Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly

Here we use whole-genome de novo assembly of second-generation sequencing reads to map structural variation (SV) in an Asian genome and an African genome. Our approach identifies small- and intermediate-size homozygous variants (1-50 kb) including insertions, deletions, inversions and their precise breakpoints, and in contrast to other methods, can resolve complex rearrangements. In total, we identified 277,243 SVs ranging in length from 1-23 kb. Validation using computational and experimental methods suggests that we achieve overall <6% false-positive rate and <10% false-negative rate in genomic regions that can be assembled, which outperforms other methods. Analysis of the SVs in the genomes of 106 individuals sequenced as part of the 1000 Genomes Project suggests that SVs account for a greater fraction of the diversity between individuals than do single-nucleotide polymorphisms (SNPs). These findings demonstrate that whole-genome de novo assembly is a feasible approach to deriving more comprehensive maps of genetic variation.     

Plasma paraoxonase-1, oxidized low-density lipoprotein and lipid peroxidation levels in gout patients

Gout patients have a high incidence of atherosclerotic coronary heart disease. Low serum paraoxonase (PON) activity is considered a risk factor for atherosclerosis. The relationships among paraoxonase-1 (PON1) activity, oxidative stress parameters, and atherosclerosis in gout is not known. Therefore, we determined the plasma levels of malondialdehyde (MDA), oxidized low-density lipoprotein (Ox-LDL), and activities of PON1/superoxide dismutase (SOD) activities in 49 gout patients (mean age 44.2 ± 7.0 years) and 42 healthy, age-matched controls (mean age 45.0 ± 9.3 years). PON1 was measured spectrophotometrically, MDA by thiobarbituric acid method, SOD by Griess reaction, and Ox-LDL by sandwich ELISA. Lipid and other biochemical parameters were determined by routine laboratory methods. In gout patients, PON1/SOD activities and MDA/Ox-LDL levels were 131.3 ± 25.3/75.3 ± 28.9 kU l⁻¹ and 6.12 ± 1.67 nmol ml⁻¹/690.1 ± 180.2 μg l⁻¹, respectively. In controls, these were 172.5 ± 27.8/94.0 ± 26.3 kU l⁻¹ and 4.10 ± 1.25 nmol ml⁻¹/452.3 ± 152.1 μg l⁻¹, respectively. Thus, in gout patients, there was a significant decrease in PON1 (P < 0.01) and SOD (P < 0.05) activities, and an increase in MDA (P < 0.01) and Ox-LDL (P < 0.01) levels compared with controls. PON1 activity correlated positively with SOD (P < 0.05), and negatively with MDA (P < 0.01) and Ox-LDL (P < 0.01). These results suggest that gout patients were in a state of oxidative stress and the protective effects of HDL against atherosclerosis maybe dependent on PON1 activity. These findings may explain in part the reported increase in cardiovascular mortality in gout patients.     

IL-1 polymorphisms in children with peptic symptoms in South China

Background: Polymorphisms of IL‐1 gene cluster are reported to be associated with histological changes and IL‐1β expression in the gastric mucosa in adults, especially in Helicobacter pylori–infected subjects. As H. pylori infecting adults and children own different virulence genotypes, the aim of this study was to investigate whether IL‐1 polymorphisms are risk factors in young children in South China. Materials and Methods: A total of 128 children with peptic symptoms were enrolled in this study. Polymorphisms of IL‐1B‐511 and IL‐1B‐31 were identified by dual fluorescence PCR. Variable number of tandem repeat region in IL‐1RN was detected by conventional PCR and IL‐1β mRNA expression by real‐time PCR ddCT assay. Results: IL‐1B‐31T and IL‐1B‐511C were completely linked in this study. Significant differences of IL‐1B‐511/‐31 genotypes were observed among different clinical outcomes (p = .001). The IL‐1B‐511TT/‐31CC was mostly found in the moderate gastritis and the above (severe gastritis or gastric ulcer) groups, with percentage of 60.7%. While no association was observed between IL‐1RN genotypes and the gastric mucosal histological changes (p = .128). Also no relationships were found between IL‐1 polymorphisms and H. pylori infection or gastric mucosal IL‐1β mRNA expression level. Conclusion: Children with IL‐1B‐511TT/‐31CC may have a risk to develop relatively severe gastric mucosal histological changes in South China.     

A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin

Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels.     

A comparative study of DNA binding and cell cycle phase perturbation by the dinuclear complex of Cd(II) with the condensation product of 2-acetylpyridine and malonic acid dihydrazide N',N'(2) -bis[(1E)-1-(2-pyridyl)ethylidene]propanedihydrazide

Organometallic Cd(II) compounds have recently attracted attention for their anticancer activity. The interaction of the dinuclear complex of Cd(II) with the condensation product of 2-acetylpyridine and malonic acid dihydrazide, N',N'(2) -bis[(1E)-1-(2-pyridyl)ethylidene]propanedihydrazide (Cd(II)H(2) L), with calf thymus DNA (CT-DNA) was monitored by blue shift in UV-vis spectra of the complex. The binding constant of Cd(II)H(2) L complex with CT-DNA was determined (K(B) = 1.8 × 10(4) M(-1) ) and was indicative of minor groove binding. Agarose gel electrophoretic changes in mobility of supercoiled and circular forms of pBR322 and pUC18 plasmids in the presence of the complex suggest that conformational changes in the plasmids occur upon binding of the Cd(II)H(2) L complex. The Cd(II)H(2) L complex induced perturbation of the cell cycle phase distribution and an increase in the percentage of cells in the sub-G1 phase of human cervical cancer HeLa cell line and murine melanoma B16 cell line. Immunoblotting analysis showed the overexpression of Bcl-2 protein with the Cd(II)H(2) L complex.     

Screening for group B streptococcus: a private laboratory experience

We examined group B streptococcus (GBS) isolates colonizing women at the 35-37 weeks of pregnancy. A total of 257 group B streptococcus (GBS) isolates for serotyped using direct agglutination with a set of commercially available antisera (Ia, Ib, II, III, IV, and V) and tested for susceptibility to antimicrobials (penicillin, macrolides, lincosamides, fluoroquinolones and tetracyclines). Fourteen isolates could not be serotyped with the antisera set used in the study. Serotype III was the predominant serotype (33%), followed by serotypes V (23%), and Ia (20%). Whereas all isolates were susceptible to penicillin, the rates of susceptibility to the other antimicrobials tested were the following: 91% for ofloxacin, 80% for clindamycin, 77% for erythromycin, and 4% for tetracycline. More than half (67%) of the macrolide resistant isolates belonged to serotypes V and III. A systematic surveillance of the autochthonous GBS serotypes, performed at the level of laboratories processing a high number of human specimens, is mandatory for strengthening the national epidemiological GBS surveillance. While penicillin remains the drug of choice for intrapartum prophylaxis, the resistance of autochthonous GBS isolates to other antibiotics should be actively monitored.     

Continuous preparation of (S)-3-hydroxy-3-phenylpropionate by asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> CGMCC No.2266 in a membrane reactor

In this study, (S)-3-hydroxy-3-phenylpropionate was prepared continuously by coupling microbial transformation and membrane separation. The effect of several factors on membrane flux, reactor capacity, and reaction conversion were investigated. A kinetic model of the continuous reduction process was also developed. The appropriate molecular weight cut-off of the ultrafiltration membrane was 30 kDa. The reactor capacity reached a maximum of 0.136/h at a biomass concentration and membrane flux of 86 g/L (dry weight/reaction volume) and 20 mL/h, respectively. The (S)-3-hydroxy-3-phenylpropionate yield was 3.68 mmol/L/day after continuous reduction over seven days. The enantiometric excess of (S)-3-hydroxy-3-phenylpropionate reached above 99.5%. The kinetic constants of continuous reduction were as follows: r _m = 3.00 × 10⁻³ mol/L/h, k _cat = 3.49 × 10⁻⁴ mol/L/h, k ₁ = 3.09 × 10⁻² mol/L, and k ₂ = 5.00 × 10⁻⁷ mol/L. The kinetic model was in good agreement with the experimental data obtained during continuous reduction. Compared with batch reduction, continuous reduction can significantly improve the catalytic efficiency of microbial cells and increase the reactor capacity.     

Enzymatic transformation of 2-O-α-D-glucopyranosyl-L-ascorbic acid by α-cyclodextrin glucanotransferase from recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The study aimed to produce 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) via the transglycosylation reaction by α-cyclodextrin glucanotransferase (α- CGTase) from recombinant Escherichia coli with L-ascorbic acid (AA) and β-cyclodextrin (β-CD) as the substrates. Liquid chromatography-tandem mass spectrometry analysis was conducted for AA-2G identification, and the glucoamylase treatment was carried out to produce AA-2G from AA-2-oilgosaccharides. The optimal temperature and pH for the enzymatic AA-2G production were 37°C and 5.5, respectively, and the optimal α-CGTase concentration and substrate mass ratio (AA:β-CD) for AA-2G synthesis were 160 U/mL and 1:1, respectively. At these optimal process conditions, maximal AA-2G production reached 13 g/L. This is the first report regarding the process optimization of enzymatic AA-2G production by α-CGTase from recombinant E. coli. The results may be useful for the industrial scale production of AA-2G.     

Proteomic analysis of sericin in <Emphasis Type="Italic">Bombyx mori</Emphasis> cocoons

Cocoon sericin plays an important role in the reeling of silk and serves as a valuable biomaterial in the field of biomedicine, skincare, and food industries; however, knowledge about cocoon sericin proteins has been limited. For a comprehensive study on sericin, cocoons of eight varieties of silkworm of different geographic origin and with varied cocoon color were analyzed utilizing proteomics and bioinformatics approaches. The electrophoresis pattern demonstrated some common protein bands for all silkworm varieties and distinctive protein bands for some of those examined in the present study. The Ser2 protein, a new Ser3 protein, and four other novel sericin proteins were identified in cocoons for the first time. Products of both Ser1 and Ser3 genes appear to be ubiquitous in the cocoon shell of Bombyx mori. In addition, cocoons with especially high-reelability produced by the mutant strain B84 had an unique protein product of the Ser2 gene, indicating that the protein may play an important role in cocoon reelability. A series of sequence conflicts and post-translational modifications (PTMs) were also revealed in sericin proteins. Lipid modifications of sericin proteins, which promote waterproofing of the cocoon shell, were observed. Further, hydroxylation was identified, which provided evidence for intermolecular bonds among neighboring molecules of sericin as found in collagens. The sericin proteome data obtained from this study illuminated the molecular complexity of cocoon sericin and contributed to our understanding of the properties of sericin in filature and biomaterials.     

Mixotrophy in the newly described phototrophic dinoflagellate Woloszynskia cincta from western Korean waters: feeding mechanism, prey species and effect of prey concentration

Woloszynskia species are dinoflagellates in the order Suessiales inhabiting marine or freshwater environments; their ecophysiology has not been well investigated, in particular, their trophic modes have yet to be elucidated. Previous studies have reported that all Woloszynskia species are photosynthetic, although their mixotrophic abilities have not been explored. We isolated a dinoflagellate from coastal waters in western Korea and established clonal cultures of this dinoflagellate. On the basis of morphology and analyses of the small/large subunit rRNA gene (GenBank accession number=FR690459), we identified this dinoflagellate as Woloszynskia cincta. We further established that this dinoflagellate is a mixotrophic species. We found that W. cincta fed on algal prey using a peduncle. Among the diverse prey provided, W. cincta ingested those algal species that had equivalent spherical diameters (ESDs) ≤12.6 μm, exceptions being the diatom Skeletonema costatum and the dinoflagellate Prorocentrum minimum. However, W. cincta did not feed on larger algal species that had ESDs≥15 μm. The specific growth rates for W. cincta increased continuously with increasing mean prey concentration before saturating at a concentration of ca. 134 ng C/ml (1,340 cells/ml) when Heterosigma akashiwo was used as food. The maximum specific growth rate (i.e. mixotrophic growth) of W. cincta feeding on H. akashiwo was 0.499 d(-1) at 20 °C under illumination of 20 μE/m(2) /s on a 14:10 h light-dark cycle, whereas its growth rate (i.e. phototrophic growth) under the same light conditions without added prey was 0.040 d(-1). The maximum ingestion and clearance rates of W. cincta feeding on H. akashiwo were 0.49 ng C/grazer/d (4.9 cells/grazer/d) and 1.9 μl/grazer/h, respectively. The calculated grazing coefficients for W. cincta on co-occurring H. akashiwo were up to 1.1 d(-1). The results of the present study suggest that grazing by W. cincta can have a potentially considerable impact on prey algal populations.