector pQE-80L for the expression of HSP110 protein, which was purified using Ni-NTA column. SDS-PAGE and Western-blot were conducted to confirm the purified mHSP110 protein, which was subsequently incubated with E749-57 peptide under heat shock condition, and high-performance liquid chromatography (HPLC) was used to evaluate the binding efficiency of the recombinant protein and E749-57 peptide. Twenty mice were divided into 4 groups to be immunized with mHSP110 protein, E749-57 peptide, mHSP110-E749-57 complex and phosphate buffered saline (PBS),respectively. Two weeks after the last immunization, spleen cells were collected from the immunized mice and divided into 2 parts: one were stimulated by E749-57 peptide followed by the detection of CD8+ INF-γ+ T cells with flow cytometry; the other one were subjected to MTT analysis for the estimation of cell proliferation. The mHSP110-E749-57 complex was also used to immunize TC-1 tumor bearing mice to observe its anti-tumor effect.Results The full-length 2577 bp-sized mHSP110 gene was amplified from mouse liver cDNA and cloned into pQE-80L vector. Direct sequencing confirmed the correctness of the cloning. SDS-PAGE and Western-blot demonstrated the successful purification of mHSP110. HPLC assay showed that the purified mHSP110 protein could bind with E749-57 to form a relatively stable protein complex. The percentage of IFN-γ+ CD8+ T cells in and proliferation index of spleen cells from the complex-immunized mice were statistically higher than those from the other 3 groups of mice. Moreover, the complex could obviously inhibit the growth of TC-1 tumor in mice. Conclusion The mHSP110-E749-57 complex could enhance the generation of specific cytotoxic T lymphocytes and exert anti-tumor effects in mice.

OBJECTIVETo investigate the changes in the functional activity of glycogen synthase kinase-3 (GSK-3) in the hepatic tissue after endotoxin (lipopolysaccharide, LPS) tolerance and explore the effects of LPS-induced GSK-3 inhibition on glycogen metabolism in the liver.

METHODSMale SD rats were randomly divided into normal control, endotoxin pretreatment and GSK-3 inhibitor (lithium chloride) groups with corresponding pretreatments prior to a large dose of LPS challenge (10 mg/kg) to induce liver injury. Glycogen deposition and content in the hepatic tissue was detected using periodic acid-Schiff (PAS) staining and a glycogen quantification kit, respectively. Western blotting was performed for semi-quantitative analysis of protein level and inhibitory phosphorylation of GSK-3, and a Coomassie brilliant blue G-250-based colorimetric assay was used to detect calpain activity in the liver.

RESULTSGlycogen content in the liver decreased significantly after LPS challenge in all the 3 groups (P<0.05) but showed no significant difference among the groups (P>0.05). Both LPS and lithium chloride pretreatments caused a significant increase of liver glycogen content (P<0.05). LPS pretreatment induced inhibitory phosphorylation of GSK-3β (P<0.05) and partial cleavage of GSK-3α but did not affect the expression of GSK-3 protein (P>0.05). Large-dose LPS challenge significantly increased the activity of calpain in the liver tissue (P<0.05) to a comparable level in the 3 groups (P>0.05).

CONCLUSIONEndotoxin pretreatment induces inhibitory phosphorylation of GSK-3β and partial cleavage of GSK-3α and promotes the deposition of liver glycogen but does not affect the activity of calpain, which may contribute to an increased glycogen reserve for energy supply in the event of large-dose LPS challenge.

Animals ; Calpain ; metabolism ; Glycogen ; metabolism ; Glycogen Synthase Kinase 3 ; antagonists & inhibitors ; metabolism ; Lipopolysaccharides ; adverse effects ; Lithium Chloride ; pharmacology ; Liver ; drug effects ; metabolism ; pathology ; Male ; Rats ; Rats, Sprague-Dawley

Objective To summarize the clinical experience of the treatment for complex aortic coarctation with extra anatomic bypass and anatomic correction techniques. Methods The clinical data of patients with complex aortic coarctation treated in the First Affiliated Hospital of Nanjing Medical University and Friendship Hospital of Ili Kazakh Autonomous Prefecture between April 2012 and November 2020 were retrospectively reviewed. Results A total of 12 patients were enrolled, including 5 males and 7 females aged 11-54 (34.3±16.2) years. Extra anatomic bypass grafting was performed in 8 patients and anatomic correction was performed in 4 patients. The operations were successful in all patients. There was no perioperative death. The average cardiopulmonary bypass time was 203.0±46.0 min (7 median incision patients), and the average intraoperative blood loss was 665.0±102.0 mL. The average postoperative ventilator support time was 32.3±7.5 h, and the average postoperative hospital stay time was 10.2±4.3 d. The mean drainage volume of median incision was 1 580.0±360.0 mL, and the mean drainage time was 9.3±2.7 d. The mean drainage volume of left thoracotomy was 890.0±235.0 mL, and the mean drainage time was 4.8±2.5 d. One patient had a transient hoarse after operation and recovered 6 months later. The follow-up period ranged from 2 to 10 years with an average time of 81.0±27.0 months. All patients had a recovery of hypertension, cardiac afterload after 2 years postoperatively. One patient who received an artificial blood vessel replacement in situ was examined stenosis recurrence at the third year after discharge. Others were asymptomatic during the follow-up period. There were no death or other complications. Conclusion The treatment strategy for complex aortic coarctation should be individualized according to the anatomical features and concomitant heart diseases. Extra anatomic bypass technique is a safe and feasible choice.