Case study on user knowledge and design knowledge in product form design

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One-step fabrication of biocompatible chitosan-coated ZnS and ZnS:Mn2+ quantum dots via a γ-radiation route

Biocompatible chitosan-coated ZnS quantum dots [CS-ZnS QDs] and chitosan-coated ZnS:Mn2+ quantum dots [CS-ZnS:Mn2+ QDs] were successfully fabricated via a convenient one-step γ-radiation route. The as-obtained QDs were around 5 nm in diameter with excellent water-solubility. These QDs emitting strong visible blue or orange light under UV excitation were successfully used as labels for PANC-1 cells. The cell experiments revealed that CS-ZnS and CS-ZnS:Mn2+ QDs showed low cytotoxicity and good biocompatibility, which offered possibilities for further biomedical applications. Moreover, this convenient synthesis strategy could be extended to fabricate other nanoparticles coated with chitosan

Genome Expression Profile Analysis of the Immature Maize Embryo during Dedifferentiation

Maize is one of the most important cereal crops worldwide and one of the primary targets of genetic manipulation, which provides an excellent way to promote its production. However, the obvious difference of the dedifferentiation frequency of immature maize embryo among various genotypes indicates that its genetic transformation is dependence on genotype and immature embryo-derived undifferentiated cells. To identify important genes and metabolic pathways involved in forming of embryo-derived embryonic calli, in this study, DGE (differential gene expression) analysis was performed on stages I, II, and III of maize inbred line 18-599R and corresponding control during the process of immature embryo dedifferentiation. A total of ∼21 million cDNA tags were sequenced, and 4,849,453, 5,076,030, 4,931,339, and 5,130,573 clean tags were obtained in the libraries of the samples and the control, respectively. In comparison with the control, 251, 324 and 313 differentially expressed genes (DEGs) were identified in the three stages with more than five folds, respectively. Interestingly, it is revealed that all the DEGs are related to metabolism, cellular process, and signaling and information storage and processing functions. Particularly, the genes involved in amino acid and carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis and signal transduction mechanism have been significantly changed during the dedifferentiation. To our best knowledge, this study is the first genome-wide effort to investigate the transcriptional changes in dedifferentiation immature maize embryos and the identified DEGs can serve as a basis for further functional characterization

Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism

Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols

Genetic studies of hypertrophic cardiomyopathy in Singaporeans identify variants in TNNI3 and TNNT2 that are common in Chinese patients

Background - To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry. Methods - We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3,634), compared findings with additional populations and Caucasian HCM cohorts (n=6,179) and performed in vitro functional studies. Results - Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (Pathogenic (P)/Likely Pathogenic (LP):18%, p<0.0001) but an excess of variants of unknown significance (exVUS: 24%, p<0.0001), as compared to Caucasians (P/LP: 31%, exVUS: 7%). Two missense variants in thin filament encoding genes were commonly seen in Singaporean HCM (TNNI3:p.R79C, disease allele frequency (AF)=0.018; TNNT2:p.R286H, disease AF=0.022) and are enriched in Singaporean HCM when compared with Asian controls (TNNI3:p.R79C, Singaporean controls AF=0.0055, p=0.0057, gnomAD-East Asian (gnomAD-EA) AF=0.0062, p=0.0086; TNNT2:p.R286H, Singaporean controls AF=0.0017, p<0.0001, gnomAD-EA AF=0.0009, p<0.0001). Both these variants have conflicting annotations in ClinVar and are of low penetrance (TNNI3:p.R79C, 0.7%; TNNT2:p.R286H, 2.7%) but are predicted to be deleterious by computational tools. In population controls, TNNI3:p.R79C carriers had significantly thicker left ventricular walls compared to non-carriers while its etiological fraction is limited (0.70, 95% CI: 0.35-0.86) and thus TNNI3:p.R79C is considered a VUS. Mutant TNNT2:p.R286H iPSC-CMs show hypercontractility, increased metabolic requirements and cellular hypertrophy and the etiological fraction (0.93, 95% CI: 0.83-0.97) support the likely pathogenicity of TNNT2:p.R286H. Conclusions - As compared to Caucasians, Chinese HCM patients commonly have low penetrance risk alleles in TNNT2 or TNNI3 but exhibit few clinically actionable HCM variants overall. This highlights the need for greater study of HCM genetics in non-Caucasian populations

Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship

Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into (i) probabilistic, persistent and (ii) deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, which supports the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, a result underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila

Laboratory-Evolved Mutants of an Exogenous Global Regulator, IrrE from Deinococcus radiodurans, Enhance Stress Tolerances of Escherichia coli

The tolerance of cells toward different stresses is very important for industrial strains of microbes, but difficult to improve by the manipulation of single genes. Traditional methods for enhancing cellular tolerances are inefficient and time-consuming. Recently, approaches employing global transcriptional or translational engineering methods have been increasingly explored. We found that an exogenous global regulator, irrE from an extremely radiation-resistant bacterium, Deinococcus radiodurans, has the potential to act as a global regulator in Escherichia coli, and that laboratory-evolution might be applied to alter this regulator to elicit different phenotypes for E. coli.To extend the methodology for strain improvement and to obtain higher tolerances toward different stresses, we here describe an approach of engineering irrE gene in E. coli. An irrE library was constructed by randomly mutating the gene, and this library was then selected for tolerance to ethanol, butanol and acetate stresses. Several mutants showing significant tolerances were obtained and characterized. The tolerances of E. coli cells containing these mutants were enhanced 2 to 50-fold, based on cell growth tests using different concentrations of alcohols or acetate, and enhanced 10 to 100-fold based on ethanol or butanol shock experiments. Intracellular reactive oxygen species (ROS) assays showed that intracellular ROS levels were sharply reduced for cells containing the irrE mutants. Sequence analysis of the mutants revealed that the mutations distribute cross all three domains of the protein.To our knowledge, this is the first time that an exogenous global regulator has been artificially evolved to suit its new host. The successes suggest the possibility of improving tolerances of industrial strains by introducing and engineering exogenous global regulators, such as those from extremophiles. This new approach can be applied alone or in combination with other global methods, such as global transcriptional machinery engineering (gTME) for strain improvements

Oligomeric states in sodium ion-dependent regulation of cyanobacterial histidine kinase-2

IMI thanks Queen Mary University of London for a graduate teaching studentship. LW thanks the China Scholarship Council (CSC) and Queen Mary University of London for financial support. SP held a Leverhulme Trust early-career post-doctoral research fellowship. JN is grateful for the continued support of the JST CREST Grant Number JPMJCR13M4, Japan. JFA acknowledges the support of research grant F/07 476/AQ and fellowship EM-2015-068 of the Leverhulme Trust