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Abstract Coal is the major source of power in India and world over. Coal mining is an essential industry which has a major role in the economic development of the country. Most major mining activities contribute directly or indirectly to air pollution. Coal dust is a major air pollutant which affects the personal working in the mines and also people residing in villages near the mines. Air pollution due to coal particulates can affect human health and cause damages to the environment. Hence effective pollution control mechanisms are needed to keep the pollution levels within permissible levels. The easiest and most common method employed for dust suppression worldwide is sprinkling of water. In majority of mines, water sprinklers are operated manually and can lead to wastage of water due to over sprinkling. It can also prove to be ineffective in dust suppression if sprinkling is not done properly. The paper proposes a system which can be deployed to automate the dust suppressions sprinklers. The system will monitor the concentration of PM10 and PM2.5 in the air and initiate sprinkling operation when the particulate matter content exceeds preconfigured limits.

Dark-field X-ray imaging is sensitive to the microstructure of a material. Here, the authors combine this with a neural network algorithm to provide efficient material discrimination, e.g., of explosives vs non-threat materials.

We build H i absorption spectra toward Supernova Remnant (SNRs) G16.7+0.1 and G15.9+0.2 using the THOR survey data. With the absorption spectra, we give a new distance range of 7–16 kpc for G15.9+0.2. We also resolve the near/far-side distance ambiguity of G16.7+0.1 and confirm its kinematic distance of about 14 kpc. In addition, we analyze the CO (J = 3–2) spectra toward G16.7+0.1 and find obvious CO emission at the 20 km s−1 OH 1720 MHz maser site. This supports Reynoso & Mangum suggestions that the velocity difference between the maser and southern molecular cloud is caused by the shock acceleration. We discuss the impact of the distances on other physical parameters of the two SNRs. [ABSTRACT FROM AUTHOR]

We simulate the evolution of supernova remnant (SNR) W51C. The simulation shows the existence of a new northeast edge. We present the magnetic field structure of the W51 complex (SNR W51C and two H ii regions W51A/B) by employing the 11 cm survey data of Effelsberg. This new edge is identified and overlaps with W51A along the line of sight, which gives a new angular diameter of about 37′ for the quasi-circular remnant. In addition, we assemble the OH spectral lines (1612/1665/1720 MHz) toward the complex by employing the newly released THOR (The HI OH Recombination line survey of Milky Way) data. We find that the known 1720 MHz OH maser in the W51B/C overlap area is located away from the detected 1612/1665 MHz absorption region. The latter is sitting at the peak of the H ii region G49.2-0.35 within W51B. [ABSTRACT FROM AUTHOR]

Objective: To determine the distributions of serum HBsAg level in treatment-naive or treatment-experienced chronic hepatitis B (CHB) patients. Methods: Based on the China Registry of Hepatitis B (CR-HepB), a nationwide hospital-based electronic platform, treatment-naive or treatment-experienced CHB patients who receive nucleos(t)ide analog (NA) therapy were enrolled in our study. We collected patients' clinical characteristics, including demographic, virological and biochemical data. The distributions of HBsAg levels in those two groups were summarized with counts and percentages. Continuous variables were expressed as median (interquartile range, IQR) or mean±SD. Results: A total of 13 505 treatment-naive CHB patients and 6 309 patients with previous NAs therapy were enrolled in the final analysis. The proportion of patients in those two groups with serum HBsAg<100 IU/ml, <500 IU/ml, <1 500 IU/ml or HBsAg level not lower than 1 500 IU/ml, 3 000 IU/ml or 8 000 IU/ml were 10.51% vs. 12.88%, 28.47% vs. 29.84%, 46.85% vs. 52.07%, 53.15% vs. 47.93%, 38.17% vs. 31.77%, 15.62% vs. 10.39%, respectively. HBsAg levels tended to decrease gradually with the duration of antiviral therapy: the percentage of patients with HBsAg levels below 100 IU/ml increased from 12.73% at less than 3 years of treatment to 26.92% at≥10 years of treatment, whereas the percentage of patients with HBsAg levels of 3 000 IU/ml or 8 000 IU/ml and above decreased from 34.66% to 23.08% and from 12.19% to 5.77%, respectively. Conclusions: Our investigation manifested the distribution of the serum HBsAg in treatment-naive CHB patients and patients who received previous NAs treatment. As the duration of antiviral therapy is prolonged, the HBsAg level tends to decrease gradually.

Objective: This study aims to compare the antiviral treatment similarities and differences in the population covered by the 2024 version of the World Health Organization's (WHO) hepatitis B prevention and treatment guidelines and the current Chinese hepatitis B prevention and treatment guidelines, so as to explore their impact on the indications for antiviral therapy in Chinese patients with chronic hepatitis B (CHB). Methods: The information of patients with chronic hepatitis B virus infection who did not receive antiviral treatment was collected through the registration database of the China Clinical Research Platform for Hepatitis B Elimination. Descriptive statistics were conducted on the demographic, blood, biochemical, and virological levels of patients according to the treatment recommendations of the two versions of the guidelines. The Mann-Whitney U test and χ2 test were used to compare the differences and proportional distribution of the treatment populations covered by the two guidelines. The χ2 test was used to analyze the coverage rate of different antiviral treatment indications. Results: A total of 21,134 CHB patients without antiviral treatment were enrolled. 69.4% of patients met the 2024 versions of the WHO guidelines' recommendations. 85.0% of patients met the current Chinese hepatitis B prevention and treatment guidelines. The WHO guidelines for antiviral therapy indications were met in younger patients with higher levels of ALT, AST, and APRI scores, as well as greater proportion of patients with higher viral loads (P<0.001). The WHO guidelines recommended a cut-off value of APRI>0.5, which raised the proportion of patients on antiviral therapy from 6.6% to 30.9%. 45.7% of patients met the antiviral indications for HBV DNA >2000 IU/ml with abnormal transaminase (ALT>30 U/L for males and ALT>19 U/L for females). The reduced APRI diagnostic cut-off value and ALT treatment threshold had further increased the treatment coverage rate by 91.6% in patients with chronic HBV infection in line with the 2024 versions of WHO guidelines. Conclusion: The reduction of the APRI diagnostic cut-off value and the ALT treatment threshold, based on the current hepatitis B guidelines of China, will further improve the treatment coverage of CHB patients.

Objective: To understand ten-year changes in clinical characteristics and antiviral treatment patterns of chronic hepatitis B in China. Methods: Patients with chronic HBV infection:demographic, virologic, hematologic, blood biochemistry, and antiviral treatment data were extracted from the China Registry of Hepatitis B (CR-HepB) database between 2012 and 2022 for descriptive statistics and change trend analysis. Multiple group comparisons were conducted using the Kruskal Wallis H test, while counting data was compared between groups using χ (2) test. Results: A total of 180 012 patients with chronic HBV infection were included, with a median age of 40 years old, and a male proportion accounting for 60.2%. The HBeAg positive rate was 43.3%. Over time, the median age of new patients each year increased from 39 to 47 years, while the HBeAg positive rate decreased from 51.3% to 32.8%. The initial diagnosis of patients was mainly CHB (71.4%), followed by hepatitis B cirrhosis (11.8%), inactive HBsAg carrier status (10.6%), and chronic HBV carrier status (6.2%). Among the newly registered patients every year from 2012 to 2022, the proportion of hepatitis B cirrhosis remained stable, but after 2019, the proportion of CHB increased and the proportion of other diagnoses decreased. The proportion of patients with cirrhosis increased with age in different age groups, with 3.5%, 19.3%, and 30.4% in the < 40, 40-69, and≥70 age groups, respectively. The proportion of women in patients with cirrhosis also increased with age, from 16.1% in those < 30 years old to 44.3% in those≥80 years old. From 2012 to 2022, the proportion of patients receiving first-line nucleos(t)ide analog antiviral treatment increased year by year, from 51.0% in 2012-2013 to 99.8% in 2022. Conclusion: The CR-HepB registration data reflect the changes in clinical characteristics and antiviral treatment patterns in patients with chronic HBV infection in China over the past ten years and can thus provide a reference to promote hepatitis B diagnosis and treatment practice, as well as scientific research.

Purpose: To investigate the differences in higher-order aberrations between non-toric or toric implantable collamer lens (ICL or TICL) V4c implantation and simulated spectacle correction., Methods: Patients with high myopia who underwent ICL/TICL V4c implantation were enrolled. The "total no defocus" pattern of iTrace aberrometry to simulate the condition of spectacle correction was measured before ICL/TICL implantation, and higher-order aberrations in this condition were compared to those 3 months after surgery. Related factors with changes in coma were comprehensively analyzed., Results: A total of 89 right eyes of 89 patients were included. Compared to simulated spectacle correction, total-eye coma (P<0.0001 ICL, P<0.0001 TICL) and internal coma (P<0.0001 ICL, P<0.001 TICL) decreased in the ICL- and TICL-treated groups after surgery. Total-eye secondary astigmatism (P<0.0001 ICL, P=0.007 TICL) and internal secondary astigmatism (P<0.0001 ICL, P=0.009 TICL) were also decreased in both groups postoperatively. Spherical error showed positive correlations with variation in total-eye coma (r=0.37, P=0.004 ICL; r=0.56, P=0.001 TICL) and internal coma (r=0.30, P=0.02 ICL and r=0.45, P=0.01 TICL). Axial length revealed negative correlations with changes in total-eye coma (r=-0.45, P<0.001 ICL; r=-0.39, P=0.03 TICL) and internal coma (r=-0.28, P=0.03 ICL and r=-0.42, P=0.02 TICL)., Conclusions: Both ICL- and TICL-treated groups demonstrated a decrease in coma and secondary astigmatism after 3 months, postoperatively. ICL/TICL may confer a compensatory effect on coma aberration and secondary astigmatism. Patients with a higher myopia achieved a greater improvement in coma and may benefit more from ICL/TICL implantation than from spectacle correction implantation than from spectacle correction., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Adenovirus infection can occur in all regions or countries of the world, with no obvious seasonality, but pandemics mostly occur in winter or early spring. Adenovirus infection is self-limited among immunocompetent host with supportive care, however fatal infection can occur among immunocompromised patients, mainly affecting respiratory, gastrointestinal tract and adjunctiva and very rarely causing hepatitis, cholecystitis, pancreatitis, hemorrhagic cystitis, myocarditis, meningitis or encephalitis. Adenovirus hepatitis mainly occur in malignant tumors or organ transplantation patients, but acute severe hepatitis can occur even in immunocompetent children or adults. On 5 April 2022, WHO was notified of 10 cases of severe acute hepatitis of unknown etiology in children. As of 21 April 2022, at least 169 cases of acute hepatitis of unknown origin have been reported from 12 countries (including 11 WHO European Region countries and the United States). Adenovirus has been detected in at least 74 cases; SARS-CoV-2 was identified in 20 cases of those that were tested. Furthermore, 19 were detected with a SARS-CoV-2 and adenovirus co-infection. At present, the etiology has not been fully elucidated. The leading hypotheses center around adenovirus, and the relationship with SARS-CoV-2 needs to be further ruled out.

Liver involvement is often observed in hematological disorders, resulting in liver abnormality, including unconjugated hyperbilirubinemia, monoclonal hyperglobulinemia, portal vein, or hepatic vein thrombosis or portal hypertension, hepatosplenomegaly, or iron accumulation in the liver. Here we summarize the major hematological diseases that often affect the liver: hemolytic anemia, defect in coagulation or anti-coagulation factors, myeloproliferative neoplasm, hemophagocytic lymphohistiocytosis, multiple myeloma, leukemia, and lymphoma. We hope this review will help clinicians diagnose and manage the patients with liver involvement by hematological disorders.

Drought is the main factor restricting peanut growth, but the molecular mechanism underlying peanut drought tolerance remains unclear. Herein, the seedling stage of drought-resistant peanut cultivar J11 was subjected to drought stress, and its proteomic profile was systematically analysed by isobaric tags for relative and absolute quantification (iTRAQ), the results of which were further complemented with our previous transcriptome results. A total of 4,018 proteins were identified by proteomic analysis, which revealed that the expression levels of 69 proteins were altered under drought stress. Among the differentially expressed proteins (DEPs), 50 were upregulated, and 19 were downregulated. The most enriched metabolic pathways for these DEPs were those involving phenylpropanoid biosynthesis, flavonoid biosynthesis, and plant hormone signal transduction. The proteomic data and previous transcriptome results revealed 44 groups of genes/proteins with the same expression trend, including a LEA (Late embryogenesis abundant) gene, AhLEA2. Our present study showed that overexpression of the AhLEA2 gene enhanced the drought resistance of transgenic Arabidopsis plants, and the activities of related antioxidant enzymes in the transgenic plants significantly changed. The AhLEA2 gene was found to be located in the cytoplasm and cell membrane by subcellular localization experiments. This work systematically analysed the differentially expressed proteins in peanut in response to drought stress, providing important candidates for further functional analysis of the stress response of peanut. Our results also indicated that AhLEA2 plays an important role in the peanut response to drought stress., (© 2021 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

Overdose acetaminophen (APAP) can result in severe liver injury, which is responsible for nearly half of drug-induced liver injury in western countries. Previous studies have found that there existed massive hepatocellular necrosis and severe inflammatory response in APAP-induced liver injury. However, the mechanistic linkage between necroptosis and NLRP3 inflammasome pathway in APAP-induced hepatotoxicity remains poorly understood. In order to investigate the relationship between inflammation and hepatocytes death in APAP hepatotoxicity, a time-course model for APAP hepatotoxicity in C57/BL6 mice was established by intraperitoneal (i.p) injection of 300 mg/kg APAP in this study. The activity of serum enzymes and pathological changes of APAP-treated mice were evaluated, and the critical molecules in necroptosis and NF-κB-NLRP3 inflammasome signaling pathway were determined by immunoblot and immunofluorescence analysis. The results demonstrated that APAP overdose resulted in a severe liver injury. Furthermore, the expression of critical molecules in NLRP3 inflammasome and necroptosis pathways peaked at 12-24 h, and then was decreased gradually, which is consistent with the pattern of pathological injury induced by APAP. Our further investigation found that the level of IL-1β in mouse liver was closely correlated with the level of phosphorylated MLKL following exposure to APAP. Furthermore, inhibition of necroptosis with necrostatin-1 significantly suppressed the activation of NLRP3 inflammasome signaling. Taken together, our results highlighted that the cross-talk between necroptosis and NLRP3 inflammasome played a critical role for promoting APAP-induced liver injury. Inhibition of the interaction of inflammation and necroptosis by pharmaceutical methods may represent a promising therapeutic strategy for APAP-induced liver injury.

Pyroptosis is a programmed death mode dependent on Caspase-1/4/5/11, which is caused by activation of inflammasome, accompanied by cell membrane rupture, pore formation and cell content release. As a new type of cell death, it is widely involved in respiratory diseases, such as pulmonary fibrosis, acute lung injury, bronchopulmonary dysplasia, chronic obstructive pulmonary disease and asthma. This article reviews the mechanism of pyroptosis and its role in different respiratory diseases, in order to provide new ideas for the treatment of respiratory diseases.

Background and Objective: Since December 2019, increasing cases of novel coronavirus disease 2019 (COVID-19) are being detected worldwide. The purpose of this paper is to provide a scientific reference for the global prevention and control of COVID-19., Methods: General demographic characteristics, epidemiological history, and clinical symptoms of COVID-19 were collected that had been reported on the websites of multiple Municipal Health Commissions in China. We herein describe distributions in time, place, and population of COVID-19., Results: As of midnight on February 10, 2020, the number of confirmed cases of COVID-19 in China was 42,638, and the province with the largest number of confirmed cases was Hubei (31728), followed by Guangdong (1177), Zhejiang (1117), and Henan (1105) province. The number of cases and the speed of confirmed cases in provinces other than Hubei were more moderate than those of the Hubei province. The median (interquartile range) age of patients with COVID-19 was 44 (33, 54) years, with a range of 10 months to 89 years., Conclusions: The COVID-19 epidemic should be considered a global threat and the steps for control include early diagnosis and treatment, as well as isolation., (© 2020 Elsevier España, S.L.U. and Sociedad Española de Medicina Interna (SEMI). All rights reserved.)

Objective: To describe the current status of registration and design characteristics of clinical trials of new drugs for curing hepatitis B through domestic and foreign websites, so as to provide references for the follow-up clinical trials of new hepatitis B drugs. Methods: A search was conducted on the US Clinical Trials Database and the Chinese Clinical Trial Registry Center. The search date was from the establishment of the database to May 26, 2020, and the registration trials of new drugs for curing hepatitis B at home and abroad were included. Two researchers independently searched and screened the literature and extracted the data. Results: A total of 106 registered clinical trials of new drugs for curing hepatitis B were included (94 English registration websites and 12 Chinese registration websites), and the number of registrations had increased year by year. Among them, the proportion of therapeutic vaccines and core protein inhibitors were the highest, accounting for 27.4% ( n = 29) and 22.6% ( n = 24), respectively. The vast majority of clinical trials ( n = 96, 90.6%) were in the early stages (Phase I and II). The subjects in phase I clinical trial were mainly healthy people and treated CHB patients, while the subjects in phase II clinical trial were mainly CHB patients who had achieved viral suppression after initial or post-treatment. The main evaluation indicators of Phase I clinical trials were the safety and tolerability of new drugs. The main evaluation indicators in about half of Phase II clinical trials were HBsAg negative conversion/quantitative decline. Overall, the number of clinical trials with the new design was small, accounting for 3.8% (4 / 106). There were relatively few trials of new drugs for curing hepatitis B on domestic registration websites, and the information provided was incomplete. Conclusion: The number of clinical trials of new hepatitis B drugs at home and abroad is increasing year by year, but most of them are in phase I and II, with few adopting new designs. In addition, the information integrity of the domestic website registration center needs to be improved.

Objective: To investigate the role of long non-coding RNA Cancer Susceptibility Candidate 15 (CASC15) in cervical cancer and its potential molecular mechanism., Patients and Methods: The CASC15 expression was measured in cervical cancer tissues and cell lines by using quantitative Real-time polymerase chain reaction (qRT-PCR) analysis. Cell counting kit-8 (CCK8), flow cytometry analysis and transwell cell invasion assays were employed to detect the capacities of cell proliferation and cell invasion. Furthermore, Western blot analysis was applied to detected the E-cadherin and N-cadherin expression in EMT pathway., Results: We demonstrated that lncRNA CASC15 expression was higher in cervical cancer tissues compared to adjacent normal tissues. Higher lncRNA CASC15 expression associated with lymph node metastasis and FIGO stage. Moreover, our results showed that higher lncRNA CASC15 expression predicted poor prognosis of cervical cancer. Functional assays showed that knockdown of lncRNA CASC15 suppressed cell proliferation and cell cycle progression in cervical cancer. Moreover, we also found that knockdown of lncRNA CASC15 inhibited cell invasion ability and Epithelial-Mesenchymal Transition (EMT) signaling pathway by upregulating E-cadherin and downregulating N-cadherin expression in cervical cancer., Conclusions: These results indicated that lncRNA CASC15 expression may be a prognostic biomarker and contributed to cell proliferation and invasion in cervical cancer.

Abstract Objective: To study the effect of obstructive sleep apnea(OSA) on the life quality of children. Method: Forty-four OSA children diagnosed with overnight polysomnography were selected as the research object, 35 cases of pediatric no snoring healthy children were as control. The inventory of subjective life quality and children's self-rating anxiety scale were used to assess children' s life quality. Result: Depending on the data of the inventory of subjective life quality questionnaire, each level and dimension of children' s life quality satisfaction scores in OSA group were significantly lower than that in healthy controls( P <0.05), especially in the cognitive component of school life(3.78±1.73) and the affective component of depression experience(3.70±2.18). The score of anxiety disorder in OSA group (21.39±10.49) was higher than that in healthy control group(14.21±9.69)( P <0.05). The rate of anxiety disorder in OSA group was higher than that in healthy control group( P <0.05). Conclusion: Children in OSA group had higher incidence of unsatisfactory life quality in all levels and dimensions. OSA group children dissatisfied with the life quality in each level, each dimensions have significant statistical difference, especially in the cognitive component of school life and affective components of depression experience. The OSA group had higher anxiety disorders than healthy ones., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Odorant receptors (ORs) expressed in the antennae of parasitoid wasps are responsible for detection of various lipophilic airborne molecules. In the present study, 107 novel OR genes were identified from Microplitis mediator antennal transcriptome data. Phylogenetic analysis of the set of OR genes from M. mediator and Microplitis demolitor revealed that M. mediator OR (MmedOR) genes can be classified into different subfamilies, and the majority of MmedORs in each subfamily shared high sequence identities and clear orthologous relationships to M. demolitor ORs. Within a subfamily, six MmedOR genes, MmedOR98, 124, 125, 126, 131 and 155, shared a similar gene structure and were tightly linked in the genome. To evaluate whether the clustered MmedOR genes share common regulatory features, the transcription profile and expression characteristics of the six closely related OR genes were investigated in M. mediator. Rapid amplification of cDNA ends-PCR experiments revealed that the OR genes within the cluster were transcribed as single mRNAs, and a bicistronic mRNA for two adjacent genes (MmedOR124 and MmedOR98) was also detected in female antennae by reverse transcription PCR. In situ hybridization experiments indicated that each OR gene within the cluster was expressed in a different number of cells. Moreover, there was no co-expression of the two highly related OR genes, MmedOR124 and MmedOR98, which appeared to be individually expressed in a distinct population of neurons. Overall, there were distinct expression profiles of closely related MmedOR genes from the same cluster in M. mediator. These data provide a basic understanding of the olfactory coding in parasitoid wasps., (© 2017 The Royal Entomological Society.)

Objective: To investigate metformin's effect on chemosensitivity of chemotherapeutic drug 5-fluorouracil in laryngocarcinoma Hep-2 cells. Investigate the variation trend of protein expression of AMPK pathway in the combined effect. Method: Laryngocarcinoma Hep-2 cells were treated with different concentrations of 5-fluorouracil in vitro together with or without metformin for 72 h. Use MTT assay to investigate the influence on the inhibition rate to Hep-2 cells. Hep-2 cells were treated with cisplatin, 5-fluorouracil or paclitaxel with or without metformin. Use Western blot assay to investigate the expression level of AMPKα, P21 or Cyclin D1 protein. Result: 5-fluorouracil and metformin could inhibit the proliferation of Hep-2 cells. 5-fluorouracil in low concentration combined with metformin could increase the proliferation inhibition rate of Hep-2 cells. In the circumstances of using 5-fluorouracil in high concentration with metformin , the cell proliferation inhibition rate of combining group makes no differences with the single-drug group. The combination of metformin and 5-fluorouracil produced an antagonism action in Hep-2 cells.Western blot assay showed that metformin, cisplatin, 5-fluorouracil could have caused the increase of expression level of AMPK-α, P21 and Cyclin D1 in Hep-2 cells while Paclitaxel could have cause the decrease of expression level of Cyclin D1. Using combined drug could cause the change of protein expression. Conclusion: 5-fluorouracil has been found to inhibit the proliferation of Hep-2 cells. Metformin has an antagonism on the anticancer effect to 5-fluorouracil in Hep-2 cells, and this antagonistic effect occurred partially through molecular signal pathways of AMPK-α, P21 and Cyclin D1 and it's significantly related to the cell cycle arrest., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Objective: To investigate the possibility of the Protein Phosphatase 2A (PP2A) inhibitor, LB100, in reversing acquired resistance to gefitinib in lung adenocarcinoma with epidermal growth factor receptor (EGFR) gene mutation. Methods: Cell line NCI-H1975 and established primary culture cell line 44-1 with gefitinib resistance were sequenced to determine the mutation type of EGFR gene. Cells were treated with gefitinib alone or combined with LB100 to determine the half maximal inhibitory concentration (IC50), and sensitivity of 44-1 and NCI-1975 to gefitinib alone or combined with LB100 was compared. The volume of NCI-H1975 xenografts with different drug treatments was observed to determine the efficiency of gefitinib with or without LB100 in tumor growth inhibition. Results: Both 44-1 and NCI-1975 cells had double EGFR mutation (sensitive L858R mutation and resistant T790M mutation). Both cells showed significant gefitinib resistance (IC50: 23.0 μmol/L in 44-1, 16.7 μmol/L in NCI-1975). When combined with LB100, IC50 of gefitinib decreased to 6.9 μmol/L in 44-1 cell and decreased to 3.4 μmol/L in NCI-H1975 cells. In NCI-1975 xenografts experiments, LB100 enhanced the ability of gefitinib in tumor growth inhibition ( P <0.05). Conclusion: LB100 reverses acquired resistance to gefitinib in lung adenocarcinoma cell lines.

Objective: To investigated the effect of metformin on growth inhibition of hypopharyngeal carcinoma xenograft in nude mice and to study the theoretical basis of metformin for the treatment of hypopharyngeal carcinoma in clinical. Method: ①To observe the course of metformin treatment, there were no severe adverse effects among the xenograft mice. ②After 28 days treatment, the average volumes of hypopharyngeal carcinoma xenografts of the metformin treated groups were significantly low compared with that of the control group.〖JP2〗The inhibition ratio of metformin at the concentration of 40 mg/(kg•d) and 200 mg/(kg•d) to the growth of tumor was 23.30%±7.17% and 40.51%±9.25%, respectively.③The aver ageoptical density(OAD) of AMPK in tumors of the control, met40 and met200 group was 0.246±0.038, 0.262±0.046, and 0.308±0.087, respectively. The OAD of p21 was 0.288±0.065, 0.305±0.091, and 0.477±0.114, respectively. And the OAD of cyclin D1 was 1.608±0.101, 0.781±0.055, and 0.711±0.046, respectively. Both the OAD of AMPK and p21 in met40 and met200 group were observably higher than those of the control group ( P < 0.05). But the OAD of cyclin D1 in met40 and met200 group were remarkably lower than the control group ( P < 0.05). ④The Western Blot drawed the consistant trend variation with immunohistochemical analyses. The gray value of AMPK in tumors of the control,met40 and met200 group was 0.641±0.042, 1.124±0.085, and 1.783±0.128; The gray value of p21 was 0.474±0.027, 0.594±0.063, and 0.970±0.079; The gray value of cyclin D1 was 1.608±0.101,0.781±0.055, and 0.711±0.046. Result: ①To observe the course of metformin treatment, there were no severe adverse effects among the xenograft mice. ②After 28 days treatment, the average volumes of hypopharyngeal carcinoma xenografts of the metformin treated groups were significantly low compared with that of the control group.〖JP2〗The inhibition ratio of metformin at the concentration of 40 mg/(kg•d) and 200 mg/(kg•d) to the growth of tumor was 23.30%±7.17% and 40.51%±9.25%, respectively.③The aver ageoptical density(OAD) of AMPK in tumors of the control, met40 and met200 group was 0.246±0.038, 0.262±0.046, and 0.308±0.087, respectively. The OAD of p21 was 0.288±0.065, 0.305±0.091, and 0.477±0.114, respectively. And the OAD of cyclin D1 was 1.608±0.101, 0.781±0.055, and 0.711±0.046, respectively. Both the OAD of AMPK and p21 in met40 and met200 group were observably higher than those of the control group ( P < 0.05). But the OAD of cyclin D1 in met40 and met200 group were remarkably lower than the control group ( P < 0.05). ④The Western Blot drawed the consistant trend variation with immunohistochemical analyses. The gray value of AMPK in tumors of the control,met40 and met200 group was 0.641±0.042, 1.124±0.085, and 1.783±0.128; The gray value of p21 was 0.474±0.027, 0.594±0.063, and 0.970±0.079; The gray value of cyclin D1 was 1.608±0.101,0.781±0.055, and 0.711±0.046. Conclusion: Metformin can suppress the growth of hypopharyngeal carcinoma xenograft in nude mice and inducing tumor cell apoptosis without any severe adverse effects., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Objective: To explore the molecular mechanism of neural plasticity in the reconstitution following impairment of auditory system,we studied the changing levels of GAP-43 expression in the auditory cortex(AC) in the developing rats and in ototoxicity deafened rats before and after electrical intracochlear stimulation. Method: Auditory brain stem response(ABR) measurements were performed to rats of OAD,NC1,CAD and NC2 group to evaluate hearing function.We studied the changing levels of GAP-43-positive neurons expression in the auditory cortex of the OAD,NC1,EIS1 and EIS2 group and RT-PCR was performed to do semiquantitative analysis. Result: The early electrical intracochlear stimulation elevated GAP-43 level(EIS1,120.00±5.59/HP) in ipsilateral AC instead of in the contralateral AC compared with the non-stimulation group (OAD4W,93.25±4.30/HP),and the two groups had statistical difference(EIS1/OAD4W,129.17%±3.33%, P <0.05).After the late electrical intracochlear stimulation,similar to the early group,the staining level of GAP-43(EIS2,102.50±4.02/HP) in the AC was also higher than the unaffected group(OAD8W,81.67±3.76/HP),and the two groups also had statistical difference(EIS2/OAD8W,123.74%±2.70%, P <0.05).Similarly,the expression of GAP-43 mRNA had the synchronous change with the GAP-43 in the AC. Conclusion: GAP-43 has close correlation with the growth and plasticity of the AC,which can be a significant marker for auditory plasticity., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Aim: This study aimed to determine the degree of discrepancy between clinical measurement of furcation involvement (FI) and cone beam computed tomography image analysis of multirooted teeth., Methods: FI measurements obtained from clinical records were compared to CBCT images of the same teeth to determine the degree of discrepancy between CBCT FI grading and clinical FI grading., Results: Of the hundred and fifty-four sites analysed, 22% of FI measurements from probing and CBCT were in agreement. Fifty-eight percent of clinical FI recordings were overestimated, and 20% were underestimated when compared to CBCT analysis., Conclusion: Clinical recording of FI is both over and underestimated compared to CBCT analysis. This was highest for probing recording grade I furcation involvement where it was highly overestimated. The occurrence of over and under estimation of FI will affect the assignment of prognosis to multirooted teeth, which can influence treatment planning for periodontal therapy and may result in inappropriate treatment., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Interferon (IFN) activation signaling and T helper 17 (Th17)-cell/B-cell regulation play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Several studies have provided convincing evidence that polymorphisms in IRF5, STAT4, IKZF1 and ETS1 from these pathways may be involved in SLE by affecting gene expression or epistasis. We analyzed the genetic interaction in known SLE susceptibility loci from the four genes in northern Han Chinese. A total of 946 northern Han Chinese participated in this study (370 unrelated SLE patients and 576 healthy controls). Subjects underwent genotyping for the single-nucleotide polymorphisms (SNPs) rs2004640 in IRF5, rs7574865 in STAT4, rs4917014 in IKZF1 and rs1128334 in ETS1 by use of a TaqMan SNP genotyping assay and direct sequencing. Gene-gene interaction analysis involved direct counting, multifactor dimensionality reduction (MDR) and linear regression analysis. SLE patients and controls differed in allele frequencies of rs7574865, rs1128334 (P < 0.001) and rs4917014 (P < 0.01). Direct counting revealed that the frequency of risk homozygote combinations was higher for SLE patients than controls (P < 0.01). Furthermore, 2-, 3- and 4-way gene-gene epistasis in SLE was confirmed by parametric methods and MDR analysis. Gene expression analysis partially supported the findings. Our study confirmed the association of the IFN pathway or Th17/B-cells and the pathogenesis of SLE, and gene-gene interaction in this pathway may increase the risk of SLE., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Systemic lupus erythematosus (SLE) is an autoimmune disease of uncertain etiology that affects multiple tissues and organs. Arsenic trioxide (ATO) has been used in lupus-prone mice with a regulatory effect on immune abnormality. Tetra-arsenic tetra-sulfide (As4S4), a traditional Chinese medicine, is effective on acute promyelocytic leukemia with mild side effects than ATO. In this study, a pilot study was performed to investigate the effects and the mechanism of As4S4 on the lupus-prone BXSB mice. Improvement of monocytosis (p<0.05) in spleen and decreased serum interleukin-6 (IL-6) (p=0.0277) were observed with As4S4 treatment. As4S4-treated mice exhibited amelioration of skin, liver and renal disease with mild side effects. Histological analysis revealed that As4S4 suppressed immune complex deposition, mesangial proliferation and inflammatory cell infiltration in kidney and liver. Our study support that As4S4 selectively suppresses cutaneous lupus and nephritis in BXSB mice and might be a potential treatment for SLE.

Two recent genome-wide association studies of East Asian populations revealed three genetic variants in WDFY4/LRRC18 associated with systemic lupus erythematosus (SLE). To identify the gene contributing to this disease susceptibility, we examined the mRNA expression of WDFY4 and LRRC18 in patients with SLE and healthy controls. WDFY4 was significantly downregulated in SLE patients as compared with controls. We used allelic expression and dual-luciferase assays to identify the functional variant. Transcriptional activity was lower for the rs877819A than -G allele. Electrophoretic mobility shift and supershift assays revealed that the transcription factor Yinyang1 (YY1) binds to rs877819, with lower affinity to the A allele, which explained the reduced transcriptional activity. This effect was further confirmed by YY1 small interfering RNA knockdown, overexpression and chromatin immunoprecipitation experiments. rs877819 in WDFY4 might be the functional site associated with SLE by reduced binding of YY1 and downregulating WDFY4 expression.

Noble-transition metal (noble=Pt,Au; transition=Co,Ni,Cu) alloy particles with sizes of about 5 nm have been studied by in situ high-energy x-ray diffraction while subjected to oxidizing (O(2)) and reducing (H(2)) gas atmospheres at elevated temperatures. The different gas atmospheres do not affect substantially the random alloy, face-centered-cubic structure type of the particles but do affect the way the metal atoms pack together. In an O(2) atmosphere, atoms get extra separated from each other, whereas, in an H(2) atmosphere, they come closer together. The effect is substantial, amounting to 0.1 Å difference in the first neighbor atomic distances, and concurs with a dramatic change of the particle catalytic properties. It is argued that such reactive gas induced "expansion shrinking" is a common phenomenon that may be employed for the engineering of "smart" nanoparticles responding advantageously to envisaged gas environments.

Objective: To explore the effects of islet neogenesis- associated protein pentadecapeptide (INGAP-PP) on proliferation and secretion function of β-cells., Methods: Islets of adult Sprague Dawley rats were isolated by collagenase digestion and treated with 10 μg/ml INGAP-PP, after 12, 24, 48 h, glucose-stimulated insulin secretion (GSIS) and acridine orange/pro pidium iodide (AO/PI) staining were used to detect the secretion function and cell viability. The INS-1 cells were treated with 0, 1, 10, 25, 50, 100, 250, and 500 μg/ml INGAP-PP for 24 or 48 h, MTT cell proliferation assay was adopted to survey the dose-response relationship between INGAP-PP and cell proliferation. The mRNA expression of roliferating cell nuclear antigen (PCNA), Cyclin D1, Cdk4, P27, p38MAPK, and JNK in INS-1 cells were examined by RT-PCR, and the protein expression of PCNA was examined by Western blot. The statistical significance was determined by Student's t-test or one-way analysis of variance., Results: The insulin secreted by islets and the cell viability were increased by INGAP-PP. MTT indicated a dose-response relationship between INGAP-PP and quantity of INS-1 cells, and treatment for 48 h had a stronger effect on cell proliferation than the 24 h. INGAP-PP up-regulated the mRNA expression of PCNA, Cyclin D1, Cdk4 and downregulated P27, p38MAPK, and JNK. Moreover, the protein expression of PCNA was up-regulated by 45% after INGAPPP exposure for 48 h., Conclusions: INGAP-PP increased the insulin secretion, enhanced the proliferation and might reduce apop tosis of β-cells. The mechanism may contribute to the changed expression of some genes related to cell cycle.

Soft cellular structures that comprise a solid matrix with a square array of holes open avenues for the design of novel soft and foldable structures. Our results demonstrate that by simply changing the shape of the holes the response of porous structure can be easily tuned and soft structures with optimal compaction can be designed., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Solid rationales are still present for the identification of synthetic ligands to simultaneously target multiple PPAR subtypes for the treatment of T2DM. The purpose of this study was to characterize the in vitro and in vivo differential effects of chiglitazar, a non-TZD type of PPAR pan-agonist currently in phase III clinic development in China, from PPARγ-selective agonist like rosiglitazone. Chiglitazar showed transactivating activity in each PPARα, γ, and δ subtype and upregulated the expression of PPARα and/or PPARδ downstream genes involved in the key processes of lipid metabolism and thermogenesis. Comparable blood glucose lowering effect was observed between chiglitazar and rosiglitazone, but chiglitazar did not significantly increase the body weight in KKAy and fat pad weight in db/db mice. Chiglitazar had high distribution in liver, pancreas, and skeleton muscles but was less present in kidney, heart, and adipose in rats. Heart weight increase was not observed in rats treated with chiglitazar for 6 months at a dose as high as 45 mg kg(-1). The in vitro and in vivo differential features of chiglitazar are informative and encouraging for the further development of this synthetic ligand for the potential use in T2DM.

The pollen of Brassica napus L. has been used in China to treat benign prostatic hyperplasia (BPH) for over decades. In this study, the pollen of Brassica napus L. was extracted successively with chloroform, ethyl acetate and ethanol. The ethyl acetate extract showed strong activity in decreasing the secretion of prostate specific antigen (PSA) in LNCaP cells as compared to two other extracts, measured by ELISA with finasteride as positive control in the assay. Five flavonoids were subsequently isolated from the active extract using bioassay-guided fractionation. They were Naringenin (1); Luteolin (2); Kaempferol (3); Kaempferol 3-(3-E-p-coumaroyl-alpha-L-rhamnopyranoside) (4); and Kaempferol 3-(2,3-di-E-p-coumaroyl-alpha-L-rhamnopyranoside) (5). All these compounds inhibited PSA secretion significantly, with IC50 values in the range of 5-50 microM. Compounds 2, 4 and 5 showed moderate cytotoxicity to LNCaP cells within the active concentration range, while compounds 1 and 3 showed no cytotoxicity. Further studies on the mechanism action of these compounds were performed by evaluating their activation of estrogen receptor (ER) and antagonistic activities on androgen receptor (AR) in cell-based reporter gene assays. All compounds described here were first isolated from the pollen of Brassica napus L.

Tumour hypoxia has been shown to increase mutation rate, angiogenesis, and metastatic potential, and decrease response to conventional therapeutics. Improved tumour oxygenation should translate into increased treatment response. Exogenous recombinant erythropoietin (rEpo) has been recently shown to increase tumour oxygenation in a mammary carcinoma model. The mechanism of this action is not yet understood completely. The presence of Epo and its receptor (EpoR) have been demonstrated on several normal and neoplastic tissues, including blood vessels and various solid tumours. In addition, rEpo has been shown in two recent prospective, randomized clinical trials to negatively impact treatment outcome. In this study, we attempt to characterize the direct effects of rEpo on tumour growth and angiogenesis in two separate rodent carcinomas. The effect of rEpo on R3230 rat mammary adenocarcinomas, CT-26 mouse colon carcinomas, HCT-116 human colon carcinomas, and FaDu human head and neck tumours, all of which express EpoR, was examined. There were no differences in tumour growth or proliferation (measured by Ki-67) between placebo-treated and rEpo-treated tumours. In the mammary window chamber, vascular length density (VLD) measurements in serial images of both placebo-treated and Epo-treated rats revealed no difference in angiogenesis between the Epo-treated tumours and placebo-treated tumours at any time point. These experiments are important because they suggest that the recent clinical detriment seen with the use of Epo is not due to its tumour growth effects or angiogenesis. These studies also suggest that further preclinical studies need to examine rEpo's direct tumour effects in efforts to improve the therapeutic benefits of Epo in solid tumour patients.

Leflunomide is a low molecular weight immunosuppressive drug which inhibits the enzymes dehydroorotate dehydrogenase and protein tyrosine kinase, both of which are important components in the immune response. As the mechanisms of action of leflunomide and tacrolimus are different, we postulated an additive or synergistic effect of the two drugs and investigated the effects of leflunomide alone, or in combination with a suboptimal dose of tacrolimus, on xenogeneic islet transplantation in a rat-to-mouse model. A total of 1200-1500 rat islets were transplanted under the left kidney capsule of streptozotocin-induced diabetic BALB/c mice. The median survival time (MST) of the untreated group was 6 days. Leflunomide at 5, 10 and 20 mg/kg/d administrated for 10 days significantly prolonged MST to 10, 16 and 20 days. A dose of tacrolimus (2 mg/kg/d) was associated with a graft survival of 9 (range 6-12) days; most grafts rejected during ongoing therapy. When tacrolimus (2 mg/kg/d) was combined with leflunomide (10 mg/kg/d), the survival time of the islet xenografts was increased further to 22 days, significantly longer than with leflunomide or tacrolimus alone. In summary, our findings demonstrate that leflunomide prolonged xenogeneic islet graft survival, and that its immunosuppressive effect was improved when combined with tacrolimus.

The purpose of this report is to present an overview of the use of fluorescence imaging in vivo, with particular emphasis on oncology. It is important to note, however, that many of the methods described herein have been applied to the study of non-malignant tissues as well. Modern medicine and biology research has benefited greatly from an ever-expanding assortment of fluorescent markers and labels. These markers and labels have allowed investigators to observe the behavior and properties of cell and molecular entities of interest in the context of complicated biological systems such as a mammalian cell or a whole mouse. Methods developed to image fluorescence in whole mice have been valuable in studying patterns of tumor growth and metastases. Alternatively, more detailed information and a wide variety of endpoints can be obtained using "intravital" preparations. This review focuses on use of fluorescence imaging for intravital preparations. For detail on fluorescence imaging of whole animals, refer to reviews on this subject [1,2]. For oncologic applications, studies have focused primarily on window chamber preparations that allow for real-time visualization of tumor growth, vascularity, vascular responses to stimulation, vascular permeability, vascular orientation, flow instability, and the like. These endpoints have been used to show that there are functional differences between tumor and normal tissues with respect to these functions under baseline conditions and after therapeutic manipulation. Examples of some of these differences are provided in this review as a means to illustrate how they can be used.

Hybridomas secreting a monoclonal antibody against glycyrrhizin were produced by fusing splenocytes from a mouse immunized against a glycyrrhizin-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very weak cross-reaction with glycyrrhetinic acid monoglucuronide and glycyrrhetic acid occurred. An enzyme-linked immunosorbent assay (ELISA) that had an effective measuring range of 20 -200 ng/mL of glycyrrhizin was established using this monoclonal antibody. In addition, a method named eastern blotting for the detection of glycyrrhizin was investigated. In this method, we developed a new way to separate the glycyrrhizin molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts were oxidized by sodium periodate to give dialdehydes, which reacted with amino groups on the protein and covalently bound to the adsorbent membrane. The monoclonal antibody bound to the aglycone part of the glycyrrhizin molecule for immunostaining. This method was validated by immunocytolocalization of glycyrrhizin in fresh Glycyrrhiza root.

The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using intravital microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using N(omega)-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to <5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to approximately 160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.

BTO-956 [methyl-3,5-diiodo-4-(4'-methoxyphenoxy)benzoate], a novel tubulin-binding drug and thyroid hormone analogue, was originally found to inhibit human carcinoma cell proliferation in vitro and to have potent growth delay activity in human breast and ovarian carcinoma xenografts in nude mice. Here we report that BTO-956 given to Fischer 344 rats also inhibits corneal angiogenesis and the growth and neovascularization of the R3230Ac rat mammary carcinoma tumor implanted in skin-fold window chambers. Hydron pellets containing recombinant human basic fibroblast growth factor (50 ng) and Sucralfate (20 microg) were implanted into surgically created corneal micropockets (day 0). BTO-956 was administrated by oral gavage (500 mg/kg, twice a day for 6 days) on days 1-6 (controls received vehicle alone). On day 7, rats received retrograde infusions of India ink via the thoracic aorta to visualize the corneal vasculature. Digitized images of slide-mounted corneas from control and treated animals were taken with a microscope. For the tumor growth and angiogenesis study, small pieces of R3230Ac tumor from a donor rat were implanted into surgically prepared window chambers (day 0). BTO-956 was given during days 5-11, and images of the tumors and their vasculature were recorded on day 12. No body weight loss was observed in either study. BTO-956 significantly inhibited corneal angiogenesis (by 50-80%), as assessed by measurements of limbal circumference displaying neovascularization, vessel length, vascularized area, and vascular area density. In the window chamber assay, tumors from treated animals were >50% smaller than tumors in control animals. In addition, vascular length densities in peripheral tumor zones were 30% less in treated compared with control animals. Together, these findings demonstrate that BTO-956 can inhibit angiogenesis induced by a growth factor in the rat cornea and in the peripheral area of implanted tumors, where tumor angiogenesis is most active.

Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with the 3'- and 2'-substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the nonbridging pro-S(P) oxygen of the reactive phosphoryl group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-S(P) oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-S(P) oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion/substrate interactions for an RNA or protein enzyme catalyzing phosphoryl transfer.

Inhibition of tumor angiogenesis is a therapeutic strategy that can inhibit tumor growth and metastases. The aim of this study was to determine whether the estrogen receptor (ER) ligand drug tamoxifen has antiangiogenic effects. We used three different models of angiogenesis, including measurement of microvessel densities in murine tumors, ex vivo aortic ring assays, and corneal pocket assays. ER-negative fibrosarcoma tumors in tamoxifen-treated ovariectomized rats had significantly less vessel formation compared with untreated animals (median microvessel density, 53.6 versus 94.3 counts/per x 200 field; P = 0.002). Rat aortic rings treated with tamoxifen at several different concentrations demonstrated significantly less vascular sprouting than control rings (P = 0.0001). Corneal pocket assays performed in tamoxifen-treated rats compared with control and estrogen-treated rats demonstrated decreased vascular length (0.88 mm versus 1.26 mm versus 1.47 mm; P = 0.022) and vessel area (21% versus 34% versus 47%; P = 0.018). These three animal models all showed significant inhibition of angiogenesis by tamoxifen and suggest a possible contributory mechanism of ER-independent manipulation by tamoxifen in the treatment and prevention of breast cancer. These studies raise the question as to whether or not newer ER ligand drugs might possess even more potent antiangiogenic effects, which in turn could lead to the broadening of the clinical usefulness of these compounds in a number of diseases. More importantly, these studies suggest that the antiangiogenic effects of tamoxifen are due, in part, to ER-independent mechanisms.

Bcl-2 and related proteins are key regulators of apoptosis or programmed cell death implicated in human disease including cancer. We recently showed that cell-permeable Bcl-2 binding peptides could induce apoptosis of human myeloid leukemia in vitro and suppress its growth in severe combined immunodeficient mice. Here we report the discovery of HA14-1, a small molecule (molecular weight = 409) and nonpeptidic ligand of a Bcl-2 surface pocket, by using a computer screening strategy based on the predicted structure of Bcl-2 protein. In vitro binding studies demonstrated the interaction of HA14-1 with this Bcl-2 surface pocket that is essential for Bcl-2 biological function. HA14-1 effectively induced apoptosis of human acute myeloid leukemia (HL-60) cells overexpressing Bcl-2 protein that was associated with the decrease in mitochondrial membrane potential and activation of caspase-9 followed by caspase-3. Cytokine response modifier A, a potent inhibitor of Fas-mediated apoptosis, did not block apoptosis induced by HA14-1. Whereas HA14-1 strongly induced the death of NIH 3T3 (Apaf-1(+/+)) cells, it had little apoptotic effect on Apaf-1-deficient (Apaf-1(-/-)) mouse embryonic fibroblast cells. These data are consistent with a mechanism by which HA14-1 induces the activation of Apaf-1 and caspases, possibly by binding to Bcl-2 protein and inhibiting its function. The discovery of this cell-permeable molecule provides a chemical probe to study Bcl-2-regulated apoptotic pathways in vivo and could lead to the development of new therapeutic agents.

Bcl-2 is a potent suppressor of apoptosis, and its overexpression contributes to tumorigenesis in many types of human cancers. To test the possibility of modulating Bcl-2 function as an anticancer strategy, a cell permeable Bcl-2 binding peptide, cell permeable moiety (cpm)-1285, was designed by chemically attaching a fatty acid to a peptide derived from the proapoptotic protein Bad. cpm-1285 entered HL-60 tumor cells, bound Bcl-2 protein, and induced apoptosis in vitro. In contrast, cpm-1285 had little effect on normal human peripheral blood lymphocytes. Furthermore, cpm-1285 had in vivo activity in slowing human myeloid leukemia growth in severe combined immunodeficient mice. These results demonstrate a novel approach for therapeutic intervention of tumor growth in vivo with small molecule inhibitors of Bcl-2.