Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli

λ-Red system-based recombinogenic engineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. Here, we report the use of a single selectable marker to enhance the usefulness of this approach. The strategy is to utilize the thymidylate synthase A (thyA) gene, which encodes an enzyme involved in the synthesis of thymidine 5′-triphosphate, for both positive and negative selection. With this approach, we successfully created point mutations in plasmid and bacterial artificial chromosome (BAC) DNA containing the mouse Col10a1 gene. The results showed that the thyA selection system is highly efficient and accurate, giving an average of >90% selection efficiency. This selection system produces DNA that is free from permanent integration of unwanted sequences, thus allowing unlimited rounds of modifications if required

Macrophage migration inhibitory factor stimulated by Helicobacter pylori increases proliferation of gastric epithelial cells

AIM: Helicobacter pylori (H pylori) is associated with increased gastric inflammatory and epithelial expression of macrophage migration inhibitory factor (MIF) and gastric epithelial cell proliferation. This study aimed at determining whether H pylori directly stimulates release of MIF in monocytes, whether the cag pathogenicity island (PAI) is involved for this function, and whether MIF stimulated by H pylori increases gastric epithelial cell proliferation in vitro. METHODS: A cytotoxic wild-type H pylori strain (TN2)and its three isogenic mutants (TN2△cag, TN2△cagA and TN2△cagE) were co-cultured with cells of a human monocyte cell line, THP-1, for 24 h at different organism/cell ratios. MIF in the supernatants was measured by an ELISA. Cells of a human gastric cancer cell line, MKN45, were then co-cultured with the supernatants, with and without monoclonal anti-MIF antibody for 24 h. The cells were further incubated for 12 h after addition of (3)H-thymidine, and the levels of incorporation of (3)H-thymidine were measured with a liquid scintillation counter. RESULTS: The wild-type strain and the isogenic mutants, TN2△cagA and TN2△cagE, increased MIF release at organism/cell ratios of 200/1 and 400/1, but not at the ratios of 50/1 and 100/1. However, the mutant TN2△cag did not increase the release of MIF at any of the four ratios. (3)H-thymidine readings for MKN-45 cells were significantly increased with supernatants derived from the wild-type strain and the mutants TN2△cagA and TN2△cagE, but not from the mutant TN2△cag. Moreover, in the presence of monoclonal anti-MIF antibody, the stimulatory effects of the wild-type strain on cell proliferation disappeared. CONCLUSION: H pylori stimulates MIF release in monocytes, likely through its cag PAI, but not related to cagA or cagE. H pylori-stimulated monocyte culture supernatant increases gastric cell proliferation, which is blocked by anti-MIF antibody, suggesting that MIF plays an important role in H pylori-induced gastric epithelial cell proliferation

Star Poly( N

New star poly(N-isopropylacrylamide)-b-polyhedral oligomeric silsesquioxane (PNIPAm-b-POSS) copolymers were synthesized from octa-azido functionalized POSS (N3-POSS) and alkyne-PNIPAm, which was prepared using an alkyne-functionalized atom transfer radical polymerization (ATRP) initiator (propargyl 2-bromo-2-methylpropionamide), via click chemistry. These star PNIPAm-b-POSS copolymers undergo a sharp coil-globule transition in water at above 32°C changing from a hydrophilic state below this temperature to a hydrophobic state above it, which is similar to linear PNIPAm homopolymers. More interestingly, we found that these star polymers exhibited strong blue photoluminescence in water above a lower critical solution temperature (LCST). This photoluminescence was likely due to the constrained geometric freedom and relatively rigid structure caused by intramolecular hydrogen bonding within the star PNIPAm polymers, which exhibit an intrinsic fluorescent behavior

Interest-driven creator theory: towards a theory of learning design for Asia in the twenty-first century

Asian education is known for its examination-driven orientation, with the downsides of distorting the processes of learning and teaching, diminishing students’ interest in learning, and failing to nurture twenty-first century competencies among students. As a group of Asian researchers, we have been developing Interest-Driven Creator (IDC) Theory, a design theory based on three anchored concepts, namely interest, creation, and habit. Each of these anchored concepts is represented by a loop composed of three components. In the interest loop, the three components are triggering, immersing, and extending. The components of the creation loop are imitating, combining, and staging. The habit loop consists of cuing environment, routine, and harmony. These three loops are interconnected in various ways, with their characteristics revealed by the design process. We hypothesize that technology-supported learning activities that are designed with reference to IDC Theory will enable students to develop interest in learning, be immersed in the creation process, and, by repeating this process in their daily routines, strengthen habits of creation. Furthermore, students will excel in learning performance, develop twenty-first century competencies, and become lifelong interest-driven creators. To sharpen our understanding and further the development of the theory, we need more discussion and collaborative efforts in the community. Hypotheses arising from this theory can be tested, revised, or refined by setting up and investigating IDC Theory-based experimental sites. By disseminating the framework, foundations, and practices to the various countries and regions of Asia, we hope that it will bring about compelling examples and hence a form of quality education for the twenty-first century, which is an alternative to the examination-driven education system. In this paper, we present an overall introduction to IDC Theory and its history, and discuss some of the steps for advancing it in the future

Uncertainty-based Traffic Accident Anticipation with Spatio-Temporal Relational Learning

Traffic accident anticipation aims to predict accidents from dashcam videos as early as possible, which is critical to safety-guaranteed self-driving systems. With cluttered traffic scenes and limited visual cues, it is of great challenge to predict how long there will be an accident from early observed frames. Most existing approaches are developed to learn features of accident-relevant agents for accident anticipation, while ignoring the features of their spatial and temporal relations. Besides, current deterministic deep neural networks could be overconfident in false predictions, leading to high risk of traffic accidents caused by self-driving systems. In this paper, we propose an uncertainty-based accident anticipation model with spatio-temporal relational learning. It sequentially predicts the probability of traffic accident occurrence with dashcam videos. Specifically, we propose to take advantage of graph convolution and recurrent networks for relational feature learning, and leverage Bayesian neural networks to address the intrinsic variability of latent relational representations. The derived uncertainty-based ranking loss is found to significantly boost model performance by improving the quality of relational features. In addition, we collect a new Car Crash Dataset (CCD) for traffic accident anticipation which contains environmental attributes and accident reasons annotations. Experimental results on both public and the newly-compiled datasets show state-of-the-art performance of our model. Our code and CCD dataset are available at https://github.com/Cogito2012/UString.Comment: Accepted by ACM MM 202

The Yuan-Tseh Lee Array for Microwave Background Anisotropy

The Yuan-Tseh Lee Array for Microwave Background Anisotropy (AMiBA) is the first interferometer dedicated to studying the cosmic microwave background (CMB) radiation at 3mm wavelength. The choice of 3mm was made to minimize the contributions from foreground synchrotron radiation and Galactic dust emission. The initial configuration of seven 0.6m telescopes mounted on a 6-m hexapod platform was dedicated in October 2006 on Mauna Loa, Hawaii. Scientific operations began with the detection of a number of clusters of galaxies via the thermal Sunyaev-Zel'dovich effect. We compare our data with Subaru weak lensing data in order to study the structure of dark matter. We also compare our data with X-ray data in order to derive the Hubble constant.Comment: accepted for publication in ApJ (13 pages, 7 figures); a version with high resolution figures available at http://www.asiaa.sinica.edu.tw/~keiichi/upfiles/AMiBA7/pho_highreso.pd

Wolfberry genomes and the evolution of Lycium (Solanaceae)

AbstractWolfberry Lycium, an economically important genus of the Solanaceae family, contains approximately 80 species and shows a fragmented distribution pattern among the Northern and Southern Hemispheres. Although several herbaceous species of Solanaceae have been subjected to genome sequencing, thus far, no genome sequences of woody representatives have been available. Here, we sequenced the genomes of 13 perennial woody species of Lycium, with a focus on Lycium barbarum. Integration with other genomes provides clear evidence supporting a whole-genome triplication (WGT) event shared by all hitherto sequenced solanaceous plants, which occurred shortly after the divergence of Solanaceae and Convolvulaceae. We identified new gene families and gene family expansions and contractions that first appeared in Solanaceae. Based on the identification of self-incompatibility related-gene families, we inferred that hybridization hotspots are enriched for genes that might be functioning in gametophytic self-incompatibility pathways in wolfberry. Extremely low expression of LOCULE NUBER (LC) and COLORLESS NON-RIPENING (CNR) orthologous genes during Lycium fruit development and ripening processes suggests functional diversification of these two genes between Lycium and tomato. The existence of additional flowering locus C-like MADS-box genes might correlate with the perennial flowering cycle of Lycium. Differential gene expression involved in the lignin biosynthetic pathway between Lycium and tomato likely illustrates woody and herbaceous differentiation. We also provide evidence that Lycium migrated from Africa into Asia, and subsequently from Asia into North America. Our results provide functional insights into Solanaceae origins, evolution and diversification.</jats:p

Regulation of High-Temperature Stress Response by Small RNAs

Temperature extremes constitute one of the most common environmental stresses that adversely affect the growth and development of plants. Transcriptional regulation of temperature stress responses, particularly involving protein-coding gene networks, has been intensively studied in recent years. High-throughput sequencing technologies enabled the detection of a great number of small RNAs that have been found to change during and following temperature stress. The precise molecular action of some of these has been elucidated in detail. In the present chapter, we summarize the current understanding of small RNA-mediated modulation of high- temperature stress-regulatory pathways including basal stress responses, acclimation, and thermo-memory. We gather evidence that suggests that small RNA network changes, involving multiple upregulated and downregulated small RNAs, balance the trade-off between growth/development and stress responses, in order to ensure successful adaptation. We highlight specific characteristics of small RNA-based tem- perature stress regulation in crop plants. Finally, we explore the perspectives of the use of small RNAs in breeding to improve stress tolerance, which may be relevant for agriculture in the near future