Genotyping performance assessment of whole genome amplified DNA with respect to multiplexing level of assay and its period of storage

Whole genome amplification can faithfully amplify genomic DNA (gDNA) with minimal bias and substantial genome coverage. Whole genome amplified DNA (wgaDNA) has been tested to be workable for high-throughput genotyping arrays. However, issues about whether wgaDNA would decrease genotyping performance at increasing multiplexing levels and whether the storage period of wgaDNA would reduce genotyping performance have not been examined. Using the Sequenom MassARRAY iPLEX Gold assays, we investigated 174 single nucleotide polymorphisms for 3 groups of matched samples: group 1 of 20 gDNA samples, group 2 of 20 freshly prepared wgaDNA samples, and group 3 of 20 stored wgaDNA samples that had been kept frozen at -70°C for 18 months. MassARRAY is a medium-throughput genotyping platform with reaction chemistry different from those of high-throughput genotyping arrays. The results showed that genotyping performance (efficiency and accuracy) of freshly prepared wgaDNA was similar to that of gDNA at various multiplexing levels (17-plex, 21-plex, 28-plex and 36-plex) of the MassARRAY assays. However, compared with gDNA or freshly prepared wgaDNA, stored wgaDNA was found to give diminished genotyping performance (efficiency and accuracy) due to potentially inferior quality. Consequently, no matter whether gDNA or wgaDNA was used, better genotyping efficiency would tend to have better genotyping accuracy.     

An ORMOSIL-Containing Orthodontic Acrylic Resin with Concomitant Improvements in Antimicrobial and Fracture Toughness Properties

Global increase in patients seeking orthodontic treatment creates a demand for the use of acrylic resins in removable appliances and retainers. Orthodontic removable appliance wearers have a higher risk of oral infections that are caused by the formation of bacterial and fungal biofilms on the appliance surface. Here, we present the synthetic route for an antibacterial and antifungal organically-modified silicate (ORMOSIL) that has multiple methacryloloxy functionalities attached to a siloxane backbone (quaternary ammonium methacryloxy silicate, or QAMS). By dissolving the water-insoluble, rubbery ORMOSIL in methyl methacrylate, QAMS may be copolymerized with polymethyl methacrylate, and covalently incorporated in the pressure-processed acrylic resin. The latter demonstrated a predominantly contact-killing effect on Streptococcus mutans ATCC 36558 and Actinomyces naselundii ATCC 12104 biofilms, while inhibiting adhesion of Candida albicans ATCC 90028 on the acrylic surface. Apart from its favorable antimicrobial activities, QAMS-containing acrylic resins exhibited decreased water wettability and improved toughness, without adversely affecting the flexural strength and modulus, water sorption and solubility, when compared with QAMS-free acrylic resin. The covalently bound, antimicrobial orthodontic acrylic resin with improved toughness represents advancement over other experimental antimicrobial acrylic resin formulations, in its potential to simultaneously prevent oral infections during appliance wear, and improve the fracture resistance of those appliances.     

MetaCluster 4.0: a novel binning algorithm for NGS reads and huge number of species

Next-generation sequencing (NGS) technologies allow the sequencing of microbial communities directly from the environment without prior culturing. The output of environmental DNA sequencing consists of many reads from genomes of different unknown species, making the clustering together reads from the same (or similar) species (also known as binning) a crucial step. The difficulties of the binning problem are due to the following four factors: (1) the lack of reference genomes; (2) uneven abundance ratio of species; (3) short NGS reads; and (4) a large number of species (can be more than a hundred). None of the existing binning tools can handle all four factors. No tools, including both AbundanceBin and MetaCluster 3.0, have demonstrated reasonable performance on a sample with more than 20 species. In this article, we introduce MetaCluster 4.0, an unsupervised binning algorithm that can accurately (with about 80% precision and sensitivity in all cases and at least 90% in some cases) and efficiently bin short reads with varying abundance ratios and is able to handle datasets with 100 species. The novelty of MetaCluster 4.0 stems from solving a few important problems: how to divide reads into groups by a probabilistic approach, how to estimate the 4-mer distribution of each group, how to estimate the number of species, and how to modify MetaCluster 3.0 to handle a large number of species. We show that Meta Cluster 4.0 is effective for both simulated and real datasets. Supplementary Material is available at www.liebertonline.com/cmb.     

Cyclic 3′,5′-nucleotide phosphodiesterase: A continuous titrimetric assay

A direct and continuous assay for cyclic 3′,5′-nucleotide phosphodiesterase has been developed. This method is based on the fact that the phosphate group of adenosine 3′,5′-phosphate has one titratable species whereas that of 5′-adenosine monophosphate has two. Hydrolysis of cyclic AMP to 5′-AMP by phosphodiesterase is accompanied by a stoichiometric generation of protons. The rate of addition of an alkaline solution to the reaction mixture to maintain a constant pH with a pH stat is thus stoichiometrically related to the rate of cyclic AMP hydrolysis. A reaction producing 10 mμmoles of H⁺ or more per minute in 1.5 ml of reaction mixture is accurately measured by this technique. Duplicates are usually within 5% of each other. Results obtained by the titrimetric method correlate well with those obtained by conventional methods. This technique has been successfully used to assay phosphodiesterase of bovine brain in the purified as well as the crude stage.     

苍耳子对速发型过敏反应的抑制作用

苍耳子 70 %醇提物 (5 0 0 ,10 0 0mg kg)剂量依赖性抑制compound 4 8 80引起的小鼠过敏性休克、卵白蛋白所致大鼠被动皮肤过敏反应 ;在体外 ,苍耳子 (2 0 ,5 0 μg ml)浓度依赖性减少大鼠肥大细胞释放组胺和 β -氨基己糖酶 ;苍耳子 (5 0 0 ,10 0 0mg kg)对组胺或 5 -羟色胺所致大鼠皮肤反应无明显作用。上述结果表明苍耳子通过稳定肥大细胞膜 ,减少组胺等过敏介质的释放 ,抑制肥大细胞依赖性速发型过敏反应     

Waterlogging tolerance of Welsh onion (Allium fistulosum L.) enhanced by exogenous spermidine and spermine

Soil flooding is a seasonal factor that negatively affects plant performance and crop yields. In order to investigate the effects of spermidine (Spd) and spermine (Spm) on the waterlogging stress, it was checked that the content of relative water content (RWC), proline, chlorophyll and malondialdehyde (MDA), net photosynthesis, the rate of hydrogen peroxide (H₂O₂) and superoxide radicals (O₂^?) generation and the antioxidant enzyme activities of superoxide dismutase (SOD) (EC 1.15.1.1), catalase (CAT) (EC 1.11.1.6), ascorbate peroxidase (APX) (EC 1.11.1.11) and glutathione reductase (GR) (EC 1.6.4.2) in Welsh onion (Allium fistulosum L) plants. Pretreatment with 2 mM of Spd and Spm effectively maintained the balance of water content in plant leaves and roots under flooding stress. In addition, the data indicate that the protective role of proline should be considered minimal, as its accumulation was found to be inversely correlated with tolerance to stress; it also significantly retarded the loss of chlorophyll, enhanced photosynthesis, decreased the rate of O₂^? generation and H₂O₂ content, and prevented flooding-induced lipid peroxidation. Spd and Spm helped to maintain antioxidant enzyme activities under flooding; however, APX activity was found to increase slightly in response to Spm. The antioxidant system, an important component of the water-stress-protective mechanism, can be changed by PAs, which are able to moderate the radical scavenging system and to lessen in this way the oxidative stress. The results suggest that pretreatment with Spd and Spm prevents oxidative damage, and the protective effect of Spd was found to be greater than that of Spm.     

Still stable after 11 years: A Catharanthus roseus Hairy root line maintains inducible expression of anthranilate synthase

Hairy root cultures generated using Agrobacterium rhizogenes are an extensively investigated system for the overproduction of various secondary metabolite based pharmaceuticals and chemicals. This study demonstrated a transgenic Catharanthus roseus hairy root line carrying a feedback‐insensitive anthranilate synthase (AS) maintained chemical and genetic stability for 11 years. The AS gene was originally inserted in the hairy root genome under the control of a glucocorticoid inducible promoter. After 11 years continuous maintenance of this hairy root line, genomic PCR of the ASA gene showed the presence of ASA gene in the genome. The mRNA level of AS was induced to 52‐fold after feeding the inducer as compared to the uninduced control. The AS enzyme activity was 18.4 nmol/(min*mg) in the induced roots as compared to 2.1 nmol/(min*mg) in the control. In addition, the changes in terpenoid indole alkaloid concentrations after overexpressing AS were tracked over 11 years. The major alkaloid levels in induced and control roots at 11 years are comparable with the metabolite levels at 5 years. This study demonstrates the long term genetic and biochemical stability of hairy root lines, which has important implications for industrial scale applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:66–69, 2017     

OMSV enables accurate and comprehensive identification of large structural variations from nanochannel-based single-molecule optical maps

We present a new method, OMSV, for accurately and comprehensively identifying structural variations (SVs) from optical maps. OMSV detects both homozygous and heterozygous SVs, SVs of various types and sizes, and SVs with or without creating or destroying restriction sites. We show that OMSV has high sensitivity and specificity, with clear performance gains over the latest method. Applying OMSV to a human cell line, we identified hundreds of SVs >2 kbp, with 68 % of them missed by sequencing-based callers. Independent experimental validation confirmed the high accuracy of these SVs. The OMSV software is available at http://yiplab.cse.cuhk.edu.hk/omsv/.