Baseline trace metals in water and sediment of the Baleh River-a tropical river in Sarawak, Malaysia.

Quantitative indices are classically employed to evaluate the contamination status of metals with reference to the baseline concentrations. The baselines vary considerably across different geographical zones. It is imperative to determine the local geochemical baseline to evaluate the contamination status. No study has been done to establish the background concentrations in tropical rivers of this region. This paper reports the background concentrations of metals in water and sediment of the Baleh River, Sarawak, derived based on the statistical methods where the areas possibly disturbed are distinguished from the undisturbed area. The baseline levels of six elements in water determined were Al (0.34 mg/L), Fe (0.51 mg/L), Mn (0.12 mg/L), Cu (0.01 mg/L), Pb (0.03 mg/L), and Zn (0.05 mg/L). Arsenic and selenium were below the detection limit. For sediment, the background values were established according to statistical methods including (mean + 2σ), iterative 2σ, cumulative distribution frequency, interquartile, and calculation distribution function. The background values derived using the iterative 2σ algorithm and calculated distribution function were relatively lower. The baseline levels calculated were within the range reported in the literatures mainly from tropical and sub-tropical regions. The upper limits proposed for nine elements in sediment were Al (100,879 mg/kg), Cr (75.45 mg/kg), Cu (34.59 mg/kg), Fe (37,823 mg/kg), Mn (793 mg/kg), Ni (22.88 mg/kg), Pb (27.26 mg/kg), Zn (70.64 mg/kg), and Hg (0.33 mg/kg). Quantitative indices calculated suggest low risk of contamination at the Baleh River.

PITX2: a promising predictive biomarker of patients' prognosis and chemoradioresistance in esophageal squamous cell carcinoma.

The paired-like homeodomain transcription factor 2 (PITX2), a downstream effector of wnt/ß-catenin signaling, is well known to play critical role during normal embryonic development. However, the possible involvement of PITX2 in human tumorigenesis remains unclear. In this study, we extend its function in human esophageal squamous cell carcinoma (ESCC). The real-time PCR, Western blotting and immunohistochemistry (IHC) methods were applied to examine expression pattern of PITX2 in two different cohorts of ESCC cases treated with definitive chemoradiotherapy (CRT). Receiver operating characteristic (ROC) curve analysis was used to determine the cutoff point for PITX2 high expression in the training cohort. The ROC-derived cutoff point was then subjected to analyze the association of PITX2 expression with patients' survival and clinical characteristics in training and validation cohort, respectively. The expression level of PITX2 was significantly higher in ESCCs than that in normal esophageal mucosa. There was a positive correlation between PITX2 expression and clinical aggressiveness of ESCC. Importantly, high expression of PITX2 was observed more frequently in CRT resistant group than that in CRT effective group (p < 0.05). Furthermore, high expression of PITX2 was associated with poor disease-specific survival (p < 0.05) in ESCC. Then, the MTS, clonogenic survival fraction and cell apoptosis experiments showed that knockdown of PITX2 substantially increased ESCC cells sensitivity to ionizing radiation (IR) or cisplatin in vitro. Thus, the expression of PITX2, as detected by IHC, may be a useful tool for predicting CRT resistance and serves as an independent molecular marker for poor prognosis of ESCC patients treated with definite CRT.

[An indirect ELISA for the detection of Babesia caballi in equine animals].

OBJECTIVE: To clone and express BC48 gene of Babesia caballi, and to establish an indirect ELISA for the diagnosis of B. caballi in equine animals. METHOD: The genomic DNA of B. caballi was extracted from the infected donkey blood. BC48 gene was amplified by PCR. The PCR product was cloned into expression plasmid pET28a, and expressed in E. coli BL21 with IPTG induction. The recombinant protein was purified by Ni-NTA affinity chro-matography and was used as a diagnostic antigen to establish an indirect ELISA. The reaction conditions of the indirect ELISA were optimized. Specificity and sensitivity of this method were evaluated. RESULT: BC48 gene of B. caballi was 1 272 bp. The recombinant protein was expressed in E. coli BL21 as a soluble protein with a molecular weight of about M, 46 000 under induction of IPTG. The concentration of purified protein was 12.98 mg/ml. The best conditions were obtained for the ELISA when the antigen concentration was 65 microg/ml with the serum dilution of 1:80. The protein specifically reacted with serum from donkey infected by B. caballi, but did not react with serum from donkey infected by Theileria equi (B. equi). Both ELISA and microscopy were applied to examine 17 donkeys in the field, 3 were positive by ELISA and 2 were found parasite-positive, respectively. CONCLUSION: The indirect ELISA method may be used to detect B. caballi infection in equine animals.

[Construction of cDNA expression library of unfed female Haemaphysalis longicornis and immuno-screening].

OBJECTIVE: To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes. METHODS: Total RNA was isolated from unfed female ticks, mRNA was purified and a library of oligo (dT) -primed cDNA with added directional EcoR I /Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I /Hind III arms of the lambda SCREEN vector. Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids that were subcloned to E. coli BM25.8 were isolated and transformed into E. coli JM109. Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing. RESULTS: The recombinant phage DNA was packaged by using phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.8 x 10(6) pfu. Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4 x 10(9) pfu/ml. Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library. Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H. longicornis tropomyosin mRNA, Rhipicephalus annulatus unknown larval protein mRNA, chromosome 2R of Drosophila melanogaster, mitochondrial DNA of H. flava, clones HqL09 unkown mRNA and Hq05 mRNA of H. qinghaiensis, and myosin alkali light chain protein mRNA. CONCLUSION: The cDNA expression library from unfed female H. longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.