Isolation of axenic cyanobacterium and the promoting effect of associated bacterium on axenic cyanobacterium.

BACKGROUND: Harmful cyanobacterial blooms have attracted wide attention all over the world as they cause water quality deterioration and ecosystem health issues. Microcystis aeruginosa associated with a large number of bacteria is one of the most common and widespread bloom-forming cyanobacteria that secret toxins. These associated bacteria are considered to benefit from organic substrates released by the cyanobacterium. In order to avoid the influence of associated heterotrophic bacteria on the target cyanobacteria for physiological and molecular studies, it is urgent to obtain an axenic M. aeruginosa culture and further investigate the specific interaction between the heterotroph and the cyanobacterium. RESULTS: A traditional and reliable method based on solid-liquid alternate cultivation was carried out to purify the xenic cyanobacterium M. aeruginosa FACHB-905. On the basis of 16S rDNA gene sequences, two associated bacteria named strain B905-1 and strain B905-2, were identified as Pannonibacter sp. and Chryseobacterium sp. with a 99 and 97% similarity value, respectively. The axenic M. aeruginosa FACHB-905A (Microcystis 905A) was not able to form colonies on BG11 agar medium without the addition of strain B905-1, while it grew well in BG11 liquid medium. Although the presence of B905-1 was not indispensable for the growth of Microcystis 905A, B905-1 had a positive effect on promoting the growth of Microcystis 905A. CONCLUSIONS: The associated bacteria were eliminated by solid-liquid alternate cultivation method and the axenic Microcystis 905A was successfully purified. The associated bacterium B905-1 has the potentiality to promote the growth of Microcystis 905A. Moreover, the purification technique for cyanobacteria described in this study is potentially applicable to a wider range of unicellular cyanobacteria.

Inhibitory effect of piperlonguminine/ dihydropiperlonguminine on the production of amyloid beta and APP in SK-N-SH cells.

The study was undertaken to explore whether piperlonguminine/dihydropiperlonguminine could inhibit the production of amyloidbeta (Abeta) in human neuroblastoma cells (SK-N-SH) and to examine the underlying mechanism of this effect. Piperlonguminine/dihydropiperlonguminine components (1:0.8) were extracted from Futokadsura stem, and then used to treat SK-N-SH cells at three different concentrations: 3.13 microg/ml, 6.25 microg/ml and 12.50 microg/ml. Subsequently, the production of Abeta42 and Abeta40 were measured by Western blot analysis and enzyme linked immunosorbent assay (ELISA). On the other hand, the expressions of amyloid precursor protein (APP), Notch1 (Notch intracellular domain) and beta-site amyloid precursor protein cleavage enzyme (BACE-1) were also examined by Western blot assay. The activities of beta-secretase and gamma-secretase were detected at the same time. Furthermore, Abeta42 level was detected by immunocytochemistry staining. We demonstrated that the treatment of piperlonguminine/dihydropiperlonguminine could significantly decrease the levels of APP, Abeta42 and Abeta40 peptide in SK-N-SH cells, despite the fact that the activities of beta-secretase and gamma-secretase were not affected significantly. These data suggest that piperlonguminine/dihydropiperlonguminine components could significantly inhibit the level of APP, Abeta42 and Abeta40 peptide without affecting the activity of beta-secretase and gamma-secretase in SK-N-SH cells.

[DRB1 * 1454: the common allele of human leukocyte antigen-DRB1 * 14 in Chinese Han populations].

OBJECTIVE: To investigate and compare the distribution of HLA-DRB1 * 14 alleles between the southern and northern Chinese Han populations. METHODS: Human leukocyte antigen (HLA)-DRB1 alleles of 436 southern and 713 northern Chinese Han bone marrow volunteers were genotyped by polymerase chain reaction (PCR)-sequence-based-typing (SBT) method, among them the DRB1 * 1401/1439/1454 ambiguous allele pairs were identified using DRB1 * 14 high-resolution PCR-sequence specific primer (SSP) kits. Also, the clinic samples previously reported as DRB1 * 1401 were re-genotyped using the same PCR-SSP kits. The allelic distribution of DRB1 * 14 in southern and northern Chinese Han population were compared by chi-square test method. RESULTS: Eighty-one ambiguous allele pairs of DRB1 * 1401/1439/1454 and 54 clinic samples previously reported as DRB1 * 1401 were all identified as DRB1 * 1454. Among the 436 Southern Han donors, six subtypes of DRB1 * 14 allele were observed including DRB1 * 1454 (69.57%), DRB1 * 1402 (1.45%), DRB1* 1403 (1.45%), DRB1 * 1404 (4.35%), DRB1 * 1405 (20.29%) and DRB1 * 1407 (2.90%). In the 713 northern Han donors, a total of seven subtypes were observed including DRB1 * 1454 (35.48%), DRB1 * 1403 (12.90%), DRB1 * 1404 (6.45%), DRB1 * 1405 (37.63%), DRB1 * 1407 (4.30%), DRB1 * 1411 (1.08%) and DRB1 * 1412 (2.15%). CONCLUSION: DRB1 * 1454 and DRB1 * 1405 were the most common alleles of HLA-DRB1 * 14 in Chinese Han populations. The distribution of HLA-DRB1 * 14 differ significantly between the southern and northern Chinese Han population, while DRB1 * 1405 showed similar distribution pattern in the two populations but DRB1 * 1454 had higher frequency in southern than in northern Chinese Han population.

[Clinical investigation on the compliance and the validity of ventilator bundle].

OBJECTIVE: To investigate the compliance of ventilator bundle implementation and its preventive effect on ventilator associated pneumonia (VAP). METHODS: A before and after design was used in this single center study. Patients aged from 18 to 80 years, with mechanical ventilation (MV) duration over 48 hours were recruited during 1 year before (control group) and 2 years after bundle implementation (intervention group). Measurements included the rate of successful ventilator bundle implementation in intervention group, incidence of VAP, duration of MV and mortality within 28 days in both groups. RESULTS: A total number of 237 patients, including 71 patients in control arm and 166 patients in intervention arm, were recruited in this study. There was no statistical significance in ratio of sex, mean age, category of diseases or mean acute physiology and chronic health evaluation II (APACHE II) score between two groups (all P>0.05). Significant changes were not found in MV duration [(5.9+/-5.6) days vs. (5.2+/-6.1) days], incidence of VAP (21.1% vs. 20.5%) and mortality within 28 days (16.9% vs. 19.8%) between control and intervention group as well. In intervention group, 57 of 166 (34.3%) patients were successfully implemented all of four ventilator bundle items. The successful rate of ventilator bundle implementation were 62.5% (35/56), 22.1% (21/95) and 6.7% (1/15) in patients received MV duration < or =3 days, 4-7 days and >7 days respectively. Among the four items of the bundle, head of bed elevation > or =30 degree angle had the lowest successful rate [43.4% (72/166)]. But it was much better in the implementation of daily wake-up plus weaning, prevention of peptic ulcer and prevention of deep vein thrombosis formation [92.2% (153/166), 88.0% (146/166) and 83.1% (138/166) respectively]. CONCLUSION: The poor compliance of ventilator bundle is an important factor in impacting the efficacy of ventilator bundle.

[Construction and expression of vector with aroA-in gene and its transformation in tobacco].

Inteins are intervening protein sequences that undergo self-excision from precursor protein with concomitant joining of flanking sequences. Here, we demonstrated intein cis-splicing in Nicotiana tabacum nuclear genomes by using artificial cis Ssp DnaB and Rma DnaB intein. We want to test whether protein splicing can occur in higher eucaryotic cell,which would play an important role in transgene containment in transgenic plants. Glyphosate-resistant Salmonella typhimurium aroA gene was divided at position 235/236 aa within EPSPS by inverse PCR from pLEPSPS. Amplified gene products with artificial cis-Ssp DnaB/Rma DnaB intein and split-Ssp DnaB/Rma DnaB intein were inserted at position 235 of EPSPS respectively to construct plasmid pLEBC, pLERC, pLEBT and pLERT. Above four aroA-In gene fusions were ligated into pET-32 to obtain E. coli expression vectors termed pETLEBC, pETLEBT, pETLERC and pETLERT. E. coli DE3 cells containing individual recombinant plasmids described above were induced by IPTG to produce corresponding protein products. Detectable spliced EPSPS and unspliced precursor demonstrated that splicing occurred in bacteria. aroA-cis SSp DnaB and aroA-cis Rma DnaB were ligated into Agrobacterium tumefaciens binary vector pLYM. Then A. tumefaciens containing EPSPS-(cis) intein cassettes were used for leaf disk transformation in N. tabacum. Integration of aroA-In gene into plant genome was confirmed by genomic PCR analyses. To verify the expression of fusion genes at transcriptional level, RT-PCR analyses were performed and the expected products were identified. These results suggested that plant cells support expression of S. typhimurium aroA-In fusion gene in nulear genomes. Thus,we speculated the existence of protein-splicing activity in plant cells. This opens the possibility of applying intein trans-splicing technique to reduce/prevent gene transfer by way of pollen in transgenic plants.

Understanding the recurrent large-scale green tide in the Yellow Sea: temporal and spatial correlations between multiple geographical, aquacultural and biological factors.

The coast of Jiangsu Province in China - where Ulva prolifera has always been firstly spotted before developing into green tides - is uniquely characterized by a huge intertidal radial mudflat. Results showed that: (1) propagules of U. prolifera have been consistently present in seawater and sediments of this mudflat and varied with locations and seasons; (2) over 50,000 tons of fermented chicken manure have been applied annually from March to May in coastal animal aquaculture ponds and thereafter the waste water has been discharged into the radial mudflat intensifying eutrophication; and (3) free-floating U. prolifera could be stranded in any floating infrastructures in coastal waters including large scale Porphyra farming rafts. For a truly integrated management of the coastal zone, reduction in nutrient inputs, and control of the effluents of the coastal pond systems, are needed to control eutrophication and prevent green tides in the future.

Determination of 17beta-oestradiol by fluorescence immunoassay with streptavidin-conjugated quantum dots as label.

A sensitive and selective method for the determination of 17beta-oestradiol by fluorescence immunoassay (FIA) was established on the basis of quantum dots (QDs) as label. The complex of biotin-labelled anti-rabbit IgG and strepavidin conjugated by quantum dots (QD-SA) was regarded as a probe in this system and the strepavidin-biotin system as signal amplification system. After optimising the conditions of the immunoreaction, such as the concentration of the reagent and the pH of the buffer solution, the linear range and the limit of detection of 17beta-oestradiol were 0.01-10,000ngml(-1) and 0.00542ngml(-1), respectively. This method was applied to determine oestradiol in water samples, with the percent recoveries in the range of 86-113%.

[Application value of allele frequencies in direct identification of ambiguous HLA genotypes].

This study was aimed to investigate the application value of allele frequencies in direct identification of the ambiguous HLA genotypes. The HLA-A, HLA-B and HLADRB1 loci in 658 Chinese Han donor were detected by PCR-SBT method, the ambiguous genotyping samples were identified by using high resolution PCR-SSP and heterozygous ambiguity resolution primers (HAPRs) methods. The relative probability of true genotypes was calculated by using allele frequencies and was compared with true results. The results indicated that the relative probability of true genotype > 95% in 220 HLA-A ambiguous samples, 238 HLA-B ambiguous samples and 107 HLA-DRB1 ambiguous samples were 99.5% (221/222), 95.8% (228/238) and 97.7% (104/107) respectively. As compared with phenotyping results detected by PCR-SSP and HARP methods, the matching ratios for HLA-A, HLA-B and HLA-DRB1 loci were 100% (222/222), 99.6% (237/238) and 99.1% (106/107) respectively, while the mismatch genotypes were observed only in B*3501/5501 and DRB1*1241/1504, the relative probability of them were 40.3% and 2.1% respectively. It is concluded that the detection method using allele frequencies to directly identify the ambiguous HLA genotypes in large scale PCR-SBT genotyping of donors not only can give higher accurate and reliable results, but also is a simple, rapid and cost-saving method. This method has to be used with great care in the identify-test of patient-donor pair before the transplantation.