[Research Advances in the Monitoring of New Psychoactive Substances in Municipal Wastewater].

ABSTRACT: In recent years, as the third-generation of drugs, new psychoactive substances （NPS） have expanded rapidly and become a serious concern for China's anti-drug prevention and control system. As a new drug monitoring technology in the current anti-drug field, wastewater analysis is an objective, real-time, accurate, convenient and effective drug monitoring method. In recent years, it has gradually been applied to the monitoring of NPS. This study summarizes wastewater sample collection, target stability research, wastewater sample pretreatment, wastewater sample analysis methods, target NPS consumption calculations and actual monitoring applications, with a view to the construction of a monitoring system for NPS in wastewater, real-time and accurate grasp of information on the use of NPS in cities, the reflection of the actual consumption of different types of NPS and consumption trends in a short period of time, and prediction of the development trend of abused use, which is of great significance for combating NPS crimes, serving and guaranteeing the personal safety of the people, and maintaining social stability.

[Analysis of Arsenic Compounds in Blood and Urine by HPLC-ICP-MS].

OBJECTIVES: To establish an analysis method for the detection of 6 arsenic compounds [AsC, AsB, As（Ⅲ）, DMA, MMA and As（V）] in blood and urine by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry （HPLC-ICP-MS）, and apply it to real cases. METHODS: Triton was used to damage cells, and then EDTA·2Na·2H2O was used to complex arsenic compounds in cells, and sonication and protein deposition by acetonitrile were performed for sample pretreatment. With the mobile phase consisted of ammonium carbonate and ultrapure water, gradient elution was performed for obtaining the arsenic compounds in samples, which were analysed by ICP-MS with Hamilton PRP-X100 column. RESULTS: The limits of detection in blood were 1.66-10 ng/mL, while the lower limits of quantitation in blood ranged from 5 to 30 ng/mL. The limits of detection in urine were 0.5-10 ng/mL, while the lower limits of quantitation in urine were 5-30 ng/mL. The relative standard deviation of inter-day and intra-day precisions was less than 10%. This method had been successfully applied to 3 cases. CONCLUSIONS: This study has established an analysis method for detecting 6 common arsenic compounds in blood and urine, which can be used to detect the arsenic compounds in the blood and urine from arsenic poisoning cases as well as the patients under arsenic treatment.

Role of the LXCXE binding site in Rb function.

Oncoproteins from DNA tumor viruses such as adenovirus E1a, simian virus 40 T antigen, and human papillomavirus E7 contain an LXCXE sequence, which they use to bind the retinoblastoma protein (Rb) and inhibit its function. Cellular proteins such as histone deacetylases 1 and 2 (HDAC1 and -2) also contain an LXCXE-like sequence, which they use to interact with Rb. The LXCXE binding site in Rb was mutated to assess its role in Rb function. These mutations inhibited binding to HDAC1 and -2, which each contain an LXCXE-like sequence, but had no effect on binding to HDAC3, which lacks an LXCXE-like sequence. Mutation of the LXCXE binding site inhibited active transcriptional repression by Rb and prevented it from effectively repressing the cyclin E and A gene promoters. In contrast, mutations in the LXCXE binding site did not prevent Rb from binding and inactivating E2F. Thus, the LXCXE mutations appear to separate Rb's ability to bind and inactivate E2F from its ability to efficiently recruit HDAC1 and -2 and actively repress transcription. In transient assays, several of the LXCXE binding site mutants caused an increase in the percentage of cells in G(1) by flow cytometry, suggesting that they can arrest cells. However, this effect was transient, as none of the mutants affected cell proliferation in longer-term assays examining bromodeoxyuridine incorporation or colony formation. Our results then suggest that the LXCXE binding site is important for full Rb function. Mutation of the LXCXE binding site does not inhibit binding of the BRG1 ATPase component of the SWI/SNF nucleosome remodeling complex, which has been shown previously to be important for Rb function. Indeed, overexpression of BRG1 and Rb in cells deficient for the proteins led to stable growth inhibition, suggesting a cooperative role for SWI/SNF and the LXCXE binding site in efficient Rb function.

Chromatin remodeling and transcriptional regulation.

Extensive studies in the past few years have begun to demonstrate that chromosome structure plays a critical role in transcriptional regulation. Two highly conserved mechanisms for altering chromosome structure have been identified: 1) post-translational modification of histones and 2) adenosine triphosphate (ATP)-dependent chromosome remodeling. Acetylation of histone lysine residues has been known for three decades to be associated with transcriptional activation. Recent discoveries, however, show that a number of transcriptional regulators are histone acetylases or histone deacetylases. Specific DNA-binding transcription factors recruit histone acetylases and deacetylases to promoters to activate or repress transcription. These results strongly support the notion that histone acetylation and deacetylation play an important role in transcriptional regulation. Recent findings have also provided insight into the molecular mechanisms by which ATP-dependent chromosome-remodeling activities participate in transcriptional regulation. Furthermore, some ATP-dependent chromosome-remodeling activities have been shown to complex with histone deacetylases. In the complexes studied to date, the ATP-dependent chromosome-remodeling activity enhances the histone deacetylase activity. Therefore, the two mechanisms appear to work in concert to achieve precise control of transcription. Disruption of chromosome remodeling has been linked to a number of diseases, and a complete understanding of the complex chromosome-remodeling machinery may lead to the development of new therapies.

Comparison of continuous and split-course radiotherapy for nasopharyngeal carcinoma--an analysis of 1446 cases with squamous cell carcinoma grade 3.

PURPOSE: An analysis of 1446 cases with squamous cell nasopharyngeal carcinoma Grade 3 was made for the comparison of treatment results of continuous (CG) and split-course (SG) radiotherapy regimens. METHODS AND MATERIALS: The patients were given their first definitive radiotherapy at our department from July 1977 to December 1980, 1093 cases by continuous treatment regimen, and 353 cases by split-course regimen. The interval of the split was 10-45 days, mean 28.6 days. Trials were nonrandomized, but the treatment conditions for both groups were the same. RESULTS: The 5-year survival rate was 33.6% for CG, and 26.9% for SG. For Stage III and IV patients, the 5-year survival rate was 31% (290/933) for CG, and 21.5% (36/107) and 18.8% (17/90) for those with a split of 21-30 and 31-45 days, respectively (both p < 0.05); the 5-year local control rate was 27.4% (256/933) for CG, and 15.5% (26/167) (p < 0.01), and 15.5% (14/90) (p < 0.05) for those with a split of 21-30 and 31-45 days, respectively. The metastatic rate in 5-years was 21.59% vs. 27.48% for CG to SG (p < 0.01), but not different statistically for any subgroup. No relation was confirmed between local control rate and radiation dose. CONCLUSION: According to the data obtained in this series, we believe that the treatment results of CG were superior to those of SG, especially for Stage III and IV patients, and those with a split of 21 days or more.

Comparison of continuous and split-course radiotherapy for nasopharyngeal carcinoma.

From July 1977 through December 1980, a series of 1882 cases with nasopharyngeal carcinoma (NPC) had their first definitive radiotherapy course at our department, 1424 cases by continuous treatment regimen, and 458 cases by a split-course regimen. The interval of the split was 11-45 days, with an average of 28.6 days. Trials were non-randomized, but the treatment conditions for both groups were the same. The overall 5-year survival rate was 34.6%, 35.4% for the continuous group and 31.8% for the split-course group. The 5-year survival rate for Stage IV patients (squamous cell carcinoma Grade III) was 25.7% (69/268) for the continuous group and 14.1% (10/71) for the split-course group. Breaking down the cases by primary and cervical metastatic sites, for T3 cases, the 5-year local control rate for the primary site was 29.4% (133/451) for the continuous group, and 18.6% (22/118) for the split-course group. These differences are statistically significant (p less than 0.05). However, according to the data shown by this series, we can conclude that the split-course treatment regimen had no benefit over the continuous one when overall 5-year survival is considered.

Indoor burning coal air pollution and lung cancer--a case-control study in Fuzhou, China.

A case-control study on risk factors for lung cancer was carried out in Fuzhou, China. One-hundred and two newly-diagnosed primary lung cancer cases in urban Fuzhou (78 male and 24 female cases) were matched with 306 population-based controls. The primary histological types were adenocarcinomas (57 cases, 55.9%) and squamous cell carcinomas (39 cases, 38.2%). Controls were obtained from the general population by random, stratified sampling and consisted of noncancer cases matched for sex, ethnicity and age. Cases and controls were interviewed by trained professionals using a standardized questionnaire. Information was obtained on: smoking habits, living conditions, history of respiratory diseases, air pollution, and 40 other variables. The data were evaluated by conditional logistic regression analysis. The major risk factors for lung adenocarcinoma were: indoor air pollution from burning coal, chronic bronchitis, and high economic income. The risk factors for lung squamous cell carcinoma were: amount of cigarettes smoked per day, "deep inhalation", a history of exposure to environmental tobacco smoke (ETS) before 20 years of age, burning coal indoors, and high economic income. The results showed that the major risk factors for lung cancer in Fuzhou were: burning coal indoors, smoking, exposure to ETS before 20 years of age, chronic bronchitis, and high economic income. Cigarette smoking significantly increased the risk of lung squamous cell carcinoma, but had no significant association with the risk of lung adenocarcinoma. In summary, our research supports the hypothesis that smoking and indoor air pollution are the major risk factors for lung cancer in Fuzhou. Burning coal indoors and smoking were associated with lung cancer mortality in a major city in southern China.

Serological diagnosis of nasopharyngeal carcinoma by enzyme linked immunosorbent assay: optimization, standardization and diagnostic criteria.

OBJECTIVES: To produce an enzyme linked immunosorbent assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18 kD EBV matrix protein, and to determine and optimize its sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma (NPC). METHODS: We used a combination of highly purified glutathione transferase fusion proteins of the 40 kD carboxy domain of EBNA1 and the 18 kD EBV matrix protein for coating ELISA plates. In three separate studies, we tested for IgA antibodies in serum specimens from 28 EBV seronegative donors, 284 EBV seropositive donors and 160 newly diagnosed NPC patients. By comparing the sensitivity and specificity of diagnosis obtained for different cutoff values, we derived several quantitative parameters to evaluate assay performance, establish objective diagnostic criteria which optimize the intrinsic diagnostic capability of the assay and assess the significance of individual test results, respectively. Optimum cutoff optical density (OD) is defined as the cutoff OD where sensitivity of the assay equals its specificity, and resolution of the assay is indicated by the value of sensitivity (or specificity) determined at the optimum cutoff OD. Diagnosis of NPC was achieved by setting a cutoff zone at +/- 20% of this value. RESULTS: All the EBV seronegative donors tested were not reactive, and most of the EBV seropositive donors were weakly reactive, while the majority of NPC patients were moderately or strongly reactive. While the assay was thus shown to be specific for EBV, there was an overlap in the level of these serum antibodies between few individuals of the two latter groups. It was shown that the assay performed equally well in two separate studies conducted under different testing conditions and using different collections of sera in that assay resolution determined on these occasions were 86% and 87% respectively. Diagnosis of NPC can be achieved at the same expected sensitivity of 89% and 83% determined at the lower and upper limits of the cutoff zones, with the corresponding values of specificity being 78% and 91%. It was further shown in the third study that resolution of the assay can be increased to 90% using an assay produced with a higher concentration of the same antigens, and that diagnosis of NPC can be achieved at a higher sensitivity ranging between 86% and 95% at a corresponding specificity of 93% and 86%. CONCLUSIONS: After optimization and standardization, the ELISA can achieve a sensitivity ranging from 86% to 95%, with corresponding specificities of 93% and 86% respectively for the diagnosis of NPC.

Rb interacts with histone deacetylase to repress transcription.

Previously, we found that Rb can actively repress transcription of cell cycle genes by binding and inactivating transcription factors at the promoter. Here, we demonstrate that Rb can also repress transcription of endogenous cell cycle genes containing E2F sites through recruitment of histone deacetylase, which deacetylates histones on the promoter, thereby promoting formation of nucleosomes that inhibit transcription. These two mechanisms of repression by Rb are selective-some promoters and transcription factors are blocked by this recruitment of histone deacetylase, whereas others are resistant to histone deacetylase activity and are repressed directly by inhibition of transcription factors.

Mechanism of active transcriptional repression by the retinoblastoma protein.

The retinoblastoma tumour-suppressor protein (Rb) belongs to a family that share a motif known as the pocket. The pocket was originally identified as the region of Rb required for binding to oncoproteins from DNA tumour viruses, which disrupt the binding of Rb to the E2F family of cell-cycle transcription factors (referred to collectively here as E2F). Rb switches E2F sites from positive to negative elements, suggesting that Rb-E2F is an active complex that blocks transcription. Here we report that Rb is selectively recruited to promoters through E2F, where it in turn inactivates surrounding transcription factors by blocking their interaction with the basal transcription complex. We suggest that this repressor activity is essential for inhibiting promoters that contain enhancers in addition to E2F sites.

Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1.

We present evidence that phosphorylation of the C-terminal region of Rb by Cdk4/6 initiates successive intramolecular interactions between the C-terminal region and the central pocket. The initial interaction displaces histone deacetylase from the pocket, blocking active transcriptional repression by Rb. This facilitates a second interaction that leads to phosphorylation of the pocket by Cdk2 and disruption of pocket structure. These intramolecular interactions provide a molecular basis for sequential phosphorylation of Rb by Cdk4/6 and Cdk2. Cdk4/6 is activated early in G1, blocking active repression by Rb. However, it is not until near the end of G1, when cyclin E is expressed and Cdk2 is activated, that Rb is prevented from binding and inactivating E2F.

Development and evaluation of an Epstein-Barr virus (EBV) immunoglobulin M enzyme-linked immunosorbent assay based on the 18-kilodalton matrix protein for diagnosis of primary EBV infection.

A new immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) based on the recombinant Epstein-Barr virus (EBV) matrix protein was developed. Compared to indirect immunofluorescence for the detection of IgM antibody to the EBV capsid antigen on clinical specimens, the sensitivity and specificity of the new IgM ELISA were 96 and 96%, respectively.

Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF.

We present evidence that Rb forms a repressor containing histone deacetylase (HDAC) and the hSWI/SNF nucleosome remodeling complex, which inhibits transcription of genes for cyclins E and A and arrests cells in the G1 phase of the cell cycle. Phosphorylation of Rb by cyclin D/cdk4 disrupts association with HDAC, relieving repression of the cyclin E gene and G1 arrest. However, the Rb-hSWI/SNF complex persists and is sufficient to maintain repression of the cyclin A and cdc2 genes, inhibiting exit from S phase. HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF then appear to maintain the order of cyclin E and A expression during the cell cycle, which in turn regulates exit from G1 and from S phase, respectively.