Nontargeted Analysis of Coumarins in Source Water and Their Formation of Chlorinated Coumarins as DBPs in Drinking Water.

Coumarin was detected as one of the most abundant compounds by nontargeted analysis of natural product components in actual water samples prior to disinfection. More importantly, prechlorination of humic acid generated 3-hydroxycoumarin and monohydroxy-monomethyl-substituted coumarin with a total yield of ≤10.1%, which suggested the humic substance in raw water is an important source of coumarins. 7-Hydroxycoumarin, 6-hydroxy-4-methylcoumarin, 6,7-dihydroxycoumarin, and 7-methoxy-4-methylcoumarin were identified in raw water by high-performance liquid chromatography-tandem high-resolution mass spectrometry because only some coumarin standards were commercially available. Their chlorination generated monochlorinated and polychlorinated coumarins, and their structures were confirmed by the synthesized standards. These products could form at various dosages of chlorine and pH levels, and some with a concentration of 600 ng/L can be stable in tap water for days. 3,6,8-Trichloro-7-hydroxycoumarin, 3-chloro-7-methoxy-4-methylcoumarin, and 3,6-dichloro-7-methoxy-4-methylcoumarin were first identified in finished water with concentrations of 0.0670, 78.1, and 14.7 ng/L, respectively, but not in source water, suggesting that they are new DBPs formed during disinfection. The cytotoxicity of 3-chloro-7-methoxy-4-methylcoumarin in CHO-K1 cells was comparable to those of 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone in TIC-Tox analyses, suggesting that further investigation of their occurrence and control in drinking water systems is warranted.

Quantitative Proteomics of the CDK9 Interactome Reveals a Function of the HSP90-CDC37-P-TEFb Complex for BETi-Induced HIV-1 Latency Reactivation.

Brd4 has been intensively investigated as a promising drug target because of its implicated functions in oncogenesis, inflammation, and HIV-1 transcription. The formation of the Brd4-P-TEFb (CDK9/Cyclin T1) complex and its regulation of transcriptional elongation are critical for HIV latency reactivation and expression of many oncogenes. To further investigate the mechanism of the Brd4-P-TEFb complex in controlling elongation, mass spectrometry-based quantitative proteomics of the CDK9 interactome was performed. Upon treatment with the selective BET bromodomain inhibitor JQ1, 352 proteins were successfully identified with high confidence as CDK9-interacting proteins. Among them, increased bindings of HSP90 and CDC37 to CDK9 were particularly striking, and our data suggest that the HSP90-CDC37-P-TEFb complex is involved in controlling the dynamic equilibrium of the P-TEFb complex during BETi-induced reactivation of HIV-1 latency. Furthermore, the HSP90-CDC37-P-TEFb complex directly regulates HIV-1 transcription and relies on recruitment by heat shock factor 1 (HSF1) for binding to the HIV-1 promoter. These results advance the understanding of HSP90-CDC37-P-TEFb in HIV-1 latency reversal and enlighten the development of potential strategies to eradicate HIV-1 using a combination of targeted drugs.

Isobaric Stable Isotope N-Phosphorylation Labeling (iSIPL) for Ultrasensitive Proteome Quantification.

Stable isotope chemical labeling methods have been widely used for high-throughput mass spectrometry (MS)-based quantitative proteomics in biological and clinical applications. However, the existing methods are far from meeting the requirements for high sensitivity detection. In the present study, a novel isobaric stable isotope N-phosphorylation labeling (iSIPL) strategy was developed for quantitative proteome analysis. The tryptic peptides were selectively labeled with iSIPL tag to generate the novel reporter ions containing phosphoramidate P-N bond with high intensities under lower collision energies. iSIPL strategy are suitable for peptide sequencing and quantitative analysis with high sensitivity and accuracy even for samples of limited quantity. Furthermore, iSIPL coupled with affinity purification and mass spectrometry was applied to measure the dynamics of cyclin dependent kinase 9 (CDK9) interactomes during transactivation of the HIV-1 provirus. The interaction of CDK9 with PARP13 was found to significantly decrease during Tat-induced activation of HIV-1 gene transcription, suggesting the effectiveness of iSIPL strategy in dynamic analysis of protein-protein interaction in vivo. More than that, the proposed iSIPL strategy would facilitate large-scale accurate quantitative proteomics by increasing multiplexing capability.

[Maxing Shigan Decoction improves lung and colon tissue damage caused by influenza virus infection through JAK1/2-STAT1 signaling pathway].

Based on Janus kinase 1/2-signal transducer and activator of transcription 1(JAK1/2-STAT1) signaling pathway, this study explored the immune mechanism of Maxing Shigan Decoction in alleviating the lung tissue and colon tissue damage in mice infected with influenza virus. The influenza virus infection was induced in mice by nasal drip of influenza virus. The normal group, model group, oseltamivir group, antiviral granule group, and Maxing Shigan Decoction group were designed. After intragastric administration of corresponding drugs or normal saline for 3 or 7 days, the body mass was measured, and lung index, spleen index, and thymus index were calculated. Based on hematoxylin-eosin(HE) staining, the pathological changes of lung tissue and colon tissue were observed. Enzyme-linked immunosorbent assay(ELISA) was used to detect serum levels of inflammatory factors interleukin-8(IL-8) and interferon-γ(IFN-γ), Western blot and real-time quantitative polymerase chain reaction(RT-qPCR) to determine the protein and mRNA levels of JAK1, JAK2, STAT1, interferon regulatory factor 9(IRF9), and IFN-γ in lung tissue and colon tissue. The results showed that after 3 and 7 days of administration, the body mass, spleen index, and thymus index were lower(P<0.05 or P<0.01), and the lung index was higher(P<0.01) in the model group than in the normal group. Moreover, the model group showed congestion, edema, and infiltration of a large number of lymphocytes and macrophages in the lung tissue, irregular structure of colon mucosa, ulceration and shedding of epithelial cells, and infiltration of a large number of inflammatory cells. The model group had higher levels of serum IFN-γ(P<0.01), higher protein and mRNA expression of JAK1, JAK2, STAT1, IRF9, IFN-γ in lung tissue(P<0.05 or P<0.01), higher level of JAK2 protein in colon tissue(P<0.01), and higher protein and mRNA levels of STAT1 and IRF9(P<0.05 or P<0.01) than the normal group. Compared with the model group, Maxing Shigan Decoction group had high body mass, spleen index, and thymus index(P<0.05 or P<0.01), low lung index(P<0.05 or P<0.01), and significant alleviation of pathological injury in lung and colon. Moreover, lower serum level of IFN-γ(P<0.05 or P<0.01), protein and mRNA levels of JAK1, JAK2, STAT1, IRF9, and IFN-γ in lung tissue(P<0.05 or P<0.01), JAK2 protein level in colon tissue(P<0.01), and protein and mRNA levels of STAT1 and IRF9(P<0.05 or P<0.01) were observed in the Maxing Shigan Decoction group than in the model group. After 3 days of administration, the level of serum IL-8 in the model group was significantly higher than that in the normal group(P<0.01), and the level in the Maxing Shigan Decoction group was significantly reduced(P<0.01). In conclusion, Maxing Shigan Decoction can significantly up-regulate body mass, spleen index, and thymus index, down-regulate lung index, reduce the levels of IL-8 and IFN-γ, and down-regulate protein and mRNA levels of JAK1, JAK2, STAT1, IRF9, and IFN-γ in lung tissue and protein and mRNA levels of JAK2, STAT1, and IRF9 in colon tissue, and alleviate pathological damage of lung tissue and colon tissue. The mechanism is the likelihood that it inhibits the activation of JAK1/2-STAT1 signaling pathway to alleviate the damage to lung and colon tissue damage.

Monitoring and exposure assessment of organophosphorus flame retardants in source and drinking water, Nanjing, China.

This study developed a new method to determine the residues of 13 organophosphorus flame retardants (OPFRs) in drinking water by gas chromatography-tandem mass spectrometry (GC-MS/MS) technique and investigated the chemical distribution in water samples from municipal plants along the Yangtze River in Nanjing. The linear calibration correlation coefficients R2 for all 13 OPFRs were at least 0.998 0. Three levels of spiked test were performed. Most of the recoveries were in the range of 80~120%, and the relative standard deviations (RSDs) for the 13 OPFRs were 2.1~17% (n = 6). Five OPFRs were 100% positively detected in the samples, and 3 OPFRs were positively detected in some samples. The concentrations of detected OPFR in the water ranged from 0.7 to 5780.0 ng L-1. The average concentrations of OPFRs in wet season were higher than those in dry season, and the contaminants mainly originated from the source water in the Yangtze River. The exposure assessments of individual and total OPFRs were investigated. The estimated daily intakes of total OPFRs via ingestion of drinking water reached up to 64.8 and 45.2 ng/kg bw/day in dry and wet season, respectively. This study demonstrates a profile of OPFR distribution in Nanjing municipal water and provides information on human exposure assessment via drinking water in the Nanjing District, China.

IGF-1 gene-modified muscle-derived stem cells are resistant to oxidative stress via enhanced activation of IGF-1R/PI3K/AKT signaling and secretion of VEGF.

Reactive oxygen species (ROS)-induced oxidative stress increases in skeletal muscle with aging and decreases the viability of implanted cells. Type 1 insulin-like growth factor (IGF-1) promotes the survival of skeletal muscle cells under oxidative stress. It is unknown whether IGF-1 protects muscle-derived stem cells (MDSCs) from oxidative stress. In this study, we genetically engineered rat MDSCs to overexpress IGF-1 and determined cell viability, apoptosis, and VEGF secretion under oxidative stress. Overexpression of IGF-1 prevented MDSCs from H2O2-induced caspase-dependent apoptotic cell death by upregulating the PI3K/AKT pathway, accompanied with an increase of NF-κB, p-NF-κB, Bcl-2, and VEGF, as well as a decrease of Bax. In contrast, pre-administration of picropodophyllinb, wortmannin, 1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate), or pyrrolidine-dithiocarbamate, specific inhibitors of IGF-1R, PI3K, AKT, and NF-κB, respectively, followed by treatment with H2O2, resulted in cell death of MDSCs. Our data indicated that IGF-1 suppresses apoptosis and enhances the paracrine function of MDSCs under oxidative stress via enhancing IGF-1R/PI3K/AKT signaling. Thus, IGF-1 gene-modified MDSCs present a potential application in the treatment of muscle wasting, such as urethra intrinsic sphincter deficiency.

Immune infiltration patterns and identification of new diagnostic biomarkers GDF10, NCKAP5, and RTKN2 in non-small cell lung cancer.

This study aimed to identify potential biomarkers for non-small cell lung cancer (NSCLC) and analyze the role of immune cell infiltration in NSCLC. R software was used to screen differentially expressed genes (DEGs) from NSCLC datasets obtained from the Gene Expression Omnibus (GEO) database, and functional correlation analysis was performed. The machine learning algorithms were used to screen the potential biomarkers of NSCLC. The diagnostic values were assessed through receiver operating characteristic (ROC) curves. The protein and mRNA expression levels of potential biomarkers were verified based on the Human Protein Atlas (HPA) database and qRT-PCR. CIBERSORT was used to evaluate the infiltration of immune cells in NSCLC tissues, and the correlation between potential biomarkers and infiltrated immune cell was analyzed. Finally, specific siRNAs were utilized to reduce the GDF10, NCKAP5, and RTKN2 expression in A549 and H1975 cells. The proliferation ability of A549 and H1975 cells was detected by MTT assay. A total of 848 upregulated DEGs and 1308 downregulated DEGs were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were mainly related to cell division. Disease ontology (DO) enrichment analysis showed that the diseases with these DEGs were mainly lung diseases, including NSCLC. In addition,three potential biomarkers were identified: GDF10, NCKAP5, and RTKN2. Immune cell infiltration analysis showed that resting NK cells, activated dendritic cells, and Tregs may be involved in the pathogenesis of NSCLC. Meanwhile, GDF10, NCKAP5, and RTKN2 were negatively correlated with Tregs and naïve B cells but were positively correlated with activated dendritic cells and resting NK cells. Immunohistochemical staining showed that the expression of GDF10, NCKAP5, and RTKN2 in the lung tissue of patients with NSCLC was lower than that of normal lung tissue. qRT-PCR also confirmed that the mRNA expression of three biomarkers in NSCLC cell lines A549 and H1975 were significantly lower than those in human normal lung epithelial cells BEAS-2B. An MTT assay showed that GDF10, NCKAP5, and RTKN2 knockdown significantly promoted the proliferation of A549 and H1975 cells. The in vitro experiments showed that GDF10, NCKAP5, and RTKN2 played the inhibitory effects on NSCLC cell lines proliferation. Hence, GDF10, NCKAP5, and RTKN2 can be used as diagnostic biomarkers for NSCLC.

Exosomal EphA2 promotes tumor metastasis of triple-negative breast cancer by damaging endothelial barrier.

Many evidences show that exosomes play an important role in cancer development, invasion and metastasis. This study is based on the need to explore exosomal protein that promote breast cancer metastasis. We found that tyrosine kinase EphA2 was enriched in Triple-negative breast cancer -derived exosomes and it could disrupt the endothelial monolayer barrier through downregulating tight junction proteins of endothelial cells. These mechanisms were confirmed by in vivo experiments. After periodical injection of exosomal EphA2 into mice caudal vein, we found increased vascular permeability and breast cancer metastases in distant organs, and this phenomenon decreased dramatically after exosomal EphA2 knockdown. This study provides a new mechanism of exosome promoting breast cancer metastasis and suggests a new therapeutic target for the prevention and treatment of breast cancer metastasis.

IGF-1-induced Muscle-Derived Stem Cells as a Potential Treatment of Stress Urinary Incontinence in Female Rats.

PURPOSE: Stress urinary incontinence (SUI) is prevalent among elderly women. This study aimed to discuss the potential of muscle-derived stem cells (MDSCs)-based therapy in treating SUI by exploring the effect of Insulin-like growth factor-1 (IGF-1) on transplanted MDSC and urethral sphincter function. MATERIALS AND METHODS: Bilaterally pudendal nerve-transected (PNT) female rats were divided into four groups: sham, PNT+ phosphate buffered solution (PBS) injection, PNT+IGF-1/MDSCs and PNT+ green fluorescent protein (GFP)/MDSCs. IGF-1 was expressed in MDSCs by lentiviral vector. Viable MDSCs were detected by laser scanning confocal microscopy (LSCM). The expression of Myosin heavy chain (MyHC), vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR-2), microvessel density (MID) and urethral resistance function were assessed. RESULTS: IGF-1 promoted the survival and differentiation of MDSCs. IGF-1-expressing MDSCs facilitated local angiogenesis and muscle fiber regeneration, and alleviated symptoms of SUI. CONCLUSIONS: IGF-1-expressing MDSCs may be used as a novel treatment for patients with SUI.

2,6-Dichloro-1,4-benzoquinone formation from chlorination of substituted aromatic antioxidants and its control by pre-ozonation in drinking water treatment plant.

Halobenzoquinones are frequently detected as disinfection by-products in drinking water. Among identified halobenzoquinones, 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) is particularly toxic and is frequently detected in drinking water. Synthetic aromatic antioxidants discharged to source water may increase the risk of 2,6-DCBQ formation, as many studies suggest that aromatic compounds are the most likely precursors to 2,6-DCBQ. Herein, we investigated the formation of 2,6-DCBQ from chlorination of three model aromatic antioxidants, including 3-tert-butyl-4-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-methylphenol (BHT) and bis(4-tert-butylphenyl)amine (BBPA). Only BBPA produced 2,6-DCBQ under chlorination, while chlorination of BHA and BHT formed α, ß-unsaturated C4-dicarbonyl ring-opening products and phenolic compounds. Based on mass balance and intermediate transformation analysis, mechanisms for the formation of 2,6-DCBQ from BBPA chlorination involved hydrolysis, tert-butyl group cleavage, chlorine substitution, desamination and oxidation. Mitigating aromatic compounds will be an efficient method for 2,6-DCBQ control, such as pre-ozonation, because the intermediates involved in 2,6-DCBQ formation were aromatic compounds. Real water samples from two drinking water treatment plants (DWTPs), one with pre-ozonation (DWTP 2) and the other without pre-ozonation (DWTP1), were analyzed. The two DWTPs were built along the Yangtse river in Nanjing city. Raw water parameters from the two DWTPs, including dissolved organic carbon (DOC), UV absorbance at 254 nm (UV254) and NH3-N, indicated the water quality between these sources was similar. Pre-ozonation in DWTP 2 vanished 2,6-DCBQ in raw water. Concentrations of 2,6-DCBQ in finished water from DWTP 1 (5.69 ng/L) was higher than concentrations generated from DWTP 2 (1.31 ng/L). These results demonstrate that pre-ozonation, granular activated carbon (GAC) and quartz sand treatments at DWTP 2 remove more 2,6-DCBQ precursors than the conventional quartz sand and GAC treatments in DWTP 1. These results suggest the pre-ozonation, GAC and quartz sand treatments can help minimize concentrations of 2,6-DCBQ generated in DWTPs.

ELL-associated factors EAF1/2 negatively regulate HIV-1 transcription through inhibition of Super Elongation Complex formation.

The ELL (ELL1 and ELL2)-containing Super Elongation Complex (SEC) is required for efficient HIV-1 transactivation by the viral-encoded Tat protein. EAF1 and EAF2 are ELL-associated factors and considered as positive regulators of ELL. However, their role in HIV-1 transcriptional control is unknown. In this study, we show that EAF1/2 inhibit the SEC-dependent and Tat-activated HIV-1 transcription. EAF1/2 are found to interact with the SEC components in an ELL1/2-dependent manner. Surprisingly, the depletion of EAF1/2 increases the SEC formation and occupancy on the HIV-1 proviral DNA, thereby stimulating Tat transactivation of HIV-1. Although EAF1/2 interact with members of the SEC in a ELL-dependent manner, this interaction competes with the binding of the scaffolding subunit AFF1 with ELL, thus reducing the SEC formation. Together, these data reveal how EAF1/2 regulate the SEC formation to control HIV-1 transcription.

JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes.

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)'s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.

In animals, an enzyme known as RNA polymerase II (Pol II for short) is a key element of the transcription process, whereby the genetic information contained in DNA is turned into messenger RNA molecules in the cells, which can then be translated to proteins. To perform this task, Pol II needs to be activated by a complex of proteins called P-TEFb; however, P-TEFb is usually found in an inactive form held by another group of proteins. Yet, it is unclear how P-TEFb is released and allowed to activate Pol II. Scientists have speculated that another protein called JMJD6 (Jumonji domain-containing 6) is important for P-TEFb to activate Pol II. Various roles for JMJD6 have been proposed, but its exact purpose remains unclear. Recently, two enzymes closely related to JMJD6 were found to be able to make precise cuts in other proteins; Lee, Liu et al. therefore wanted to test whether this is also true of JMJD6. Experiments using purified JMJD6 showed that it could make a cut in an enzyme called MePCE, which belongs to the group of proteins that hold P-TEFb in its inactive form. Lee, Liu et al. then tested the relationships between these proteins in living human and mouse cells. The levels of activated Pol II were lower in cells without JMJD6 and higher in those without MePCE. Together, the results suggest that JMJD6 cuts MePCE to release P-TEFb, which then activates Pol II. JMJD6 appears to know where to cut by following a specific pattern of elements in the structure of MePCE. When MePCE was mutated so that the pattern changed, JMJD6 was unable to cut it. These results suggest that JMJD6 and related enzymes belong to a new family of proteases, the molecular scissors that can cleave other proteins. The molecules that regulate transcription often are major drug targets, for example in the fight against cancer. Ultimately, understanding the role of JMJD6 might help to identify new avenues for cancer drug development.

Structural and functional insight into the effect of AFF4 dimerization on activation of HIV-1 proviral transcription.

Super elongation complex (SEC) is a positive regulator of RNA polymerase II, which is required for HIV-1 proviral transcription. AFF1/4 is the scaffold protein that recruits other components of SEC and forms dimer depending on its THD domain (TPRL with Handle Region Dimerization Domain). Here we report the crystal structure of the human AFF4-THD at the resolution of 2.4 Å. The α4, α5, and α6 of one AFF4-THD mediate the formation of a dimer and pack tightly against the equivalent part of the second molecule in the dimer of AFF-THD. Mutagenesis analysis revealed that single mutations of either Phe1014 or Tyr1096 of AFF4 to alanine impair the formation of the AFF4 dimer. In addition, transactivation assay also indicated that Phe1014 and Tyr1096 of AFF4 are critical to the transactivation activity of AFF4. Interestingly, the corresponding residues Phe1063 and Tyr1145 in AFF1 have an effect on the transactivation of HIV-1 provirus. However, such mutations of AFF1/4 have no effect on the interaction of AFF1/4 with other subunits of the SEC. Together, our data demonstrated that the dimerization of AFF1/4 is essential to transactivation of HIV-1 provirus.

Positive association between MIC gene polymorphism and tuberculosis in Chinese population.

The disease progression and morbidity of tuberculosis (TB) infections are determined by virulence of the micro-organism, host genetic factors and environmental factors. The highly polymorphic MHC class I chain-related gene (MIC) could serve as a potential host genetic candidate. To investigate the association of MIC polymorphism with TB infection, 124 patients and 191 ethnically matched controls from Hunan province, Southern China, were genotyped for the MIC polymorphism using polymerase chain reaction-sequence specific priming and sequencing-based typing. The results showed that allele frequencies of MIC-sequence and MICA-STR were different in TB patients in comparison to normal controls (both P < 0.05). MICA-A4 and MICA*012:01 alleles were positive associated (OR = 2.42, 95% CI: 1.69-3.87; OR = 3.41, 95% CI: 2.19-5.33, respectively, both Pc < 0.05) while MICA -A5 were inversely associated (OR = 0.59, 95%CI: 0.41-0.94, Pc < 0.05) with TB. Homozygote MICA*012:01/012:01 was observed to have significant risk effects on TB (OR = 4.76, 95% CI: 1.94-11.69, Pc0000-0001-5151-1853 < 0.05). Additionally, MICB*008 allele conduct a significant risk effect for TB (OR = 3.17, 95%CI: 1.80-5.61, Pc < 0.05). All the data showed that MIC polymorphism was associated with the variable susceptibility to TB in Chinese population.

[Determination of three volatile N-nitrosamine in drinking water by solid phase extraction and gas chromatography-triple quadrupole mass spectrometry with programmable temperature vaporizer-based large volume injection].

A method had been developed for the determination of three N-nitrosamines, namely, N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), and N-nitrosodiethylamine (NDEA) in drinking water by solid phase extraction (SPE) and gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS/MS) with programmable temperature vaporizer (PTV)-based large volume injection (LVI). The N-nitrosamine compounds were extracted from the water sample using a solid phase extraction (SPE) cartridge filled with coconut charcoal, and then eluted with 10 mL methylene chloride. The eluate was dried by anhydrous sodium sulfate and 10 µL was injected into the GC-MS/MS by PTV-LVI. The target compounds were detected by the multi-reaction monitoring (MRM) mode, and quantified with the external standard method. The results showed that the three compounds had good linearities in the range of 1-50 ng/L with correlation coefficients (r2) higher than 0.999. Drinking water samples were spiked with the target compounds at three concentration levels (10, 20, and 50 ng/L), and satisfactory recoveries (between 94.8% and 109%) and good reproductivities (relative standard deviation RSD<10%) were achieved. The limits of quantitation (LOQs) of the three N-nitrosamines were found to be in the range of 0.08-0.7 ng/L. The developed method was sensitive, accurate, convenient, and reliable for the determination of the three trace level N-nitrosamines in drinking water.

MICB*002 and MICB*014 protect against rheumatoid arthritis, whereas MICA*009 and MICA*A6 are associated with rheumatoid arthritis in a Hainan Han Chinese population.

AIM: Rheumatoid arthritis (RA) as an inflammatory autoimmune disease affects the synovial joints as well as other organs and tissues. Since aberrant expression of MIC molecules has been observed in RA patient, MIC genotypes might play certain roles in the development of RA. METHOD: To explore the association of MICA and MICB polymorphisms with RA in a Han Chinese population in Hainan Island, samples from 172 RA and 137 healthy controls were genotyped for MICA and MICB. RESULTS: Our results indicated that MICB*002 and MICB*014 were less frequent in RA patients than in controls (P = 0.000, 0.005) while there were higher percentages of RA patients carrying MICA*009 and MICA*A6 (P = 0.005). CONCLUSION: Different MIC variants might modulate the autoimmune reaction differently in RA disease and therefore serve as protective or risk factors.

Cellular retinaldehyde-binding protein-like (CRALBPL), a novel human Sec14p-like gene that is upregulated in human hepatocellular carcinomas, may be used as a marker for human hepatocellular carcinomas.

Sec14p-like lipid-binding domain (SEC14 domain) is an evolutionarily conserved protein domain often found in secretory proteins, such as Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p, and in lipid-regulated proteins, such as GTPase-activating proteins, guanine nucleotide exchange factors, and neurofibromin. We have cloned a novel human gene, cellular retinaldehyde-binding protein-like (CRALBPL), containing SEC14 domain from the cDNA library of human fetal brain. The RT-PCR expression pattern of 16 adult human tissues indicated that CRALBPL was only expressed in brain, while it was expressed in all of seven human carcinoma cell lines we used, especially in human gastric adenocarcinoma cell line, human rhabdomyoma cell line, human hepatocellular carcinoma (HCC) cell line, and human prostatic carcinoma cell line. Further, we found that CRALBPL has a remarkably more abundant RT-PCR expression pattern in human HCC cell lines than in normal human liver cell line, and the same result was gained when RT-PCR expression patterns between human HCC specimens and normal human liver specimens were compared. We also found that CRALBPL is located mainly in cytoplasm in human liver cell line L-02, which is consistent with the common function of Sec14p-like domain family. Our results show that CRALBPL may be used as a marker for human HCCs.

Characterization of AP3B2_v2, a novel splice variant of human AP3B2.

A novel splice variant of human AP3B2, named AP3B2_v2, was isolated through the large-scale sequencing analysis of a human fetal brain cDNA library. The AP3B2_v2 cDNA is 1171 bp in length. Sequence analysis revealed AP3B2_v2 missed 22 exons that existed in AP3B2_v1, leading to a different putative protein. The deduced proteins were 145 amino acids (designated as AP3B2_v2) and 1082 amino acids (AP3B2_v1) in length, sharing the C-terminal 145 amino acids. RT-PCR analysis showed that human AP3B2_v2 were expressed in several human adult tissues analyzed. The expression levels of AP3B2_v2 were relatively high in brain and testis. In contrast, low levels of expression were detected in kidney, pancreas, spleen, thymus, prostate, ovary and small intestine.

Sequence analysis of a human RhoGAP domain-containing gene and characterization of its expression in human multiple tissues.

Rho GTPase activating proteins (GAPs) stimulate the intrinsic GTP hydrolysis activity of Rho family proteins. Here we isolated a rhoGAP domain-containing protein gene with the same reading frame with ARHGAP19 gene, which has an ORF of 1485 bp encoding a putative protein of 494 amino acid residues with a predicted molecular mass of 55.806 kDa. Protein pattern analysis shows that it contains a bipartite nuclear localization signal (NLS) besides the rhoGAP domain, and it is consistent with the result of sub-cellular localization. ARHGAP19 is located in chromosome 10q24.1 and consists of 12 exons according to the Blastn result. Weak expression was detected in adult pancreas, spleen, thymus and ovary of the 16 adult tissues examined, while it had a more abundant expression pattern in eight important human fetal tissues. The expression pattern of ARHGAP19 shows it may have functions related to fetus development and gives us some clues on its probable functions in adult tissues.