Background and purpose: Checkpoint kinase 1 and 2 have been proposed to be potential therapeutic targets to sensitize cancers to radio- or chemo-therapeutics. However, little is known about whether Chk1/2 is also a suitable target for sensitizing cancers to curcumin. In the present study, we investigated effects of Chk1/2 siRNA on curcumin-induced apopotosis in hepatoma cell line Huh7 and evaluated the effectiveness of Chkl/2as a therapeutic target to potentiate human hepatoma to curcumin. Methods: Effect of curcumin on the cell cycle checkpoint-associated proteins was detected by Westem blot. The knockdown efficacy of Chk1/2 siRNA was measured by RT-PCR and Westem blot. Effect of Chk1/2 siRNA on curcumin-induced apoptosis in Huh7 cells was evaluated by DAPI staining. Effect of Chk1/2 siRNA on cell cycle distribution in curcumin-treated Huh7 cells was analyzed by flow cytometry. Results: Curcumin significantly inhibited phosphorylation of cell cycle checkpoint-associtaed proteins Chk1(S317), Cdc25C(S216) and Cdk1(Y15). Chk1 siRNA decreased Chk1 mRNA and protein by 95% and 92% and Chk2 siRNA decreased Chk2 mRNA and protein by 60% and 55% respectively as compared with negative control siRNA (P<0.01). Inhibition of Chk1, but not Chk2, increased apoptotic rate from (21.3±1.8)% to (29.5±2.6)% (P<0.05). Neither Chk1 nor Cbk2 siRNA had any impact on cell cycle distribution in Huh7 cells induced by curcumin. Conclusion: Chk1 siRNA sensitized Huh7 cells to curcumin-induced apoptosis, suggesting that Chk1 is a potential therapeutic target to sensitize human hepatoma to curcumin.

Background and purpose:Checkpoint kinase 1 and 2 have been proposed to be potential therapeutic targets to sensitize cancers to radioor chemo-therapeutics. However, little is known about whether Chk1/2 is also a suitable target for sensitizing cancers to curcumin. In the present study, we investigated effects of Chk1/2 siRNA on curcumin-induced apopotosis in hepatoma cell line Huh7 and evaluated the effectiveness of Chk1/2 as a therapeutic target to potentiate human hepatoma to curcumin. Methods:Effect of curcumin on the cell cycle checkpoint-associated proteins was detected by Western blot. The knockdown efficacy of Chk1/2 siRNA was measured by RT-PCR and Western blot. Effect of Chk1/2 siRNA on curcumin-induced apoptosis in Huh7 cells was evaluated by DAPI staining. Effect of Chk1/2 siRNA on cell cycle distribution in curcumin-treated Huh7 cells was analyzed by flow cytometry. Results:Curcumin significantly inhibited phosphorylation of cell cycle checkpoint-associtaed proteins Chk1(S317), Cdc25C(S216) and Cdk1(Y15). Chk1 siRNA decreased Chk1 mRNA and protein by 95% and 92% and Chk2 siRNA decreased Chk2 mRNA and protein by 60% and 55% respectively as compared with negative control siRNA (P

New registry classifications of chemical drugs redefined the concepts of new drugs and generic drugs. New drugs emphasize the international innovation including innovate new drugs and modified new drugs. Modified new drugs are improvement of drugs already on the market to achieve superior efficacy. With the advantages of high success rate,high return,low risk and long life cycle,modified new drugs have become the mainstream of new drugs research and development in the world. Modified new drugs R&D in China should be based on clinical demand and focused on the differentiation research in order to obtain new preparations with signif-icant clinical advantages to better serve the clinical practise. This paper states the concept and characteristics of modified new drugs in China,makes case analysis and shares the development history of blockbuster modified new drugs in the world,clarifies the opportuni-ties and challenges and provides ideas for the future development of modified new drugs in China.

Objective To assess the association between NOD2 gene single nucleotide polymorphisms (SNPs) and leprosy in Chinese Yi population.Methods Whole blood samples were obtained from 300 patients with leprosy and 300 healthy human controls of Yi nationality in Sichuan province.Genomic DNA was extracted,and a SNaPshot assay was performed to determine the genotypes of four single nucleotide polymorphisms (SNPs) of the NOD2 gene,including rs9302752,rs7194886,rs8057341 and rs3135499.Chi-square test was conducted to compare allele frequency,and Hardy-Weinberg equilibrium was tested.Results The genotype distribution of all the four SNPs was consistent with Hardy-Weinberg equilibrium (all P ＞ 0.05).Significant differences were observed between the patients with leprosy and healthy controls in both genotype distribution and allele frequency of the SNP rs3135499 (both P ＜ 0.01),but not in those of the other three SNPs (all P ＞ 0.05).Conclusion The SNP rs3135499 of the NOD2 gene may be associated with the development of leprosy in Chinese Yi population.

Objective To explore the expressions and distributions of glial cell line -derived neurotrophic factor (GDNF)and itstyrosine kinase receptor RET in the terminal rectums of fetal rats with congenital anorectal malfor-mations (ARM)at different gestationalage,and to explore their effects on the enteric nervous system in the terminal rectum of ARMfetal rats.Methods Thirty -five SD pregnancy rats were divided into a saline group (n =1 0)and an ethylenethiourea experiment group (n =25)by simple randomized study.The fetal rats were removed from the pregnant rats at the gestational 1 6 d,1 8 d and 20 d.The fetal rats were divided into the saline control group,the ethylenethiourea control group (fetal rats without ARM)and the ethylenethiourea malformation group (ARM fetal rats)by the naked eye and dissecting microscope.HE staining was used to observe the morphology and the intestinal ganglion cells in the terminal rectum were counted.The immunohistochemical staining and Western blot methods were used to observe the distributions of GDNF and RET in the rectum at the gestational 1 6 d,1 8 d and 20 d.The quantitative real -time poly-merase chain reaction (qRT -PCR)was used to detect the expression of GDNF mRNA in the fetal rats in the terminal rectum at the gestational 1 6 d,1 8 d and 20 d.Results HE staining:the development of anorectal terminal in 3 groups of fetal rats was unclear at the gestational 1 6 d.A small amount of scattered nerve plexuses were observed in the muscu-lar layer.The nuclei were small and sparse.The axons and cytoplasms were less.The serosal layer,muscular layer,sub-mucosa,mucosal layer and glands in the terminal rectum were gradually clear in the saline control group and the ethyle-nethiourea control group at the gestational 1 8 d and 20 d.The intermuscular submucosal nerve plexuses increased gra-dually (1 1 .400 ±3.1 34 and 1 1 .200 ±3.425 at the gestational 1 8 d;66.1 00 ±4.954 and 67.600 ±5.481 at the gesta-tional 20 d).While,the layer was unclear in the ethylenethiourea malformation group and the nerve plexus was less (7.800 ±1 .989 at the gestational 1 8 d,and 25.200 ±3.048 at the gestational 20 d),and the difference was statistical-ly significant compared with 2 control groups (F =7.591 ,271 .833,all P <0.05).Immunohistochemistry satning:the expressions of GDNF and RET in all layers of the intestinal wall in the 3 groups of fetal rats were unclear at the gesta-tional 1 6 d and only a few positive cells were observed.The GDNF and RET were expressed in the mucosal layer and submucosa of the terminal rectum as well as intermuscular nerve plexus in the saline control group and the ethylene-thioured control group at the gestational 1 8 d and 20 d.With the continuous development of the embryo,their expression intensities were gradually increased.The expressions of GDNF and RET positive cells were decreased gradually in the ethylenethiourea malformation group.The difference was significant statistically compared with 2 control groups (all P <0.05).qRT -PCR:the expressions of GDNF mRNA showed no statistical difference among 3 groups at the gestational 1 6 d (P >0.05);the expressions of GDNF and RET protein were 1 03.624 ±27.533 and 1 05.1 84 ±1 9.634 at the ges-tational 1 8 d;1 51 .496 ±33.622 and 1 50.738 ±21 .423 at the gestational 20 d in 2 control groups.Compared with the ethylenethiourea malformation group (79.1 69 ±1 1 .697 at the gestational 1 8 d;94.873 ±1 1 .309 at the gestational 20 d),and the difference were statistically significant (all P <0.05).Conclusions The expressions of GDNF and its tyrosine kinase receptor RET had a certain temporal correlation in the terminal rectum of normal fetal rats at different gestational ages and ARM.Moreover,the abnormal expressions of GDNF and its tyrosine kinase receptor RET in the dis-tal rectum of ARMfetal rats can affect the development of enteric nervous system.

OBJECTIVE: To investigate the expression of hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) and connexin43 (Cx43) in the sinoatrial node of electric shock death. METHODS: As experimental group, 34 cases of electric shock death who had definite current mark evidence were selected from pathology department of Xuzhou Medical College from 2010 to 2013. As the control group, 20 cases of fatal severe craniocerebral injury in traffic accidents were chosen. The expressions of HCN4 and Cx43 in the sinoatrial node were observed by immunohistochemical technology. RESULTS: HCN4 positive cells expressed in the cell membrane and cytoplasm of the sinoatrial node. Cx43 positive cells expressed in the cell membrane and cytoplasm of T cells and myocardial cells. The expression of HCN4 was significantly higher than that of the control group (P < 0.05) and the expression of Cx43 was significantly lower than that of the control group (P < 0.05). CONCLUSION The changes of HCN4 and Cx43 expressions in the sinoatrial node illustrate electric shock death might be related to the abnormalities of cardiac electrophysiology and conduction.
Connexin 43/metabolism* ; Cyclic Nucleotide-Gated Cation Channels ; Heart Rate ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism* ; Immunohistochemistry/methods* ; Myocardium/metabolism* ; Myocytes, Cardiac ; Sinoatrial Node/physiopathology*

Objective To investigate the effects of DMOG on the microcirculation of the choke-area and the survival of the cross-boundary flap in rats via tail vein injection.Methods Rats with ischemic three-territory perforator flaps on the dorsum were treated with DMOG at a dosage of 40 mg/kg body weight via tail vein injection at 1 day before surgery(day-1),the time of surgery(day 0),1 day after surgery(day 1),2 days after surgery(day 2) and 3 days after surgery(day 3).Control group received sterile saline at the same time points and same dosage via tail vein injection.① Draw materials from the choke-area at day 1,day 3 and day 7,HE stain was used to compare the diameter size of the artery and vein at the same site.② Western blotting to check the expression of PCNA and HIF-1α,ELISA to detect the content of PCNA,HIF-1α,SDF-1α and VEGF at day 7.③At day 7,measure the survival area of the flap and observe the vessel of the flap by lead oxide-gelatine technique.Results ① There was a greater survival rate of (96.3 ± 5.1)％ in the treatment group than in the control group with (73.9 ± 5.8)％ at day 7 (P ＜ 0.05).② The diameter size of the arterioles and venules were dilated in both groups until postoperative days 7.But the treatment group was more expanded than the control group at day 3(2.20 ± 0.26 vs.1.50 ± 0.20,P ＜ 0.05) and day 7(3.67 ± 0.35 vs.2.03 ± 0.15,P ＜ 0.05).③ The skin expression of PCNA and HIF-1α in the treatment group were greater than the control group(P ＜ 0.05) at day 7.④ The content of skin PCNA in the treatment group and control group were(8.95 ± 0.71) ng/mg and (4.15 ± 0.72) ng/mg,HIF-1α were(5.04 ± 0.50)ng/mg and (2.98 ± 0.29) ng/mg,SDF-1α were (2.91 ± 0.61) ng/mg and (1.39 ± 0.62) ng/mg,and VEGF were(2.17 ± 0.41) ng/mg and (0.95 ± 0.44) ng/mg,respectively.The treatment group was greater than the control group (P ＜ 0.05).Conclusion DMOG can improve the microcirculation of the choke area,and then increase the survival of the perforator skin flaps in rats via tail vein injection.

Mycoepoxydiene is a novel antitumor agent extracted from marine lignicolous fungi HLY-2, which is Diaporthe phaseolorum by molecule identification. The medium optimization for mycoepoxydiene by orthogonal design and the comparison of submerged fermentation and solid state fermentation were studied. The rusult is that the maximal yield of the compound is 543mg/L, which is 43 times compared to the customary half-seawater PD medium and 15 times to the best submerged condition. This optimum culture medium included potato 250g/L, seawater 300mL/L, glucose 30g/L, lactose 50g/L, KH_ 2 PO_ 4 0.65mmol/L and (NH_ 4 )_ 2 SO_ 4 1g/L in the solid state condition. Differentiation analysis between submerged and solid state fermentation, and antitumor activity of these ferment products were also studied. The antitumor activity of products of the optimum medium approached the pure compound.

Objective: To know the pre-treatment drug resistance ( PDR ) status of newly reported human immunodeficiency virus type 1 ( HIV-1 ) infected individuals in Wenzhou, so as to provide guidance for antiretroviral therapy ( ART ). Methods: Totally 232 plasma samples of newly reported HIV-1 infected individuals who had not received ART were collected in Wenzhou in 2019. Virus ( HIV-1 ) RNA was extracted, followed by reverse transcription PCR and nested PCR to amplify the pol region and sequence. Resistance mutations and resistance to non-nucleoside reverse transcriptase inhibitors ( NNRTIs ), nucleoside reverse transcriptase inhibitors ( NRTIs ) and protease inhibitors ( PIs ) was analyzed. Results: The pol region sequences from 199 infected patients were obtained and the incidence of PDR was 8.04% ( 16/199 ). Eight genotypes were detected, including circulating recombinant forms ( CRFs ) CRF07_BC ( 47.24%, 94/199 ) and CRF01_AE ( 29.15%, 58/199 ) which were the dominant types. Two unique recombinant forms ( URFs ) were detected, namely URF( CRF01_AE/BC ) and URF( B/C ) . Thirty-one cases ( 15.58% 31/199 ) had drug-resistant mutations. For NNRTIs, NRTIs and PIs, 20 cases ( 64.52% ) , 2 cases ( 6.45% ) and 9 cases ( 29.03% ) with drug resistance mutations were detected, respectively. The resistance mutations to NNRTIs included K101E, K103N/R, V106I, E138K, V179D/E/T, Y181C, G190A and H221Y. Four cases each had two resistance mutations to NNRTIs. The resistance mutations to NRTIs were V75M and M184V. The resistance mutations to PIs were M46I, L33F and Q58E. For the newly released NNRTI drug Doravirine ( DOR ), two cases were found to have mutations of resistance. Conclusions The incidence of PDR among newly reported HIV-1 patients in Wenzhou is 8.04%, mainly caused by NNRTIs drug-resistant mutation. Resistance to the new drug DOR has emerged. The surveillance of drug resistance should continue to be strengthened.

Objective Pemetrexed and β-elemene can inhibit the growth of tumor cells .This study was to investigate the effect of pemetrexed combined with β-elemene on the proliferation and apoptosis of cervical cancer HeLa cells. Methods Cervical cancer HeLa cells were treated with pemetrexed at the concentrations of 38, 76, 152, 228, and 304μg/mL, and at 24 and 48 hours of treatment subjected to MTT for detection of their proliferation .The experiment included four groups , with the cells treated with β-elemene ( 125μg/mL) , pemetrexed ( 76 μg/mL ) , β-elemene ( 125 μg/mL ) +pemetrexed (76μg/mL), and nothing (blank control) for 24 hours, followed by determination of their proliferation and apoptosis by MTT and flow cytometry, respectively. Results Pemetrexed at 38, 76, 152 and 228μg/mL inhibited the proliferation of the HeLa cells in a concentration-dependent manner, with the inhibition rates of (7.24 ±3.78), (7.94 ±4.37), (11.10 ±2.86) and (15.88 ± 3.38)%at 24 hours, and (16.69 ±0.95), (22.54 ±1.53), (24.48 ±0.92) and (25.54 ±3.61)%at 48 hours, both with statis-tically significant differences between any two groups (P<0.05).Significant differences were also found in the proliferation rate of the same concentration of pemetrexed at the two time points (P<0.05).The combination of pemetrexed and β-elemene showed an inhibi-tion rate of (49.95 ±5.76)%at 24 hours, remarkably higher than (24.36 ±5.59)%in theβ-elemene group and (10.69 ±1.37)%in the pemetrexed group (P<0.01). Conclusion Pemetrexed combined with β-elemene can significantly inhibit the proliferation and synergistically accelerate the apoptosis of HeLa cells .