Structure-activity relationship and biological evaluation of berberine derivatives as PCSK9 down-regulating agents.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein and its deficiency markedly enhanced the survival rate of patient with cardiovascular diseases (CVDs). Forty berberine (BBR) derivatives were synthesized and evaluated for their activities on down-regulating the transcription of PCSK9 in HepG2 cells, taking BBR as the lead. Structure-activity relationship (SAR) analysis revealed that 2,3-dimethoxy moiety might be beneficial for activity. Among them, 9k displayed the most potent activity with IC50 value of 9.5 ± 0.5 µM, better than that of BBR. Also, it significantly decreased PCSK9 protein level at cellular level, as well as in the liver and serum of mice in vivo. Furthermore, 9k markedly increased LDLR expression and LDL-C clearance via down-regulating PCSK9 protein. The mechanism of action of 9k is targeting HNF1α and/or Sp1 cluster modulation upstream of PCSK9, a different one from BBR. Therefore, 9k might have the potential to be a novel PCSK9 transcriptional inhibitor for the treatment of atherosclerosis, worthy for further investigation.

[Cefoxitin plus levofloxacin for prevention of severe infection after transrectal prostate biopsy].

OBJECTIVE: To evaluate the effect of cefoxitin prophylactic in reducing the incidence of severe infection after transrectal prostate biopsy (TRPB). METHODS: This retrospective study included 155 cases of TRPB with a 5-day administration of oral levofloxacin at 200 mg bid (the control group) and another 167 cases with a 3-day administration of oral levofloxacin at the same dose plus intravenous cefoxitin at 2.0 g 2 hours before TRPB (the experimental group) according to the distribution characteristics of drug-resistance bacteria in our department. The patients of the control and experimental groups were aged (68.68 ± 8.12) and (68.72 ± 7.51) years, with PSA levels of (19.78 ± 21.57) and (21.15 ± 42.63) µg/L, involving (11.68 ± 1.44) and (11.77±1.02) biopsy cores, respectively. Comparisons were made between the two groups of patients in the incidence rate of severe infection, which was defined as lower urinary track symptoms plus the systemic inflammatory response syndrome (SIRS) within 7 days after TRPB. RESULTS: The incidence rate of postoperative severe infection was significantly lower in the experimental group than in the control (0.6% ï¼»1/167ï¼½ vs 5.8% ï¼»9/155ï¼½, P < 0.05). Blood cultures revealed positive E-coli strains in 6 cases in the control group, including 5 ESBL-positive and 4 quinolone-resistant and amikacin-sensitive cases, all sensitive to cefoxitin, cefoperazone/sulbactam and imipenem. The only one case of severe infection was shown to be negative in blood culture. CONCLUSIONS: Preoperative intravenous administration of cefoxitin according to the specific distribution characteristics of drug-resistance bacteria can significantly reduce the incidence of severe infection after TRPB.

Chrebp regulates the transcriptional activity of androgen receptor in prostate cancer.

Androgen receptor (AR), a member of nuclear hormone receptor, plays an essential role in the initiation and progression of prostate cancer (PCa). In the present study, by way of immunoprecipitation followed by mass spectrometry (IP/MS) system, we found that carbohydrate-responsive element-binding protein (Chrebp), a glucose sensor in normal and cancer cells, interacted with AR in LNCaP cells. The interaction was further confirmed by coimmunoprecipitation analysis. Besides, Chrebp is required for the optimal transcriptional activity of AR in promoting the transcription of the prostate-specific antigen (PSA) promoter and messenger RNA (mRNA) expression. Consistently, knockdown of Chrebp using small interfering RNA (siRNA) in LNCaP cells reduced endogenous PSA levels. Together, our study demonstrates that Chrebp interacts with AR and regulates its transcriptional activity.

[Review of change detection methods using multi-temporal remotely sensed images].

With the development of platforms and sensors, continuous repetition of remote sensing observation of the earth surface has been realized, and a mass of multi-source, multi-scale, multi-resolution remote sensing data has been accumulated. Those images have detailedly recorded the changing process of ground objects on the earth, which makes the long term global change research, such as change detection, based on remote sensing become possible, and greatly push forward the research on image processing and application. Although plenty of successful research has been reported, there are still enormous challenges in multi-temporal imagery change detection. A relatively complete mature theoretical system has not formed, and there is still a lack of systematic summary of research progress. Firstly, the current progress in change detection methods using multi-temporal remotely sensed imagery has been reviewed in this paper. Then, the methods are classified into three categories and summarized according to the type and amount of the input data, single-phase post-classification comparison, two-phase comparison, and time series analysis. After that, the possible existing problems in the current development of multi-temporal change detection are analyzed, and the development trend is discussed finally.

[Assessment of Potential Risk of Diffuse Pollution in Haihe River Basin Based Using DPeRS Model].

Considering the Haihe River Basin as an example, the DPeRS model was used to analyze the spatial distribution characteristics and pollution sources of the diffuse pollution by remote sensing pixel scale. Combined with the evaluation standard of surface water quality, a potential risk grading method for diffuse pollution was constructed to assess the potential risk of diffuse pollution in Haihe River Basin. The results showed that, in 2016, the diffuse discharge loads of total nitrogen (TN), total phosphorus (TP), ammonia nitrogen (NH4+-N), and chemical oxygen demand (COD) were 429.2, 25.7, 288.3, and 1017.0 kg ·km-2, respectively, with the amount of river entry being 2.5×104 ton, 1597.2 ton, 1.7×104 ton, and 6.6×104 ton in Haihe River Basin, respectively. Farmland runoff is the most important source of diffuse pollution of TN, TP and NH4+-N in the Haihe River Basin. For COD index, urban life is the primary type of pollution, followed by livestock. The diffuse pollution is relatively severe in the central and southern areas of Haihe River Basin, and this area is also a high-risk concentrated distribution area of diffuse pollution in the basin. The distribution of high-risk areas of nitrogen-phosphorus diffuse pollution are relatively concentrated, and the chemical oxygen demand is relatively scattered. More than 36% of the Haihe River Basin has a nitrogen-phosphorus diffuse pollution risk, and 2.94% of the area has a chemical oxygen demand diffuse pollution risk.

[Status of Antibiotic Contamination and Ecological Risks Assessment of Several Typical Chinese Surface-Water Environments].

To learn about the status of antibiotic contamination and their ecological risks in Chinese surface-water environments, the risk quotient (RQ) and joint risk quotient (RQSUM) methods were applied to assess the ecological risks of five typical surface-water environments in China during the flood season. The results showed that the main types of antibiotic contamination in the five regions were sulfamethoxazole (SMX), sulfamethazine (SM), erythromycin (ETM), roxithromycin (RTM), tetracycline (TC), oxytetracycline (OTC), norfloxacin (NOR), and ofloxacin (OFL). Among eight types of antibiotic contamination, sulfamethoxazole (SMX) and erythromycin (ETM) occupied a dominant position. The contribution rate of SMX in the Yangtze River Delta and Chaohu Basin was 91.1% and 98.5%, respectively. Meanwhile, the contribution rates of ETM in Jianghan Plain, Pearl River Delta, and Yellow River Delta were 94.4%, 81.8%, and 60%, respectively. Based on the joint risk quotients (RQSUM), the order of ecological risks in the research areas was:Jianghan Plain (20.204) > Yangtze River Delta (8.769) > Chaohu Basin (2.692) > Yellow River Delta (1.943) > Pearl River Delta (1.222).

Inhibitory effects of the recombinant human serine protease inhibitor Hespintor on the proliferation, migration and invasion of hepatocellular carcinoma cells.

Hespintor is a new Kazal-type serine proteinase inhibitor (Serpin) screened from the HepG2 hepatoblastoma cell line using the suppression subtractive hybridization (SSH) technique. Seprin is closely associated with the progression and remission of malignant tumors, and has certain significance in the diagnosis and treatment of tumors. Investigations on the antitumor activity of Serpin are expected to aid in the development of a new method for tumor treatment based on the serine protease inhibitor. Although the Hespintor prokaryotic expression strain and recombinant Hespintor protein (recombinant fusion protein of Hespintor and rHespintor) have already been obtained, the protein extraction efficiency is low due to the low initial amount of extracted protein and large number of purification steps, which affect the study of the protein function. The aim of the present study was to improve the purification method of rHespintor, increase the protein extraction efficiency, and investigate its effects on the proliferation, migration and invasion of the HepG2 hepatoblastoma cell line. The results demonstrated that the application of urea gradient washing of inclusion body of the protein may effectively remove the majority of impure proteins from the targeted protein. After one-step purification, the target protein rHespintor exhibited a high inhibitory effect of Trypsin Hydrolysis, which was exhibited in a dose-dependent manner. Hoechst 33258 staining was used to determine cell apoptosis. After treating HepG2 hepatoblastoma cells with rHespintor, the cell growth was inhibited, the proliferation ability was reduced, and the number of migrated and invaded cells were significantly decreased. Hoechst 33258 staining and flow cytometry assay results showed clear cell apoptosis. The results reveal showed that rHespintor significantly inhibited proliferation, migration and invasion of the HepG2 hepatoblastoma cell line in vitro, and induced cell apoptosis to a certain extent, indicating that the recombinant protein Hespintor exerts an antitumor effect in vitro, and has the potential and feasibility to become an antitumor drug.

UBE3C promotes growth and metastasis of renal cell carcinoma via activating Wnt/ß-catenin pathway.

Renal cell carcinoma (RCC) is the most common primary malignancy of the kidney and one of the most lethal genitourinary malignancies. Clear-cell renal cell carcinoma (ccRCC) has an extremely poor prognosis because of a high potential for tumor growth, vascular invasion, metastasis and recurrence. Unfortunately, the mechanism of RCC growth and metastasis is not well understood. In this report, we for the first time demonstrated ubiquitin protein ligase E3C (UBE3C) as a driving factor for RCC growth and metastasis. UBE3C expression was increased in ccRCC tissues compared with adjacent normal tissues. ccRCC patients with high UBE3C protein expression in tumors were associated with significantly worse postoperative survival. Knockdown of UBE3C expression in ACHN cells inhibited cell proliferation, migrations and invasiveness in vitro while overexpression of UBE3C in 786-O cells exerted the opposite effects. UBE3C up-regulated ß-catenin protein levels and promoted ß-catenin nuclear accumulation, leading to the activation of the Wnt/ß-catenin signal pathway in RCC cells. Collectively, these observations suggest that UBE3C plays an important role in RCC development and progression, and UBE3C may be a novel target for prevention and treatment of ccRCC.

Purification and identification of the Kazal domain of a novel serine protease inhibitor, Hespintor, through a bacterial (Escherichia coli) expression system.

In this study, Hespintor, a protein with unknown function, was screened and obtained from the hepatoblastoma cell line, HepG2, using suppression subtractive hybridization (SSH). Sequence analysis demonstrated that the protein is a novel secreting member of the Kazal-type serine protease inhibitor (serpin) family, and possesses the basic structure of serpin, which is highly homologous to esophageal cancer-related gene 2 (ECRG2). To further elucidate its biological functions, the Hespintor protein was expressed and purified. The coding sequence of the Hespintor Kazal domain was cloned into the prokaryotic expression vector, pET-40b(+), and was then transformed into host bacteria (Escherichia coli) Rosetta (DE3). The optimally expressed recombinant fusion protein, Hespintor-Kazal, with a molecular weight of 42 kDa was obtained by 0.25 mmol/l isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction at 30˚C for 5 h. Western blot analysis was performed to further confirm the specificity of the recombinant protein, Hespintor-Kazal. The recombinant fusion protein, Hespintor­Kazal, was expressed in the host bacteria in the form of an inclusion body. Two-step metal chelating affinity chromatography and anion exchange chromatography columns were used to purify the recombinant protein. The preliminary activity identification results revealed that the purified recombinant fusion protein, Hespintor-Kazal, specifically inhibited the hydrolysis activity of trypsin, suggesting that Hespintor has potential value as a novel antitumor drug.

Identification of paired immunoglobulin-like type 2 receptor α as hepatitis B virus DNA polymerase transactivated protein 1 interacting proteins.

Hepatitis B Virus (HBV) DNA polymerase transactivated protein 1 (HBVDNAPTP1) is a novel protein transfected by HBV DNA polymerase, which has been screened by a suppression subtractive hybridization technique. In the present study, a yeast two-hybrid system was used to screen the proteins interacting with HBVDNAPTP1 in leukocytes in order to investigate the biological function of HBVDNAPTP1. The HBVDNAPTP1 coding sequence was cloned into a pGEM-T vector. Subsequent to sequencing, the HBVDNAPTP1 was subcloned into the bait plasmid pGBKT7 and transformed into yeast AH109. Western blotting confirmed the presence of HBVDNAPTP1 expression in the AH109 yeast strains. The transformed yeast AH109 cells were mated with Y187 yeast cells containing the leucocyte cDNA library pACT2 plasmids in 2X yeast extract peptone D-glucose adenine (YPDA) medium. For selection and screening, diploid yeast was plated on synthetic dropout medium (SD/-Trp-Leu-His-Ade) containing X-α-gal. Following sequencing and the verification of the open reading frames of positive colonies, four different proteins were obtained. To further confirm the interaction between HBVDNAPTP1 and the screened proteins, paired immunoglobulin-like type 2 receptor α (PILRA), one of the positive colonies, was cloned. The glutathione S-transferase pull-down in vitro assay and a co-immunoprecipitation in vivo assay were used to examine the interaction between HBVDNAPTP1 and PILRA, respectively. HBVDNAPTP1 may be involved in the negative regulation of the PILRA­mediated Janus-activated kinase/signal tranducer and activator of transcription signaling pathway, and exert a positive effect on the initiation of monocyte apoptosis. These results contribute our knowledge of the biological functions of HBVDNAPTP1 and provide novel data to aid in the further analysis of the regulatory mechanism of this protein.

Transactivation of proto-oncogene c-Myc by hepatitis B virus transactivator MHBst167.

C-terminally truncated hepatitis B virus (HBV) middle size surface proteins (MHBst) has been shown to be a transcriptional activator and may be relevant to hepatocarcinogenesis by transactivating gene expression. In the present study, a pcDNA3.1(-)-MHBst167 vector coding for MHBst truncated at amino acid 167 (MHBst167) was constructed and transfected into the HepG2 hepatoma cell line. mRNA and protein expression of MHBst167 in the cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. A cDNA library of genes transactivated by the truncated protein in HepG2 cells was made in pGEM-T Easy using suppression subtractive hybridization. The cDNAs were sequenced and analyzed with BLAST searching against the sequences in GenBank. The results showed that certain sequences, such as that of human proto-oncogene c-Myc, may be involved in tumor development. An expression vector pCAT3/c-Myc containing the chloramphenicol acetyltransferase (CAT) gene under the control of a c-Myc promoter was generated, and the transcriptional transactivating effect of MHBst167 on the c-Myc promoter was investigated by RT-PCR and western blotting. MHBst167 was found to upregulate the transcriptional activity of the promoter, as well as transcription and translation of c-Myc. MHBst167 was also shown to transactivate SV40 immediate early promoter, and transcriptionally transactivate the expression of human c-Myc. These findings provide new directions for studying the biological functions of MHBst167, and for a better understanding of the tumor development mechanisms of HBV infection.

[Research on spatial characteristic of non-point source pollution in Liaohe River basin].

The spatial characteristic of non-point source pollution in the Liaohe River was studied. Coupling the remote sensing data and non-point source (NPS) models, a method of assessing NPS pollution by pixel unit was developed, aiming to analyse the NPS pollution characteristic of Liaohe River basin in 2010, in turn to identify the main polluted areas and prevention measures. The work will provide technical supports for pollution prevention in Liaohe River basin. The results showed that in 2010, the total discharge of total nitrogen (TN) was 1.03 x 10(5) t, the total phosphorus (TP) was 6.8 x 10(3) t, the chemical oxygen demand (COD) was 1.31 x 10(5) t and the ammonia nitrogen (NH+4 -N) was 1. 8 x 10(4) t. The main pollution source of NPS was from agriculture. The contributions of NPS pollution to water quality were 67.4% , 76.4% , 39.4% and 21.9% for TN, TP, COD and NH+4 -N, respectively. The south of Liaohe River basin was the most serious polluted area, followed by the northeast areas. In this research, a method was build to estimate the NPS loads based on remote sensing pixel and the spatial characteristic of non-point source pollution in Liaohe River in 2010 was analysed, which will provide support for pollution prevention in Liaohe River.

[Effect of nitrogen removal simulated by RIP_ N model to a riparian zone in Guangting Reservoir catchment].

An eco-hydrological model system (RIP_ N) was constructed to simulate the nitrogen (N) removal by riparian zone in reservoir catchment scale. As a case study, the N removal in Guanting Reservoir riparian zone from March to September in 2007 was estimated. At same time, the field simulated experiment was carried out in Yanqing experimental station, which lies in the northeast part of the catchment. With the experimental data and previous studies, the RIP_ N model was calibrated. RIP_ N model was consisted with two parts, which were soil chemical process modeling and plant growth modeling. Soil chemical processes considered the soil denitrification, nitrification and ammonium volatilization. Plant growth included net primary productivity (NPP) module, plant production allocation module and nutrition uptake module. The research indicated that the correlation coefficient between simulated value and monitored value was larger than 0.5, which proved the effectiveness and reliability of RIP_ N model in catchment scale simulating. The simulated results showed that the N removal loss by riparian zone in Guanting Reservoir catchment from March to September was 5.91 x 10(3) t. The model also identified the N removal functions of different land use. At present land use condition, the bottomland, forest land and grassland contributed positively environmental benefits and removed most of N. In the temporal scale, the N removal from March to September consisted 76.5% of annual removal load. On the contrary, the wetlands just removed 5.9% of N of whole watershed. By comparison, the riparian zone was recognized as critical location for non point source pollution prevention.

Nonpoint source pollution responses simulation for conversion cropland to forest in mountains by SWAT in China.

Several environmental protection policies have been implemented to prevent soil erosion and nonpoint source (NPS) pollutions in China. After severe Yangtze River floods, the "conversion cropland to forest policy" (CCFP) was carried out throughout China, especially in the middle and upper reaches of Yangtze River. The research area of the current study is located in Bazhong City, Sichuan Province in Yangtze River watershed, where soil erosion and NPS pollution are serious concerns. Major NPS pollutants include nitrogen (N) and phosphorus (P). The objective of this study is to evaluate the long-term impact of implementation of the CCFP on stream flow, sediment yields, and the main NPS pollutant loading at watershed level. The Soil and Water Assessment Tool (SWAT) is a watershed environmental model and is applied here to simulate and quantify the impacts. Four scenarios are constructed representing different patterns of conversion from cropland to forest under various conditions set by the CCFP. Scenario A represented the baseline, i.e., the cropland and forest area conditions before the implementation of CCFP. Scenario B represents the condition under which all hillside cropland with slope larger than 25 degrees was converted into forest. In scenario C and D, hillside cropland with slope larger than 15 degrees and 7.5 degrees was substituted by forest, respectively. Under the various scenarios, the NPS pollution reduction due to CCFP implementation from 1996-2005 is estimated by SWAT. The results are presented as percentage change of water flow, sediment, organic N, and organic P at watershed level. Furthermore, a regression analysis is conducted between forest area ratio and ten years' average NPS load estimations, which confirmed the benefits of implementing CCFP in reducing nonpoint source pollution by increasing forest area in mountainous areas. The reduction of organic N and organic P is significant (decrease 42.1% and 62.7%, respectively) at watershed level. In addition, this study also proves that SWAT modeling approach can be used to estimate NPS pollutants' impacts of land use conversions in large watershed.