AIM:To construct and breed infertility model mice with ATP/GTP binding carboxypeptidase 1(Agtpbp1)gene knockout homozygote(Agtpbp1-/-)by CRISPR/Cas9 technology,so as to provide an animal model for the subsequent exploration of the pathogenesis of Agtpbp1 gene in male sterility.METHODS:The CRISPR/Cas9 technology was used to obtain Agtpbp1 gene knockout heterozygote(Agtpbp1+/-)mice according to the data of the main protein functional region of Agtpbp1 gene and combined with Cas9 nuclease.The obtained Agtpbp1+/-mice were mated,and their offspring were genotyped by PCR and agarose gel electrophoresis.The expression of Agtpbp1 at different levels was detected by RT-PCR,Western blot and immunohistochemical staining to support the identification results.The HE staining was used to observe the mouse cerebellum and eyeball structure to analyze the effect of Agtpbp1 gene knockout on Purkinje cells and photoreceptor cells.The symptoms of ataxia in mice were observed in combination with behavioral tests.The growth of mice was observed,and the changes of testicular tissue volume and weight of male mice were analyzed.The HE staining was used to observe the changes of testicular structure,and PAS staining was used to observe the changes of testicular germ cell cycle.Finally,sperm analyzer was used to analyze the sperm motility,so as to analyze the growth and develop-ment of the mice.RESULTS:The male and female Agtpbp1+/-mice could continue to mate,and three genotypes,Agtpbp1 wild-type(Agtpbp1+/+),Agtpbp1+/-and Agtpbp1-/-,were obtained.The genotypes of the offspring mice were successfully identified by PCR.The results of RT-PCR,Western blot and immunohistochemical staining verified the successful con-struction of Agtpbp1-/-mouse model at different levels(P<0.05).The results of HE staining showed that Purkinje cells were lost in the cerebellum of Agtpbp1-/-mice and the number of photoreceptor cells in the eyeball was reduced.Behavioral tests confirmed that Agtpbp1-/-mice had ataxia symptoms such as motor dysfunction and uncoordinated movements.Com-pared with control group,the testicular volume and weight of Agtpbp1-/-mice were significantly reduced.The results of HE staining showed a very small amount of sperm in the testis of Agtpbp1-/-mice.Combined with the sperm analyzer,it was ob-served that the sperm motility,vitality and movement rate of Agtpbp1-/-mice were significantly lower than those of the con-trol mice.Testicular sections with PAS staining showed cell cycle arrest of the sperm from Agtpbp1-/-mice.CONCLU-SION:In this study,Agtpbp1 knockout mice were successfully bred.The deletion of Agtpbp1 caused the arrest of sper-matogenic cell differentiation and the decrease in sperm motility in adult male mice,resulting in infertility.At the same time,it provides a new experimental tool for further exploring the molecular mechanism of Agtpbp1-induced male sterility.

AIM:To construct and breed infertility model mice with ATP/GTP binding carboxypeptidase 1(Agtpbp1)gene knockout homozygote(Agtpbp1-/-)by CRISPR/Cas9 technology,so as to provide an animal model for the subsequent exploration of the pathogenesis of Agtpbp1 gene in male sterility.METHODS:The CRISPR/Cas9 technology was used to obtain Agtpbp1 gene knockout heterozygote(Agtpbp1+/-)mice according to the data of the main protein functional region of Agtpbp1 gene and combined with Cas9 nuclease.The obtained Agtpbp1+/-mice were mated,and their offspring were genotyped by PCR and agarose gel electrophoresis.The expression of Agtpbp1 at different levels was detected by RT-PCR,Western blot and immunohistochemical staining to support the identification results.The HE staining was used to observe the mouse cerebellum and eyeball structure to analyze the effect of Agtpbp1 gene knockout on Purkinje cells and photoreceptor cells.The symptoms of ataxia in mice were observed in combination with behavioral tests.The growth of mice was observed,and the changes of testicular tissue volume and weight of male mice were analyzed.The HE staining was used to observe the changes of testicular structure,and PAS staining was used to observe the changes of testicular germ cell cycle.Finally,sperm analyzer was used to analyze the sperm motility,so as to analyze the growth and develop-ment of the mice.RESULTS:The male and female Agtpbp1+/-mice could continue to mate,and three genotypes,Agtpbp1 wild-type(Agtpbp1+/+),Agtpbp1+/-and Agtpbp1-/-,were obtained.The genotypes of the offspring mice were successfully identified by PCR.The results of RT-PCR,Western blot and immunohistochemical staining verified the successful con-struction of Agtpbp1-/-mouse model at different levels(P<0.05).The results of HE staining showed that Purkinje cells were lost in the cerebellum of Agtpbp1-/-mice and the number of photoreceptor cells in the eyeball was reduced.Behavioral tests confirmed that Agtpbp1-/-mice had ataxia symptoms such as motor dysfunction and uncoordinated movements.Com-pared with control group,the testicular volume and weight of Agtpbp1-/-mice were significantly reduced.The results of HE staining showed a very small amount of sperm in the testis of Agtpbp1-/-mice.Combined with the sperm analyzer,it was ob-served that the sperm motility,vitality and movement rate of Agtpbp1-/-mice were significantly lower than those of the con-trol mice.Testicular sections with PAS staining showed cell cycle arrest of the sperm from Agtpbp1-/-mice.CONCLU-SION:In this study,Agtpbp1 knockout mice were successfully bred.The deletion of Agtpbp1 caused the arrest of sper-matogenic cell differentiation and the decrease in sperm motility in adult male mice,resulting in infertility.At the same time,it provides a new experimental tool for further exploring the molecular mechanism of Agtpbp1-induced male sterility.

OBJECTIVE: To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells. METHODS: Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level. RESULTS: Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 0.05). CONCLUSIONS TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.
Animals ; Male ; Mice ; GTP-Binding Proteins/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; RNA, Messenger ; Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism* ; Signal Transduction ; Spermatocytes ; Tubulin/genetics*

Objective To investigate the relationship of lymph node metastasis rate (LNR) with prognosis of esophageal squamous cell carcinoma after radical resection and postoperative adjuvant chemotherapy.Methods The retrospective case-control study was conducted.The clinicopathological data of 121 patients who underwent radical resection of esophageal squamous cell carcinoma in the Peking University Cancer Hospital from January 2012 to September 2016 were collected.There were 105 males and 16 females,aged from 42 to 76 years,with a median age of 58 years.All patients underwent radical resection of esophageal cancer with at least two-field lymph nodes dissection.Some patients underwent corresponding chemotherapy and radiotherapy.The thoracic and abdominal lymph nodes were grouped according to the 7th edition standard of Americau Joint Committee on Cancer (AJCC).The lymph nodes dissected were labeled in groups,and all the lymph nodes were examined by pathology test.Observation indicators:(1) follow-up;(2) effects of LNR on prognosis of patients in different AJCC N staging;(3) relationship between LNR and postoperative adjuvant chemotherapy.Follow-up was conducted by outpatient examination,telephone interview and hospital statistical office to detect postoperative survival of patients up to February 2017.The disease-free survival time was from surgery date to date of confirmation of tumor recurrence,and the overall survival time was from surgery date to death of the patient or the last follow-up date.Measurement data with skewed distribution were expressed by M (range).The Kaplan-Meier method was used to calculate the survival rate and draw the survival curve.The Log-rank test was used for survival analysis.Results (1) Follow-up:121 patients were followed up for 3.0-94.2 months,with a median follow-up time of 27.1 months.During the follow-up,98 of 121 patients had tumor recurrence and metastasis (including 64 deaths),22 had no metastasis,and 1 had unknown tumor metastasis.The mean overall survival time of patients was 30.8 months.The 1-,3-,5-year disease-free survival rates were 47.1％,20.3％,and 5.9％,respectively.The 1-,3-,5-year overall survival rates were 93.1％,48.7％,and 35.3％,respectively.(2) Effects of LNR on prognosis of patients in different AJCC N staging:of 121 patients,46 were in N0 stage,42 were in N1 stage,28 were in N2 stage,and 5 were in N3 stage.Of 42 patients in N1 stage,35 with 0 ＜ LNR ≤ 0.15 had a disease-free survival time of 12.2 months (range,1.2-82.3 months),and 7 with LNR ＞ 0.15 had a disease-free survival time of 6.9 months (range,2.1-23.1 months);the difference between the two groups was statistically significant (x2 =3.888,P＜0.05).Of the 28 patients in N2 stage,12 with 0 ＜ LNR ≤ 0.15 had a disease-free survival time of 8.5 months (range,1.2-38.8 months),and 16 with LNR ＞ 0.15 had a disease-free survival time of 4.4 months (range,1.0-52.7 months);the difference was not statistically significant (x2 =0.007,P＞0.05).Forty-six patients in N0 stage were detected no lymph node metastasis,and only 5 cases were in N3 stage,with no analysis.(3) Relationship between LNR and postoperative adjuvant chemotherapy:of the 121 patients,56 underwent postoperative adjuvant chemotherapy,which was mainly constituted by pactitaxel,platinum,and 5-fluorouracilbased regimens,58 didn't undergo postoperative adjuvant chemotherapy,and 7 had unknown data of postoperative adjuvant chemotherapy.Of 121 patients,46 had LNR =0,47 had 0 ＜ LNR ≤ 0.15,28 had LNR ＞ 0.15.Of the 46 patients with LNR =0,17 who underwent postoperative adjuvant chemotherapy had a disease-free survival time of 8.1 months (range,3.9-66.7 months) and a overall survival time of 34.0 months (range,4.7-76.0 months);29 who didn't undergo postoperative adjuvant chemotherapy had a disease-free survival time of 18.8 months (range,1.6-53.2 months),and a overall survival time of 48.6 months (range,8.3-94.2 months);there was no significant difference in the disease-free survival time and overall survival time between the two groups (x2=0.311,0.858,P＞0.05).Of the 47 patients with 0 ＜ LNR ≤ 0.15,27 who underwent postoperative adjuvant chemotherapy had a disease-free survival time of 13.3 months (range,5.0-82.3 months),and a overall survival time of 53.1 months (range,5.7-82.3 months);20 without postoperative adjuvant chemotherapy had a disease-free survival time of 8.4 months (range,1.2-39.2 months),and a overall survival time of 26.5 months (range,5.9-52.6 months).There were significant differences in the disease-free survival time and overall survival time between the two groups (x2 =10.322,4.971,P＜0.05).Of the 28 patients with LNR ＞ 0.15 (7 had unknown data of postoperative adjuvant chemotherapy),12 who underwent adjuvant chemotherapy had a diseasefree survival time of 10.3 months (range,2.9-52.7 months),and a overall survival time of 29.5 months (range,11.2-58.5 months);9 without postoperative adjuvant chemotherapy had a disease-free survival time of 2.9 months (range,1.4-35.7 months),and a overall survival time of 14.5 months (range,3.0-62.3 months);there was a significant difference in the disease-free survival time between the two groups (x2 =6.687,P＜0.05),and no significant difference in the overall survival time between the two groups (x2=2.938,P＞ 0.05).Conclusions LNR can be used as a supplementation of AJCC N staging system.In patients with 0＜ LNR ≤ 0.15,postoperative adjuvant chemotherapy can improve disease-free survival time and overall survival time.

Objective To isolate and identify the methicillin-resistant Staphylococcus aureus (MRSA) strains carrying Panton-Valentine leukocidin genes (pvl+-MRSA) from clinical samples and to further understand their molecular characteristics and infections caused by them.Methods Drug susceptibility test was performed to detect the drug resistance in 259 MRSA strains.pvl+-MRSA strains were screened out from those MRSA strains using cefoxitin slip test and mecA gene detection by PCR.Multiple PCR and multilocus sequence typing (MLST) were used for SCCmec and ST typing.Pulsed-field gel electrophoresis (PFGE) and cluster analysis were used to understand the genetic and epidemic features of the pvl+-MRSA strains.Different types of infections and diseases caused by the pvl+-MRSA strains were analyzed.ResultsAmong the 259 MRSA strains, 51 pvl+-MRSA strains were identified (19.7%, 51/259), of which 29 and 22 strains were respectively isolated from patients with community-acquired and hospital-acquired infections.ST59-SCCmecⅢ (35.3%, 18/51) was the predominant type of the 51 pvl+-MRSA strains, followed by ST59-SCCmecⅣ(25.5%, 13/51).But no predominant clone among those strains was revealed by the result of PFGE.Children, young-and middle-aged patients (≤44 years old) had a significantly higher positive rate of pvl+-MRSA than patients aged ≥45 years (P<0.05).Skin and soft tissue infection (47.1%, 24/51) was the most common disease caused by the pvl+-MRSA strains (P<0.05), followed by pneumonia (17.6%, 9/51).The pvl+-MRSA strains showed lower resistance to levofloxacin, gentamycin and rifampicine (7.8%-21.6%).No moxifloxacin-, nitrofurantoin-or linezolid-resistant pvl+-MRSA strains were identified.Conclusion The rate of pvl+-MRSA infection is high in the local population.ST59-SCCmecⅢ and ST59-SCCmecⅣ are the predominant types of pvl+ MRSA strains.Children, young-and middle-aged persons are the susceptible population.Skin and soft tissue infection and pneumonia are the common diseases caused by pvl+-MRSA.

Objective To evaluate the effect of maternal separation stress on the behavior of neonatal rd mice.Methods Neonatal rd mice were divided into maternal separation (MS) group (n=9) and control group (n=9).MS-stress was induced in the MS group by 4-hour-separation per day for 28 days.Open field test,elevated plus maze test,forced swim test and tail suspension test were used to evaluate the anxiety-like and depression-like behavior of the neonatal rd mice.Results The stay time and distance travelled of MS group in the central zone were 0.88％ and 28.17±5.65 cm,respectively,significantly shorter than that of the control group (2.61％,109.9±9.79 cm.P =0.04,P =0.001).Compared with the control group,the stay time in open arms of the MS group was significantly decreased (P<0.01),while the immobility time in forced swim test and tail suspension test of the MS group were 126.5±10.22 s and 21.56±6.83 s,significantly longer than that of the control group (77.75±16.83 s,P =0.02,7.37±3.22 s,P =0.03).Conclutions The 28-day maternal separation stress can significantly increase the anxiety-like and depression-like behavior in neonatal rd mice.

OBJECTIVETo analyze the relationship between primary tumor location and clinical response of chemotherapy in patients with metastatic colorectal cancer(mCRC).

METHODSClinical data of 721 mCRC patients who received first-line and second-line chemotherapy in Peking University Cancer Hospital between January 1996 and December 2011 were collected. All the patients were divided into 5 groups according to primary tumor location: ileocecum in 61 patients(8.5%), ascending colon or hepatic flexure in 126 patients (17.5%), transverse colon or splenic flexure in 26 patients (3.6%), descending or sigmoid colon in 172 patients (23.9%), rectum in 336 patients (46.6%). Outcomes of chemotherapy were evaluated by Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1), including complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). The overall response rate (ORR) was counted with the total number of patients divided by the number of CR+PR. Differences in first-line and second-line chemotherapy efficacy among different primary tumor sites in metastatic colorectal cancer were compared by using Chi-square test.

RESULTSOf the 571 patients receiving first-line chemotherapy, no one patient was classified as CR, while there were 190 as PR (33.3%), 277 as SD (48.5%) and 104 as PD (18.2%), with ORR 33.3% (190/571). The ORRs of patients with primary tumor located at ileocecum, ascending colon or hepatic flexure, transverse colon or splenic flexure, descending or sigmoid colon, rectum were 21.3% (10/47), 35.3% (36/102), 14.3% (3/21), 41.3% (57/138) and 31.9% (84/263), respectively, with statistically significant difference(P = 0.028). Difference of oxaliplatin-based first-line chemotherapy efficacy among different tumor sites was statistically significant(P = 0.009), while differences in irinotecan-based or single-agent 5-fluorouracil chemotherapy efficacy were not statistically significant (all P>0.05). In patients with primary tumor located at transverse colon or splenic flexure, irinotecan-based first-line chemotherapy had higher ORR than oxaliplatin-based or single-agent 5-fluorouracil chemotherapy, and the difference was statistically significant (P=0.042). There was no significant difference in the efficacy of different first-line chemotherapy regimens in patients with primary tumor located at other sites (all P>0.05). Of the 353 patients receiving second-line chemotherapy, no one patient was classified as CR, while there were 43 as PR (12.2%), 187 as SD (53.0%) and 123 as PD (34.8%), with ORR 12.2%(43/353). The ORRs of patients with primary tumor located at the ileocecum, the ascending colon or the hepatic flexure, the transverse colon or the splenic flexure, the descending or sigmoid colon, the rectum were 4.2%(1/24), 12.1%(8/66), 8.3%(1/12), 15.2%(12/79) and 12.3%(21/171) respectively, without statistically significant difference (P=0.686). Differences in second-line chemotherapy efficacy with the same regimen among different tumor sites were not statistically significant, and there were also no significant differences of efficacy of different second-line chemotherapy regimens in patients with the same tumor site (all P>0.05).

CONCLUSIONThere are differences in first-line chemotherapy efficacy among different primary tumor sites in metastatic colorectal cancer, while their second-line chemotherapy efficacy is equivalent.

Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Camptothecin ; analogs & derivatives ; Colon, Sigmoid ; Colon, Transverse ; Colorectal Neoplasms ; drug therapy ; Female ; Fluorouracil ; therapeutic use ; Humans ; Male ; Middle Aged ; Organoplatinum Compounds ; therapeutic use ; Rectum ; Retrospective Studies

Wistar rats were intragastrically administered with different doses (2, 5 and 10 g / kg body weight) of haematitum. 1H NMR-based metabonomic analysis coupled with multivariate statistical analysis (principal component analysis and partial least squares-discriminant analysis) was used to analyze the metabolic profiles of the urine samples collected from the treated rats. Univariate analysis on the 1H NMR spectra of urine (1 d before administration, 1-5 d post administration) was used to screen out the potential features of haematitum. Significant treatment related changes were observed for the levels of citrate, tuarine, creatinine,α-ketoglutarate, succinate and dimethylglycine, which could be used as potential features of haematitum. A trend of recovery in connection with dose levels was observed overtime. Such biochemical changes indicated that haematitum treatment at the dose of 2, 5 and 10 g / kg body weight affected the Krebs cycle and glucose metabolism, energy metabolism, choline metabolism and dimethylglycine metabolism in rats. These changes may attribute to the disturbances of hepatic function in 10 g / kg body weight group.

Colorectal cancer is one of the most common malignancies. Various studies have focused on differences between colon can-cers on the left and right sides. These types of colon cancer differ in terms of their molecular features, embryologic origin, anatomy, pathogenesis to physiological functions, clinical features, treatment response, and prognosis. Therefore, the left-and right-side colon cancers are regarded as different diseases. These differences have significant effect on clinical decision-making and personalized medi-cine.

Objective To investigate the drug resistant genes against carbapenems,aminoglycosides and quinolones and the molecular epidemiology of clinical isolates of Acinetobacter baumannii.Methods Forty non-duplicate strains of Acinetobacter baumannii were collected from clinical specimens in First Affiliated Hospital of Wenzhou Medical University.The identification of strains was conducted by Vitek 2 Compact system.The susceptibilities to antimicrobials commonly used were determined by agar plate dilution method and broth microdilution method.The presence of class B metalloenzyme-encoding genes (blaIMP,blaVIM,blaNDM,blaSIM,blaGIM),class D cabapenemase-encoding genes (blaOXA-23,blaOXA-48,blaOXA-58),16S rRNA methylase genes (armA,rmtB) and quinolone resistance-determining regions (QRDR) in gyrA and parC were detected by polymerase chain reaction (PCR) and sequenced.Chromosomal or plasmid location of blaOXA-23 and armA genes were assessed by Southern blot.Multiple loci sequence classification (MLST) was performed to analyze the molecular epidemiology of these strains.Results All of the 40 isolates were multi-drug resistant Acinetobacterbaumannii (MDR-AB) and showed high level resistance to all of the tested antimicrobial agents excluding colistin and tigecycline.The positive rates of blaOXA-23 and armA were 90％ and 95％,respectively.All of the 40 isolates carried QRDR mutations in gyrA and parC genes,leading to the Ser83→ Leu and the Ser80→ Leu amino-acid substitutions,respectively.Southern blot showed the chromosomal location of blaOXA-23 and armA genes.Six different ST (ST191,ST381,ST373,ST426,ST208 and ST207) were assigned for these isolates by MLST and the most dominant clones were ST191 (23/40) and ST381 (10/40).Conclusions The predominant cabapenemase-encoding gene and 16S rRNA methylase gene of Acinetobacter baumannii isolates in First Affiliated Hospital of Wenzhou Medical University are blaOXA-23 and armA,respectively,which may be located on the chromosome and vertically transmit the drug resistance.ST191 MDR-AB with blaOXa-23 and armA gene clonally spread in this hospital.