MiR-3653 blocks autophagy to inhibit epithelial-mesenchymal transition in breast cancer cells by targeting the autophagy-regulatory genes ATG12 and AMBRA1 / 中华医学杂志(英文版)

BACKGROUND@#Metastasis is the main cause of tumor-associated death and mainly responsible for treatment failure of breast cancer. Autophagy accelerates tumor metastasis. In our work, we aimed to investigate the possibility of microRNAs (miRNAs) which participate in the regulation of autophagy to inhibit tumor metastasis.@*METHODS@#MiRNA array and comprehensive analysis were performed to identify miRNAs which participated in the regulation of autophagy to inhibit tumor metastasis. The expression levels of miR-3653 in breast cancer tissues and cells were detected by quantitative real-time polymerase chain reaction. In vivo and in vitro assays were conducted to determine the function of miR-3653. The target genes of miR-3653 were detected by a dual luciferase reporter activity assay and Western blot. The relationship between miR-3653 and epithelial-mesenchymal transition (EMT) was assessed by Western blot. Student's t -test was used to analyze the difference between any two groups, and the difference among multiple groups was analyzed with one-way analysis of variance and a Bonferroni post hoc test.@*RESULTS@#miR-3653 was downregulated in breast cancer cells with high metastatic ability, and high expression of miR-3653 blocked autophagic flux in breast cancer cells. Clinically, low expression of miR-3653 in breast cancer tissues (0.054 ± 0.013 vs . 0.131 ± 0.028, t  = 2.475, P  = 0.014) was positively correlated with lymph node metastasis (0.015 ± 0.004 vs . 0.078 ± 0.020, t  = 2.319, P  = 0.023) and poor prognosis ( P  < 0.001). miR-3653 ameliorated the malignant phenotypes of breast cancer cells, including proliferation, migration (MDA-MB-231: 0.353 ± 0.013 vs . 1.000 ± 0.038, t  = 16.290, P  < 0.001; MDA-MB-468: 0.200 ± 0.014 vs . 1.000 ± 0.043, t  = 17.530, P  < 0.001), invasion (MDA-MB-231: 0.723 ± 0.056 vs . 1.000 ± 0.035, t  = 4.223, P  = 0.013; MDA-MB-468: 0.222 ± 0.016 vs . 1.000 ± 0.019, t  = 31.050, P  < 0.001), and colony formation (MDA-MB-231: 0.472 ± 0.022 vs . 1.000 ± 0.022, t  = 16.620, P  < 0.001; MDA-MB-468: 0.650 ± 0.040 vs . 1.000 ± 0.098, t  = 3.297, P  = 0.030). The autophagy-associated genes autophagy-related gene 12 ( ATG12 ) and activating molecule in beclin 1-regulated autophagy protein 1 ( AMBRA1 ) are target genes of miR-3653. Further studies showed that miR-3653 inhibited EMT by targeting ATG12 and AMBRA1 .@*CONCLUSIONS@#Our findings suggested that miR-3653 inhibits the autophagy process by targeting ATG12 and AMBRA1 , thereby inhibiting EMT, and provided a new idea and target for the metastasis of breast cancer.

Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis

Bromodomain containing protein 4 (BRD4), as an epigenetic reader, can specifically bind to the acetyl lysine residues of histones and has emerged as an attractive therapeutic target for various diseases, including cancer, cardiac remodeling and heart failure. Herein, we described the discovery of hit 5 bearing 4-phenylquinazoline skeleton through a high-throughput virtual screen using 2,003,400 compound library (enamine). Then, structure-activity relationship (SAR) study was performed and 47 new 4-phenylquinazoline derivatives toward BRD4 were further designed, synthesized and evaluated, using HTRF assay set up in our lab. Eventually, we identified compound C-34, which possessed better pharmacokinetic and physicochemical properties as well as lower cytotoxicity against NRCF and NRCM cells, compared to the positive control JQ1. Using computer-based molecular docking and cellular thermal shift assay, we further verified that C-34 could target BRD4 at molecular and cellular levels. Furthermore, treatment with C-34 effectively alleviated fibroblast activation in vitro and cardiac fibrosis in vivo, which was correlated with the decreased expression of BRD4 downstream target c-MYC as well as the depressed TGF-β1/Smad2/3 signaling pathway. Taken together, our findings indicate that novel BRD4 inhibitor C-34 tethering a 4-phenylquinazoline scaffold can serve as a lead compound for further development to treat fibrotic cardiovascular disease.

Study on Proficiency Testing of Determination Potassium in Serum / 中国医疗器械杂志

OBJECTIVE@#To evaluate the capacity of laboratories participated in the proficiency testing (PT) of determination potassium in serum and improve the quality of testing, and put forward technical suggestions for unsatisfied laboratories.@*METHODS@#According to the requirements of CNAS related documents, the homogeneity and stability of the real PT sample were evaluated by one-way ANOVA and t test, respectively. The values of real PT samples were assigned by reference method which was used in PT results assay. It is required that the deviation of value of real PT samples (code:2, 3, 5) between the measured value and the assigned value shall be within ±15.0%. The precision of values for all samples should not be greater than 3.0%.@*RESULTS@#All the laboratories submitted valid data according to the requirements. Only one laboratory did not meet the requirements, and the satisfaction rate was 90.9%.@*CONCLUSIONS@#The ability of most of laboratories are accurate and reliable.

Characteristics of amino acid and codon usage of Env genes in HIV-1 infected individuals with highly broad cross-neutralizing activity / 中华微生物学和免疫学杂志

Objective:To study the amino acid and codon usage profile of HIV-1 Env gene in donors whose serum exhibit highly broad cross-neutralizing activity. Methods:The samples were divided into highly broad cross-neutralizing activity group (hBCN + group) and non-highly broad cross-neutralizing activity group (hBCN - group) based on whether the neutralization breadth was higher than 90% or not. Full-length Env genes were amplified by single genome amplification (SGA) method from patients′ plasma samples, and the characteristics of Env sequences in hBCN + group were compared with hBCN - group. The correspondence analysis (COA) on relative amino acid usage (RAAU), adaptability to host based on similarity index D( A, B) and relative synonymous codon usage (RSCU) values of Env genes (hBCN + and hBCN -) with respect to human host RSCU were analyzed. Results:Correspondence analysis showed that the RAAU data of hBCN + group and hBCN - group were distributed along the two main axes to form two relatively separated clusters, indicating that the Env genes of the two groups had relatively unique amino acid usage patterns; the similarity index calculation results showed that hBCN + group (0.097) was lower than the hBCN - group (0.102), in addition, the Env gene of the hBCN + group had less frequency of similarly selected codons with human host system compared to hBCN - group. Conclusions:Env genes in hBCN + group and hBCN - group may have relatively unique amino acid usage patterns, and virus strains in hBCN + group are less adaptable to the host than those in hBCN - group.

Discovery of thiosemicarbazone derivatives as effective New Delhi metallo-β-lactamase-1 (NDM-1) inhibitors against NDM-1 producing clinical isolates

@#New Delhi metallo-β-lactamase-1 (NDM-1) is capable of hydrolyzing nearly all β-lactam antibiotics, posing an emerging threat to public health. There are currently less effective treatment options for treating NDM-1 positive “superbug”, and no promising NDM-1 inhibitors were used in clinical practice. In this study, structure–activity relationship based on thiosemicarbazone derivatives was systematically characterized and their potential activities combined with meropenem (MEM) were evaluated. Compounds 19bg and 19bh exhibited excellent activity against 10 NDM-positive isolate clinical isolates in reversing MEM resistance. Further studies demonstrated compounds 19bg and 19bh were uncompetitive NDM-1 inhibitors with Ki = 0.63 and 0.44 μmol/L, respectively. Molecular docking speculated that compounds 19bg and 19bh were most likely to bind in the allosteric pocket which would affect the catalytic effect of NDM-1 on the substrate meropenem. Toxicity evaluation experiment showed that no hemolysis activities even at concentrations of 1000 mg/mL against red blood cells. In vivo experimental results showed combination of MEM and compound 19bh was markedly effective in treating infections caused by NDM-1 positive strain and prolonging the survival time of sepsis mice. Our finding showed that compound 19bh might be a promising lead in developing new inhibitor to treat NDM-1 producing superbug.

Development of the triazole-fused pyrimidine derivatives as highly potent and reversible inhibitors of histone lysine specific demethylase 1 (LSD1/KDM1A)

Histone lysine specific demethylase 1 (LSD1) has been recognized as an important modulator in post-translational process in epigenetics. Dysregulation of LSD1 has been implicated in the development of various cancers. Herein, we report the discovery of the hit compound (IC = 3.93 μmol/L) and further medicinal chemistry efforts, leading to the generation of compound (IC = 49 nmol/L, and = 16 nmol/L), which inhibited LSD1 reversibly and competitively with H3K4me2, and was selective to LSD1 over MAO-A/B. Docking studies were performed to rationalize the potency of compound . Compound also showed strong antiproliferative activity against four leukemia cell lines (OCL-AML3, K562, THP-1 and U937) as well as the lymphoma cell line Raji with the IC values of 1.79, 1.30, 0.45, 1.22 and 1.40 μmol/L, respectively. In THP-1 cell line, significantly inhibited colony formation and caused remarkable morphological changes. Compound induced expression of CD86 and CD11b in THP-1 cells, confirming its cellular activity and ability of inducing differentiation. The findings further indicate that targeting LSD1 is a promising strategy for AML treatment, the triazole-fused pyrimidine derivatives are new scaffolds for the development of LSD1/KDM1A inhibitors.

Secondary metabolites from Aspergillus petrakii / 中成药

AIM To study the secondary metabolites from Aspergillus petrakii.METHODS The ethyl acetate extract liquid of A.petrakii fermentation broth was isolated and purified by silica,ODS,Sephadex LH-20 and RP-HPLC column,then the structures of obtained compounds were identified by spectral data and physicochemical properties.RESULTS Thirteen compounds were isolated and identified as R-(-)-mellein (1),3-(1-hydroxyethyl)-7-hydroxy-l-isobenzofuranone (2),(-)-semivioxanthin (3),endocrocin (4),p-hydroxyphenylethanol (5),p-hydroxyphenylacetamide (6),hydroquinone (7),adenosine (8),cyclo (Phe-Val) (9),cyclo (Phe-Ile) (10),cyclo (Tyr-Ala) (11),cyclo (Leu-Ile) (12),cyclo (Leu-Leu) (13).CONCLUSION All the compounds are isolated from A.petrakii for the first time.

Replication kinetic properties of HIV-1 CRF_BC novel drug resistance associated mutations / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the drug sensitivity and analyze the replication kinetics of HIV-1 B and CRF07_BC subtypes with I132L or T139K/R mutations.</p><p><b>METHODS</b>The amino acids in position 132 and 139 of reverse transcriptase (RT) region of the infectious clone PNL4-3 (HIV-1 B subtype) were changed to L and T/R through site mutagenesis. Combined with the previously constructed infectious clone of HIV-1 CRF07_BC subtype with I132L and T139K/R mutations in RT region, mutated PNL4-3 infectious clones were transfected into 293T cells. The infection ability of mutated clones was detected. The drug sensitivity to NNRTIs (TMC-125, DLV, NVP, EFV) and the properties of replication kinetics were also evaluated.</p><p><b>RESULTS</b>The mutated infectious clones were constructed including PNL4-3-RT-I132L, PNL4-3-RT-T139K and PNL4-3-RT-T139R. The I132L and T139K/R mutations in HIV-1 B and CRF07_BC infectious clones reduced their drug sensitivity to NNRTIs, which accompanied with the increase of EC50 (concentration for 50% of maximal effect). In subtype CRF07_BC, I132L mutation increased EC50 by 2.55, 19.35, 28.05, 6.13 fold, T139K mutation increased EC50 by 4.67, 3.66, 7.35, 3.30 fold, and T139R mutation increased EC50 by 1.82, 4.69, 25.12, 1.89 fold, respectively. In subtype B, I132L increased EC50 by 3.91, 4.61, 6.38, 3.56 fold, T139K increased EC50 by 3.13, 1.78, 2.26, 2.10 fold, T139R increased EC50 by 5.79, 3.99, 5.78, 2.75 fold, respectively. Similar as wild type PNL4-3, the replication ability of 132L/139K/139R mutated infectious clones reached the peak in day 11. However, compared to wild type BC-WT, I132L/T139R mutations delayed the peak time to day 14 and 21.</p><p><b>CONCLUSION</b>The novel drug resistance associated mutations I132L and T139K/R can reduce the drug sensitivity to NNRTIs in subtype B and CRF07_BC, and the replication ability of CRF_07BC declined by I132L mutation.</p>

Resistance evolutionary pathway analysis of HIV-1 CRF_07BC reverse transcriptase / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study resistance evolution pathway of HIV-1 CRF_BC under drug selection pressure, and compare with B subtype.</p><p><b>METHODS</b>Based on the reverse transcriptase region of CRF_ 97BC HIV-1 from 588 treatment-naive and 274 treatment patients, selection pressure based method was used to select resistance-associated mutations, and Bayesian network was used to construct the resistance evolutionary pathway under antiretroviral therapy. Meanwhile, it was constructed that the resistance evolutionary pathway for B subtype with the same regimens using the data from HIV resistance database, and made a comparison with CRF_07BC.</p><p><b>RESULTS</b>The major resistance mutations for CRF_07BC were identified including K103N, Q197K, V179D and Y188L. While for B subtype, the major resistance mutations include M184V, K103N,Y181C, T69N,G190A, K238T,Y188H and P225H. Much difference was observed between these two classes. However, the classical TMA1 (41L, 210W and 215Y) and TMA2 (67N, 70R and 219E/Q) pathways exist in both pathways. As different from B subtype, the predicted major drug resistance mutations for CRF_07BC did not contain TAM-related mutations, and nucleoside reverse transcriptase inhibitor-related mutations and non-nucleoside reverse transcriptase inhibitor-related mutations were mutually depending on each other.</p><p><b>CONCLUSION</b>HIV-1 CRF_07BC showed distinctive resistance evolutionary pathway, the mutations K103N,Q197K,V179D and Y188L were the major resistance mutations, and different resistance evolutionary pathways were observed between HIV-1 CRF_07BC and B subtype.</p>

The biologic characteristics of human immunodeficieney virus-1 subtype B' R5 tropic strains in different disease stage / 中华传染病杂志

Objective To study biological characteristics of R5 tropic human immunodeficiency virus (HIV)-1 strains in different disease stage. Methods Primary clinical viruses were isolated from fresh peripheral blood mononuclear cells (PBMC) using co-culture methods; meanwhile, viral co receptor usage and infectivity were tested using flow cytometry on GHOST (3) cell lines,which expressed CD4 receptor and CC ehemokine receptor 5 (CCR5) or CXCR4 eoreceptor; to identified CCR5 tropic viruses(R5 tropic strains). Viral replication kinetics was detected in PBMCs. Plasma viral load was measured using an HIV-1 nucleotide fluorescence quantification assay kit. Results There were 22 individuals with HIV-1 subtype B' infection, in which 11 were CD4>0. 2 × 109/L and 11 were CD4≤0. 2 × 109/L. All isolated viruses used CCR5 coreceptor and therefore were HIV-1 R5 tropic strains. The infectivity of R5 tropic strains isolated from patients with CD4≤0.2 × 109/L was (7.392 7 ± 4. 584 2) % ; while the infectivity of R5 tropic strain from patients with CD4>0. 2 × 109/L was (2. 613 6 ± 1. 610 5)%. There were significant statistical difference(t= 3. 262, P<0.05). The possibility of viral replication became strong after the day 7 post-infection. There was a significant difference of viral replication between two groups in the day 7,10, 15 post-infection(t value was 3. 771, 2. 509 and 2. 260 respectively, P<0. 05). The possibility of viral replication was higher in CD4≤0.2 ×109/L group than that of CD4>0.2 × 109/L group. The logarithm of viral load was (5. 606 8 ± 0. 815 1 ) copies/mL in CD4≤0.2 × 109/L group and (4. 729 8 ± 0. 431 6) copies/mL in CD4> 0.2 × 109/L group. There was a significant difference between two groups(t = 3. 771 ; P<0.05). Conclusion Viral infection and replication are enhanced during progression of disease, even if viral coreceptor usage do not switch from CCR5 to CXCR4.

Viral load and neutralizing activity of plasma from HIV-1 chronic infected former blood donors in Anhui province, China / 中华微生物学和免疫学杂志

Objective To explore the association among viral load,CD4 count and neutralizing ac-tivity of plasma from chronic HIV-1 infected former blood donors in Anhui province, China. Methods 294 chronically HIV-1 B' infected individuals from former blood donors in Anhni province were enrolled, whose plasma and peripheral blood mononuelnar cells (PBMC) were isolated. The viral load and CD4 were detec-ted, and the neutralization activity of plasma against primary virus (1597) and lab-adapted strain (SF33) was detected by Luciferase Assay System based on TZM cell line, and 50% neutralizing level was calculated by the Reed-Mueneh method. Results Neutralizing activity responding against SF33 strain was higher than 1597 (P<0.05). There was a positive correlation between neutralizing concentration of plasma against SF33 and viral load (1g) (r = 0.191, P = 0.001), but there was no correlation between neutralizing concen-tration of plasma against 1597 and viral load(lg) (r = 0.097, P = 0.096). Conchtsion In chronic HIV-1 infectious people, neutralizing level against SF33 increases with viral replication, however, there is no asso-ciation of plasma neutralizing against 1597 replication with viral load.

The relationship between genotype of mouse and the immune response of hepatitis B vaccine / 中华微生物学和免疫学杂志

Objective To test the potency of hepatitis B vaccine in China. Methods Two inbred strains(DBA/1 and BALB/c) and two NIH closes-colonies of mice were typed in the H-2 region by microcy-totoxicity method and PCR. Groups of mice of the tested strains were immunized with the same hepatitis B vaccine, the titre of anti-HBsAg antibody was analyzed by microplate, and the ED50 was then estimated by Karder method for each strain. Results Significant differences were found between potency estimates de-rived from assays using different strains of mice. Conclusion It is likely that the variation of immune re-sponse to hepatitis B vaccine in mice is correlative with the H-2 haplotype. In some special case, the bet-erozygosity in H-2 region found in NIH stock could influence the accuracy in such testing even a reference preparation of hepatitis B vaccine was used. Base on our experiment, to select an appropriate NIH stocks with the H-2q haplotype for potency testing of hepatitis B vaccine in China.

Protective effects of tiopronin on liver in antitubercucar treatment / 中国药学杂志

OBJECTIVE　To study the protective effects of Tiopronin in antitubercular treatment.METHODS　The patients in the therapeutic group took Tioprooin orally and had administration of antitubercular drugs,while those in the control group used antitubercular drugs only.The changes of their ALT and TBIL were observed druing the course.RESULTS　The abnormalities of ALT and abnormalities of ALT and TBIL at the same time in the thenapeutic group were obviously lower than those in the control group.CONCLUSION　Tiopronin may prevent liver injury caused by antitubercular drugs.

STRUCTURAL ELUCIDATION OF A NEW PERYLENEQUINONE / 药学学报

AIM　To study the chemical constituents from the mycelia of Hypomyces sp. METHODS Silica gel column chromatography was employed for the isolation and purification. The structure of compound 1 was elucidated on the basis of spectral analysis. RESULTS and CONCLUSION　A new perylenequinone, named hypomycin B 1, was isolated from the mycelia of Hypomyces sp.

Protective effect of compound Danshen dripping pills on isolated rat myocardium with anoxin/reoxygenation / 中国病理生理杂志

AIM: The experiment was designed to study the effect of compound Danshen dripping pills (DSDP) on myocardium with anoxin/reoxygenation. METHODS: The myocardial anoxin/reoxygenation model was made in perfused isolated rat heart. DSDP and isosorbide dinitrate (ID) were given at the time of pre-perfusion and reperfusion, then HPLC and H-600 electron microscope were used to detect the change of high energy phosphate and the ultrastructure of myocardial cell. RESULTS: ① The contents of AMP, ADP, ATP and AN in myocardium in only anoxin/reoxygenation group were obviously lower than those in the control group (P0.05). ③ In the groups with ID, the contents of AMP, ADP, ATP and AN were distinctively lower than those in the control group (P

Effects of Fluoride on Growth and Development,Morphological Structure and Biochemistry Indices of Long Bone in Vitro / 环境与健康杂志

0.05),while the activities of SOD of bone tissue were lower(P

Changes of energy metabolism in ischemic myocardium and in reperfusion during human cardiovalvular-transplantation / 中国病理生理杂志

Ⅳ, change of phosphocreatine (PCr) content was very similar to thatof ATP. Content of ADP, AMP, Cr and HYPO was negatively correlated to ATP content. NAD content was also reduced. In summary, recently proposed hyperkalemic crystalloiddilluted blood protecting myocardium would not be sufficient enough and further improve-ment is quiet neccessary.

High performance liquid chromatography:a rapid isocratic method for determination of energy compounds in myocardial tissue / 中国病理生理杂志

To determine energy compounds in myocardial tissue simultaneously, we determined standard Creatine(Cr), Creatine phosphate(PCr), Hypoxanthine(Hypo), Nicotinamide adenine dinucleotide (NAD), Adenosine monophosphate (AMP), Adenosine diphosphate (ADP), Adenosine triphosphate (ATP), Cyclic-adenosine monophosphate(cAMP), and other compounds related to energy metabolism by a rapid isocratic method.The result showed that standard curves of the eight compounds were linear and passed through the origo for all examined concentrations (r=0.999).A recovery (88.1～117.6%) and replication (CV%=0.97～3.78%) for the present method was found to be accord with methology request.

The protection of the myocardium from injury of acute ischemia and reperfusion: the effects of salvia miltiorrniza (dan shen) / 中国病理生理杂志

The experimental materials were rabbit hearts. The adenylates were measured by HPLC, the levels of lipoperoxidation were determined by spectrophotometry, and atomic absorption spectrophotometry was used for measurement of myocardial calcium. The study showed that in the reperfused group ATP, ADP, AMP, AN (total adenylates concentration), ATP/ADP, ATP/AMP and the energy charge were much lower in the ischemic area than those in the non-ischemic area and ischemic area of the ischemic group (Ⅰ) and Dan Shen group (D) (P