α/Sulfono-γ-AA peptide hybrids agonist of GLP-1R with prolonged action both in vitro and in vivo

Peptides are increasingly important resources for biological and therapeutic development, however, their intrinsic susceptibility to proteolytic degradation represents a big hurdle. As a natural agonist for GLP-1R, glucagon-like peptide 1 (GLP-1) is of significant clinical interest for the treatment of type-2 diabetes mellitus, but its in vivo instability and short half-life have largely prevented its therapeutic application. Here, we describe the rational design of a series of α/sulfono-γ-AA peptide hybrid analogues of GLP-1 as the GLP-1R agonists. Certain GLP-1 hybrid analogues exhibited enhanced stability (t 1/2 > 14 days) compared to t 1/2 (<1 day) of GLP-1 in the blood plasma and in vivo. These newly developed peptide hybrids may be viable alternative of semaglutide for type-2 diabetes treatment. Additionally, our findings suggest that sulfono-γ-AA residues could be adopted to substitute canonical amino acids residues to improve the pharmacological activity of peptide-based drugs.

Primary culture of cat intestinal epithelial cell and construction of its cDNA library / 中国血吸虫病防治杂志

Objective To establish the primary cat intestinal epithelial cells(IECs)culture methods and construct the cD-NA library for the following yeast two-hybrid experiment,so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection ,by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMARTTM technology,and then the double-strand cDNAs were acquired by LD-PCR,which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recom-bination. Matchmaker?Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calcula-tion of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of colla-genase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1× 106 independent clones. The titer was 2.8 × 109 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research,and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

Prokaryotic expression and identification of rhoptry protein 38 of Toxoplas-ma gondii / 中国血吸虫病防治杂志

Objective To explore the biological function of rhoptry protein 38(ROP38)of Toxoplasma gondii,and to iden?tify the reactogenicity of the recombinant protein(rROP38). Methods The ROP38 was amplified by RT?PCR from T. gondii RH strain,and was cloned into prokaryotic expression vector pET?28a(+). The recombinant plasmid was transformed into E. co?li BL21(DE3)competent cells. Then the rROP38 was analyzed by SDS?PAGE and identified by Western blot. Results SDS?PAGE showed that rROP38 was efficient expression with a molecular weight of about 43 kD. Western blot showed that rROP38 reacted with antibody of His tag or human positive antibody,which indicated that ROP38 had good reactogenicity and could be a serological diagnostic antigen. Conclusion The study successfully obtains the rROP38 of T. gondii with good reactogenicity.

Development and application of rapid molecular method for detection of as-ymptomatic infection of Leishmania / 中国血吸虫病防治杂志

Objective To develop a rapid molecular biological method for detection of the asymptomatic infection of Leish?mania. Methods Two pairs of primers named RV1?RV2 and K13A?K13B were selected to be the fast diagnosis primers since they were designed according to the conserved region of Leishmania kinetoplast DNA(kDNA)minicircles. The PCR amplifica?tion products of Leishmania donovani promastigote from Shandong Province were sequenced to compare their conservatism. The method was applied to detect 105 venous blood samples from healthy home canine and 7 venous blood samples from home canine suffered from Kala?azar in Heishui County of Sichuan Province,and 75 venous blood samples from susceptible population(no leishmaniasis symptoms)and 7 venous blood samples from patients in Xinjiang Kashi area in order to verify the feasibility and accuracy of the method. Results The size of PCR products was consistent with the expected fragments with high conservative among Leishmania species. The positive rates of 105 home canine samples and 75 susceptible population samples were 37.14%(39/105)and 82.67%(62/75)rspectively,and the positive rates of home canine suffered from Kala?azar and patients were all 100%(7/7). Conclusion This rapid diagnosis method is suitable for detection of asymptomatic infection of Leishmania in Kala?azar endemic areas of China with high sensitive and specific,thus it has bright perspective to be used.

Construction and identification of pcDNA3-HBsAg-p30-ROP2 expression vec-tor / 中国血吸虫病防治杂志

Objective To construct a multi-gene recombinant pcDNA3-HBsAg-p30-ROP2 expression vector and identify it preliminarily. Methods According to recombinant pcDNA3-p30-ROP2 restriction sites,HBV HBsAg gene sequences of primers were designed and synthesized to amplify target fragment,and then cloned into pcDNA3-HbsAg-p30-ROP2 expression vector. Af-ter sequencing,it was identified finally by restriction enzyme digestion and other molecular biology techniques. Results HBV HBsAg gene segment was amplified by PCR and the multi-gene recombinant pcDNA3-HBsAg-p30-ROP2 expression vector was constructed and identified to be correct as theoretical values. The PCR and restriction enzyme digestion results showed that HBsAg and p30-ROP2 gene in recombinant plasmid were confirmed by DNA sequencing. Conclusion The multi-gene recombinant pcD-NA3-HBsAg-p30-ROP2 expression vector is successfully constructed.

Diagnosis and treatment of parathyroid carcinoma / 国际外科学杂志

Parathyroid carcinoma is a rare maligant tumor.The main clinical symptoms are primary hyperparathyroidism and all kinds of metabolism disorder and the damage of homologous tissues and organs.The consummate laboratory examination and the accurate imaging localization and the credible histopathologic feature are key points to diagnosis of parathyroid carcinoma.Parathyroid carcinoma is unsensitive to chemotherapy and radiotherapy,and en bloc resection of the carcinoma is the preferred treatment approach.AntiPTH immunization as a new treatment of parathyroid carcinoma has been paid close attention.The prognosis of parathyroid carcinoma is mainly depended on whether the tumor was cut off completely and the control as well as the monitor of the symptomatic hypercalcemian after operation.This article reviews the latest researches and introduces the latest advancement in the diagnosis and treatment of parathyroid carcinoma.

Secondary lymphoid-tissue chemokine and CCR7 in it's relation with lymphangiogenesis of pancreatic adenocarcionoma / 中华普通外科杂志

Objective To study the expression of secondary lymphoid-tissue chemokine (SLC)、 CCR7 and its correlation with clinical pathology and lymphangiogenesis in pancreatic adenocarcinoma (PAC). Methods The tissue specimens including PAC, the cancerous peripheral tissues, the normal pancreatic tissues and peripheral lymph nodes were obtained from 30 patients with PAC. The expressions of SLC and CCR7 in these tissues were assayed by immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR). MIND marked by VEGFR-3 was detected by morphometric analysis, and the relationship between MLND and clinical pathology of PAC was analyzed. Results In all the specimens, the positive rates of SLC protein in PAC, the cancerous peripheral tissues, the normal pancreatic tissues and peripheral lymph nodes were respectively 16. 7%, 43. 3%, 76. 7% and 46. 6%. The positive rates of CCR7 protein in PAC, the cancerous peripheral tissues, the normal pancreatic tissues and peripheral lymph nodes were respectively 76. 7%, 66. 7%, 30. 0% and 70. 0%. The results of RT-PCR and fluorescence quantitative real-time PCR indicated that the expression levels of CCR7 mRNA in PAC tissues, the cancerous peripheral tissues and peripheral lymph nodes were higher than that in the normal pancreatic tissues ( P <0. 01 ). There was no significant correlation between the expression of SLC protein with MLVD of PAC ( P > 0. 05 ). There was 23 specimens that the CCR7 protein was positive, and among these specimens the MIND was higher than that in negative group of CCR7 protein (P = 0.004). Conclusions The expression of SLC was not related to lymphatic metastasis and TNM stages of PAC. The expression of CCR7 was significantly associated with lymphatic metastasis and TNM stages of PAC, and the high expression of CCR7 in PAC tissues was significantly associated with lymphangiogenesis of PAC.

Significance of VEGF-C and VEGF-D in lymphatic metastasis of pancreatic cancer / 国际肿瘤学杂志

Objective To analyze the intratumoral and peritumoral microvessel density (MVD) and microlymphatic vessel density (MLVD) in pancreatic cancer and record the expression of vascular endothelial growth factor(VEGF)-C and VEGF-D. And to explore the significance of VEGF-C and VEGF-D during the lymphatic metastasis and development of pancreatic cancer. Methods The expression of VEGF-C and VEGF-D, VEGF-R3, CD34 were assayed by immunohistochemical staining in 30 cases of pancreatic cancer tissues. Results The positive rates of VEGF-C and VEGF-D protein in the central portion of tumors (30% and 16.7%) were significantly lower than those in the marginal portion (73.3% and 56.7%), P <0.01. The group with high expression of VEGF-C and VEGF-D in the marginal portion had significantly higher incidences of lymph node metastasis, lymphatic invasion and venous invasion( P <0. 01 ). MLVD in both of the VEGF-C and VEGF-D positive groups was higher than that in the negative groups( P <0. 01 ), and the lymph node me-tastasis increased. MVD in VEGF-C positive group was significantly higher than that in the negative group. MVD had no significant difference between VEGF-D positive and negative group ( P =0. 07). Conclusions The expression of VEGF-C and VEGF-D in the marginal portion of tumor is significantly correlated with lym-phatic metastasis in pancreatic cancer patients, and may induce lymphangiogenesis. VEGF-C may play an im-portant role in the regulation of angiogenesis and lymphangiogenesis in pancreatic cancer, and VEGF- D maybe only participate in the regulation of lymphangiogenesis.

Combination of frozen section and touch imprint cytology for the intraoperative diagnosis of sentinel lymph node in breast cancer / 肿瘤研究与临床

Objective Frozen section(FS)and touch imprint cytology(TIC)were common methods for intraoperative evaluation of sentinel lymph node(SLN)biopsy in breast cancer,with low sensitivity when used separately.The purpose of this study was to evaluate the value of combination of these two techniques.Methotis This study included 400 sentinel nodes from 150 patients with breast cancer.352 sentinel nodes were bisected along the long axis.Each sectioned surface of SLN was imprinted onto the surface of a slide and was analyzed by cytologist;meanwhile SLN were analyzed with intraoperative FS.The other 48 SLN were only analyzed with intraoperative PS due to their small size.Results of intraoperative P3 and TIC were compared with final pathology.Results Eighty-nine positive SLN from 55 patients were identified by final pathology.The specificity of FS and TIC were both 100%.According to the number of SLN.the sensitivity of TIC and FS was 71.9%(64/89)and 83.1%(74/89),respectively(P＞0.05).The sensitivity of TIC compared with FS was 96.6%(86/89),significantly higher than that of TIC and FS separately(both P＜0.001).According to the number of patients,the sensitivities of TIC and FS were 80.0%(44/55)and 81.8%(45/55),respectively(P＞0.05).The sensitivity of TIC compared with FS was 94.5%(52/55).significantly higher than that of TIC and FS separately (both P＜0.001).Conclusion Combination of FS and TIC for the intraoperative diagnosis of SLN biopsy in breast cancer was reliable,with hish sensitivity and specificity,and could avoid the second axillary operation efficiently.

Correlation analysis of CXCL12,CXCR4 and VEGF-C expression and clinical significance in pancreatic cancer / 中国现代普通外科进展

Objective:To investigate the expression of CXCL12-CXCR4 and VEGF-C in pancreatic cancer and relation to clinical pathology.Methods:The tissue samples including PAC,the cancerous peripheral tissues,the normal pancreatic tissues and peripheral lympho nodes were obtained from 30 patients with PAC.The expressions of CXCL12,CXCR4and VEGF-C proteins in these tissues were assayed by immunohistochemical staining.The expressions of CXCL12,CXCR4 and VEGF-C mRNA in PAC were also investigated by fluorescence quantitative real-time PCR.Results:In all the samples,the positive rates of CXCL12 protein in PAC,the cancerous peripheral tissues,the normal pancreatic tissues and peripheral lympho nodes were respectively 13.3%(4/30),46.7%(14/30),56.7%(17/30) and 50.0%(15/30).The positive rates of CXCR4 protein in PAC,the cancerous peripheral tissues,the normal pancreatic tissues and peripheral lympho nodes were respectively 80.0%(24/30),70.0%(21/30),26.7%(8/30) and 73.3%(22/30).The expression levels of CXCR4 mRNA in PAC tissues,the cancerous peripheral tissues and peripheral lympho nodes were higher than that in the normal pancreatic tissues(P

A study on the clinicopathological features and prognosis of solid-pseudopapillary tumor of the pancreas / 中国普通外科杂志

Objective To study the clinicopathological and immunohistochemical features, histogenesis and biological behavior of solid pseudopapillary tumor of the pancreas ( SPT ). Methods Routine HE and immunohistochemical ( SP) methods were used in 20 cases of SPT. Results There were 18 females and 2 males, age ranging from 13 to 48 years with mean age of 25. 3 years. Abdominal pain and palpable mass were among the main complains. Seventeen cases were followed-up from 9 to 120 monthes. Fourteen cases were alive. Tumors were encapsulated, mixed with solid and cystic tissues. Histological features were psudopapillary structure with a fibrovascular core. Immunohistogically, the tumors were positive for a-1-AT ( 17 cases) , vimentin ( 14 cases) , synaptophysin ( 10 cases) , CgA (5 cases) ,CK and insulin (2 cases) ,glucagon and S-100 (1 case) ,PR (14 cases) , ER (1 case) ,pS2 (6 cases) , but all were negative for CEA and gastrin. Conclusion SPT is of low-graded malignancy and a distinct clinicopathologic entity in young female patients with both exocrine as well as endocrine differentiation. The tumor is closely related with sex hormone receptors.