Objective To estimate the expressions of cyclin D1 and Ki67 in esophageal squamous cell carcinomas and their clinical significance. Methods The expressions of cyclin D1 and Ki67 were detected by immunohistochemistry in 28 specimens esophageal squamous cell carcinoma (ESCC) and 33 samples esophagitis tissue. Results The positive expression rate of cyclin D1 in ESCC and esophagitis tissues was 60.7% (17/28) and 33.3% (11/33) respectively (?2=4.573,P=0.032),while the mean Ki67 label index (Ki67 LI) was (49.21?25.15)% and (11.62?9.87)% respectively (t=7.908,P=0.000). The positive expression rate of cyclin D1 in TNM stage I tissues was 14.3% (1/7),which was significantly lower than that in stages Ⅲ and Ⅳ tissues[85.7% (6/7) and 100% (5/5),P=0.029 and 0.015,respectively]. In cases with lymph node metastasis,the positive rate of cyclin D1 expression was 90.9% (10/11) that was significantly higher than that in those without lymph node metastasis (P=0.016). The 28 patients with ESCC were followed up for 6 to 34 months [mean,(25.0?4.2) months],during which 19 patients survived,4 patients died of deterioration of the primary diseases (3 cases) or cerebrovascular affair (1 case),and the other 5 patients was lost to follow-up. Conclusions The expression of cyclin D1 is correlated with advanced tumor and lymph node metastasis. The Ki67 is highly expressed in ESCC tissues; no relationship exists between the level of the expression and the pathological differentiation type and TNM stage of ESCC.

Objective To study the expression of nephrin, podocin and ? actinin in glomeruli in puromycin aminonucleoside (PAN) nephrosis rat,and to disclose the possible association of these molecules with the development of proteinuria and the relation among these molecules′changes. Methods PAN nephrosis rat models were established by a single intraperitoneal injection of PAN. Indirect immunofluorescence (IF) staining and real time quantitative reverse PCR were used to study the expression of nephrin, podocin and ? actinin in glomeruli of model rats at 12 hours, 1 day, 36 hours,2 days, 5 days, 10 days, 15 days and 20 days after PAN injection. Results (1)In PAN rats, the urinary protein reached the peak level (P=0 02) at the 10th day and decreased back to control level at the 20th day. (2)The IF intensity of podocin decreased significantly at the 36th hr (P=0 04), the 2nd day(P=0 03), the 5th day(P=0 04) and the 10th day(P=0 006),while at the 15th day,the intensity began to recover (P=0 007)and reached to the control level at the 20th day. The distribution of podocin showed a linear pattern along the glomerular capillary wall (GCW) in control rats and discontinuous in PAN rats. (3)The intensity of nephrin staining decreased significantly at the 5th day (P=0 002), 10th day (P=0 007) and began to recover at the 15th day (P=0 04), while still abnormal at the 20th day(P=0 02). The distribution of nephrin in control rats showed a linear pattern along the GCW whereas a discontinuous pattern in PAN rats from the first day throughout the course. (4)The intensity of ? actinin in PAN rats increased significantly at the 20th day(P=0 009) accompanied by the increase of area ratio (P=0 007). (5)The mRNA of nephrin decreased(P =0 02)at the 5th day and recover at the 10th day. Conclusions Nephrin and podocin change prior to the presence of heavy proteinuria, which suggests their causative effects to proteinuria in PAN nephrosis. The distribution changes of nephrin and podocin occurre before the decrease of their expression level and seem to be an initial trigger factor of proteinuria. The significant decrease and recovery of podocin presents earlier than those of nephrin.The proteinuria level is correlated with the expression of podocin and nephrin.

Objective: To investigate the association between nephrin, podocin and ? actinin of the glomerular podocyte molecules, the morphometric change of podocyte foot process and the development of proteinuria. Methods: Puromycin aminonucleoside (PAN) nephrosis was established. Immunofluorescence staining, image analysis and real time quantitative PCR were employed to study the distribution and quantitation of glomerular expression of nephrin, podocin and ? actinin. Morphometric methods were applied to evaluate the morphology change of podocyte foot processes under electron microscopy. Results: (1) Before the onset of proteinuria, 2 days after PAN injection, the podocyte foot process became swollen;nephrin and podocin staining were changed into discontinuous pattern accompanied by the decrease of podocin staining intensity. The foot process became more swollen on day 5,and podocin intensity continued to decrease. Meanwhile, nephrin decreased significantly both in protein intensity and at mRNA level. (2) When heavy proteinuria [(130.8?30.7) mg/d, P =0.02]occurred, complete effacement of podocyte foot processes was revealed; both podocin and nephrin staining intensity decreased dramatically( P

Objective To establish a duplex fluorescence PCR for detection of HIV proviral DNA and to evaluate its application for early diagnosis of HIV infection in infants .Methods A duplex fluores-cence PCR system was set up based on TaqMan technology for detection of human ribonuclease P ( RNase P) gene and long terminal repeat ( LTR) region of HIV.A recombinant plasmid containing the targeted gene fragment , pTG19-T, was constructed by TA cloning technique and used as the template for evaluation of sen -sitivity of the assay .Blood samples from 11 healthy individuals and 98 HIV-infected patients were collected and detected to validate the assay specificity .The assay of duplex fluorescence PCR was then carried out to detect 96 infant blood samples collected from several maternal and child health hospitals in Zhejiang province from January 2011 to September 2012 for early diagnosis of HIV infection .The results were compared with those by using the Roche HIV DNA qualitative detection kit .Results The established duplex fluorescence PCR could specifically detect HIV proviral DNA with a specificity of 100%and a detection sensitivity of 100 cps per reaction .The coincidence rate between the established assay and the Roche HIV DNA qualitative de -tection kit was 100%in the detection of 96 blood samples .Conclusion The duplex fluorescence PCR as-say showed advantages of cost-effectiveness , convenience , good specificity and accuracy with high sensitivi-ty.It could be used for early diagnosis of HIV infection in infants and also as a general technical platform for the detection of HIV proviral DNA .

Objective To explore the molecular effect and interaction among nephrin, podocin, CD2AP and ?-actinin-4. Methods Firstly, the recombinant RNA interference (RNAi) plasmid-psiRNA-hH1GFPzeo, specifically targeting to the mRNA of nephrin, podocin, CD2AP or ?-actinin-4, was respectively tansfected into the mouse podocyte clone (MPC5) to each knockdown (KD) the expression of nephrin, podocin, CD2AP or ?-actinin-4. Molecular distributions were revealed by confocal microscopy, and the mRNA and protein expressions were detected with semi-quantitative RT-PCR and Western blotting. Results (1)In podocin KD group (siPod966 and siPod54), the mRNAs of podocin and nephrin were not detected, their protein decreased 92% and 79%, 82% and 67%, respectively. The mRNA and protein level of CD2AP increased 62% and 42%, 71% and 46%, respectively, whereas ?-actinin-4 did not change. In nephrin KD group (siNep492), the mRNA expression and protein level of nephrin were not detected, CD2AP increased 35% and 48%, respectively; and whereas podocin and ?-actinin-4 did not change. In CD2AP KD group (siCda744 and siCda21), the mRNA of expression CD2AP was not detected, and its protein level decreased 92% and 83%, the mRNA and protein of nephrin decreased 60% and 48%, 76% and 72%, respectively; podocin increased 38% and 22%, 56% and 44%, respectively; whereas ?-actinin-4 did not change. In ?-actinin-4 KD group (siAct1790 and siAct319), the mRNAs expression of ?-actinin-4 and nephrin decreased 69% and 58%, 64% and 49%, respectively; their protein level decreased 81% and 55%, 71% and 64%, respectively. However, the mRNAs of podocin and CD2AP increased 50% and 34%, 45% and 28%, respectively; and their protein level increased 64% and 46%, 65% and 42%, respectively. (2) With their expression change, the distributions of nephrin, podocin and CD2AP shifted evidently from the cell membrane surface to the nucleus circumference, whereas ?-actinin-4 showed no change, which was still localized in the cytoplasm and further extended to foot processes. Conclusion (1) Nephrin might more independently play a crucial role in the slit diaphragm complex. (2) Alpha-actinin-4 might interact direcdy or indirectly with nephrin, podocin and CD2AP. (3) The relationship among these podocyte molecules might not be spontaneous, either a single-directional or bi-directional reaction. (4) The normal localization of these podocyte molecules might depend on their normal expression quantity.

Objective To explore the molecular mechanisms underlying therapeutic responses of the anti-proteinuria drugs from the view of podocyte molecule. Methods Adriamycin (ADR) nephropathy was induced by a single tail intravenous injection of adriamycin. Lisinopril, prednisone and all-trans retinoic acid (ATRA) were administered once a day to the adriamycin-induced nephrotic rats at the first day after adriamycin injection respectively. Renal tissue samples were collected at day 3, 7, 14, and 28 after adriamycin injection respectively. The distribution, mRNA expression and protein expression of nephrin, podocin, CD2AP and ?-actinin-4 were examined by indirect immunofluorescence, real-time PCR and Western blotting, respectively. The interactions among nephrin and podocin, nephrin and CD2AP, as well as the nephrin phosphorylation were detected by immunoprecipitation, respectively. Results Compared to the control rats, 24 h urinary protein of the ADR rats increased significantly at day 14 (P

In order to study the chemical constituents in the water extract of the stem of Nauclea officinalis, column chromatography over D101 macroporous resin and silica gel and an automatic purification system were used to isolate and purify the chemical constituents from the extract. Nine compounds were obtained. By analysis of the physicochemical properties and spectral data, their structures were identified as naucleamide G (1), 3, 4-dimethoxyphenol-beta-D-apiofuranosyl (1-->6)-beta-D-glucopyranoside (2), kelampayoside A (3), 3alpha, 5alpha-tetrahydrodeoxycordifoline lactam (4), naucleamide A-10-O-beta-D-glucopyranoside (5), pumiloside (6), 3-epi-pumiloside (7), strictosamide (8) and vincosamide (9), separately. Among them, compound 1 is a new compound, compound 2 was found in plants of the genus Nauclea for the first time, and compounds 3 and 4 were isolated from this plant for the first time.

Objective To assess the characteristics of different doses of cisplatin-induced acute kidney injury,further to understand mitochondrial dysfunction and its role in acute kidney injury (AKI).Methods Male C57BL/6J mice were first randomly divided into two groups:control group (n =6) and AKI group (n =12).Then,AKI group was subsequently divided into other two groups according to different dose of cisplatin (10 mg/kg or 20 mg/kg).AKI group received intraperitoneal injection of cisplatin.All mice were sacrificed after 72 h of injection.Renal biochemical function,renal pathological changes,renal injury markers,kidney mitochondrial function and structural changes were observed.Results (1) After 72 hours of injection,the AKI group performed significant kidney injury changes compared to control group,thereinto 20 mg/kg group was more serious than 10 mg/kg group.With the cisplatin dose increasing,renal function markers such as serum creatinine,urine protein gradually increased.(2)Kidney biopsy showed tubular structural damage,the formation of protein casts,kidney injury molecule-1 (KIM-1) gradually increased(P ＜ 0.05).(3)Electron microscopy found tubular mitochondrial structural damage,mtDNA copy number decreased,the level of peroxisome proliferatoractivated receptor-gamma coactivator-1alpha (PGC-1α),ATP synthase β decreased(P ＜ 0.05),and Western blotting manifested cytochrome C was released from mitochondria to the cytoplasm.These data all exhibited significant difference between different groups(P ＜ 0.05).Conclusions Cisplatin induces acute kidney injury in dose-dependent manner.Mitochondrial dysfunction participates in kidney injury,and is also related to the kidney pathological damage.

AIM:To study whether cordycepin (Cordy) plays a role in myocardial protection by regulating the expression of microRNA-455 ( miR-455) and reducing apoptosis induced by endoplasmic reticulum stress in the ischemia -reperfusion (IR) rats.METHODS: SD rats (250 ~300 g) were randomly divided into 3 groups: control group, the chests of the rats were only opened;IR group, the rats were given myocardial ischemia for 30 min, and then reperfusion for 120 min;IR+Cordy group:before IR, the rats were given Cordy (10 mg/kg) through femoral vein injection once a day for one week, and the last injection was given 30 min before ischemia.Automated biochemical analysis was used to detect rat serum activity of LDH and CK-MB.The myocardial endothelial cell ( EC) apoptotic rate was measured by TUNEL , and the ultrastructural changes of the myocardial EC were observed under transmission electron microscope .RT-qPCR was used to detect the expression of miR-455 and the mRNA levels of glucose-regulated protein 78 ( Grp78) and caspase-12 in the myo-cardium.RESULTS:Compared with control group, EC apoptosis in IR group was significantly increased (P<0.05). Compared with IR group , EC apoptosis in IR +Cordy group decreased significantly ( P<0.05 ) .Compared with control group, swelling of mitochondria , irregular membrane , loose wrinkles with vacuoles , disappeared matrix granules , irregular nuclear membrane , chromatin condensation , disappeared nucleoli and even small apoptosis body in the EC were found in IR group.Compared with IR group , the symptoms in IR +Cordy group were greatly improved .Compared with control group, the mRNA expression of Grp78 and caspase-12 as well as the miR-455 level in IR group was increased (P<0.05). Compared with IR group , the mRNA expression of Grp78 and caspase-12, as well as the miR-455 level in IR+Cordy group was decreased (P<0.05).CONCLUSION:Cordycepin attenuates myocardial EC apoptosis , down-regulates the expres-sion of miR-455, and inhibits endoplasmic reticulum stress in the myocardium .

Telomeres are protective caps located at the ends of human chromosomes. Telomeres shorten with each successive cell di-vision in normal human cells, whereas they are continuously elongated by human telomerase in over 85%of tumors. This simple and attractive difference steers the development of anticancer drugs targeting telomeres and telomerase. Many promising current telo-mere/telomerase-targeting agents, such as GRN163L and GV1001, showed good therapeutic effect both in preclinical studies and phaseⅠ/Ⅱclinical trials. These agents have even entered phaseⅢclinical trials in patients with various tumors. Most therapeutics are more effective when used in combination with standard chemotherapies. Moreover, pharmacological interference with tumor-cell telomere biology to reduce telomere length and/or telomere stability could enhance the effectiveness and safety of radiotherapy. Therapeutics targeting telomere/telomerase may play a key role in radiotherapy in the era of personalized medicine in the future.