Evaluation of combinatorial cis-regulatory elements for stable gene expression in chicken cells

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Recent successes in biotechnological application of birds are based on their unique physiological traits such as unlimited manipulability onto developing embryos and simple protein constituents of the eggs. However it is not likely that target protein is produced as kinetically expected because various factors affect target gene expression. Although there have been various attempts to minimize the silencing of transgenes, a generalized study that uses multiple cis-acting elements in chicken has not been made. The aim of the present study was to analyze whether various cis-acting elements can help to sustain transgene expression in chicken fibroblasts. Results: We investigated the optimal transcriptional regulatory elements for enhancing stable transgene expression in chicken cells. We generated eight constructs that encode enhanced green fluorescent protein (eGFP) driven by either CMV or CAG promoters (including the control), containing three types of key regulatory elements: a chicken lysozyme matrix attachment region (cMAR), 5′-DNase I-hypersensitive sites 4 (cHS4), and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Then we transformed immortalized chicken embryonic fibroblasts with these constructs by electroporation, and after cells were expanded under G418 selection, analyzed mRNA levels and mean fluorescence intensity (MFI) by quantitative real-time PCR and flow cytometry, respectively. We found that the copy number of each construct significantly decreased as the size of the construct increased (R2 = 0.701). A significant model effect was found in the expression level among various constructs in both mRNA and protein (P < 0.0001). Transcription with the CAG promoter was 1.6-fold higher than the CMV promoter (P = 0.027) and the level of eGFP expression activity in cMAR- or cHS4-flanked constructs increased by two- to three-fold compared to the control CMV or CAG promoter constructs. In addition, flow cytometry analysis showed that constructs having cis-acting elements decreased the level of gene silencing as well as the coefficient of variance of eGFP-expressing cells (P < 0.0001). Conclusions: Our current data show that an optimal combination of cis-acting elements and promoters/enhancers for sustaining gene expression in chicken cells is suggested. These results provide important information for avian transgenesis and gene function studies in poultry

Experimental observation of hidden Berry curvature in inversion-symmetric bulk 2H-WSe2

We investigate the hidden Berry curvature in bulk 2H-WSe2 by utilizing the surface sensitivity of angle resolved photoemission (ARPES). The symmetry in the electronic structure of transition metal dichalcogenides is used to uniquely determine the local orbital angular momentum (OAM) contribution to the circular dichroism (CD) in ARPES. The extracted CD signals for the K and K' valleys are almost identical but their signs, which should be determined by the valley index, are opposite. In addition, the sign is found to be the same for the two spin-split bands, indicating that it is independent of spin state. These observed CD behaviors are what are expected from Berry curvature of a monolayer of WSe2. In order to see if CD-ARPES is indeed representative of hidden Berry curvature within a layer, we use tight binding analysis as well as density functional calculation to calculate the Berry curvature and local OAM of a monolayer WSe2. We find that measured CD-ARPES is approximately proportional to the calculated Berry curvature as well as local OAM, further supporting our interpretation.Comment: 6 pages, 3 figure

Effects of Photoperiod, Water temperature, and Exogenous Hormones on Spawning and Plasma gonadal Steroid in Starry Flounder, Platichthys stellatus

The sexual maturation and spawning of teleosts are regulated by the external environment and the endocrine system. When the environmental conditions are artificially adjusted at a fish farm, the maturity and spawning of fish can be controlled. In this study, sexual maturation and spawning of the starry flounder, Platichthys stellatus, were artificially induced by adjusting the water temperature and photoperiod at a fish farm to accelerate the species’ natural spawning period. One experimental group acted as a control and was exposed to a natural photoperiod and natural water temperature (NPNT). In contrast, another experimental group was exposed to an adjusted environment consisting of a regulated photoperiod and temperature (RPRT). Daylight time was reduced by 10 minutes every 3 days from 13 hours to a duration of 8 hours. The water temperature was first reduced by 1oC every day, starting at 22oC and ending at 8oC, and then raised to 10oC until the spawning period. Both experimental groups were treated with gonadotropin-releasing hormone analog (GnRHa) pellets to induce ovulation. The results show that when the water temperature and photoperiod were artificially controlled, ovulation could be induced 97 days earlier than the natural spawning. Plasma testosterone levels of RPRT and NPNT tended to increase and then decrease 1–2 months before spawning, and plasma levels of 17α,20β-dihydroxy-4-pregnen-3-one increased 1–2 months before spawning. The concentration of estradiol-17β (E2) in plasma was not associated with spawning

Introducing Small-World Network Effect to Critical Dynamics

We analytically investigate the kinetic Gaussian model and the one-dimensional kinetic Ising model on two typical small-world networks (SWN), the adding-type and the rewiring-type. The general approaches and some basic equations are systematically formulated. The rigorous investigation of the Glauber-type kinetic Gaussian model shows the mean-field-like global influence on the dynamic evolution of the individual spins. Accordingly a simplified method is presented and tested, and believed to be a good choice for the mean-field transition widely (in fact, without exception so far) observed on SWN. It yields the evolving equation of the Kawasaki-type Gaussian model. In the one-dimensional Ising model, the p-dependence of the critical point is analytically obtained and the inexistence of such a threshold p_c, for a finite temperature transition, is confirmed. The static critical exponents, gamma and beta are in accordance with the results of the recent Monte Carlo simulations, and also with the mean-field critical behavior of the system. We also prove that the SWN effect does not change the dynamic critical exponent, z=2, for this model. The observed influence of the long-range randomness on the critical point indicates two obviously different hidden mechanisms.Comment: 30 pages, 1 ps figures, REVTEX, accepted for publication in Phys. Rev.

A study of the photometric variability of the peculiar magnetic white dwarf WD1953-011

We present and interpret simultaneous new photometric and spectroscopic observations of the peculiar magnetic white dwarf WD1953-011. The flux in the V-band filter and intensity of the Balmer spectral lines demonstrate variability with the rotation period of about 1.45 days. According to previous studies, this variability can be explained by the presence of a dark spot having a magnetic nature, analogous to a sunspot. Motivated by this idea, we examine possible physical relationships between the suggested dark spot and the strong-field magnetic structure (magnetic "spot", or "tube") recently identified on the surface of this star. Comparing the rotationally-modulated flux with the variable spectral observables related to the magnetic "spot" we establish their correlation, and therefore their physical relationship. Modeling the variable photometric flux assuming that it is associated with temperature variations in the stellar photosphere, we argue that the strong-field area and dark, low-temperature spot are comparable in size and located at the same latitudes, essentially overlapping each other with a possible slight longitudinal shift. In this paper we also present a new, improved value of the star's rotational period and constrain the characteristics of the thermal inhomogeneity over the degenerate's surface.Comment: accepted to the Ap

Edge-Functionalization of Pyrene as a Miniature Graphene via Friedel–Crafts Acylation Reaction in Poly(Phosphoric Acid)

The feasibility of edge-functionalization of graphite was tested via the model reaction between pyrene and 4-(2,4,6-trimethylphenyloxy)benzamide (TMPBA) in poly(phosphoric acid) (PPA)/phosphorous pentoxide (P2O5) medium. The functionalization was confirmed by various characterization techniques. On the basis of the model study, the reaction condition could be extended to the edge-functionalization of graphite with TMPBA. Preliminary results showed that the resultant TMPBA-grafted graphite (graphite-g-TMPBA) was found to be readily dispersible in N-methyl-2-pyrrolidone (NMP) and can be used as a precursor for edge-functionalized graphene (EFG)

Regulation of cAMP and GSK3 signaling pathways contributes to the neuronal conversion of glioma

Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule-induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs

Grafting of 4-(2,4,6-Trimethylphenoxy)benzoyl onto Single-Walled Carbon Nanotubes in Poly(phosphoric acid) via Amide Function

Single-walled carbon nanotubes (SWCNTs), which were commercial grade containing 60–70 wt% impurity, were treated in a mild poly(phosphoric acid) (PPA). The purity of PPA treated SWCNTs was greatly improved with or without little damage to SWCNTs framework and stable crystalline carbon particles. An amide model compound, 4-(2,4,6-trimethylphenoxy)benzamide (TMPBA), was reacted with SWCNTs in PPA with additional phosphorous pentoxide as “direct” Friedel–Crafts acylation reaction to afford TMPBA functionalized SWCNTs. All evidences obtained from Fourier-transform infrared spectroscopy, Raman spectroscopy, thermogravimetric analysis, scanning electron microcopy, and transmission electron microscopy strongly supported that the functionalization of SWCNTs with benzamide was indeed feasible

Clinical Significance of Thrombosis in an Intracardiac Blind Pouch After a Fontan Operation

The univentricular heart after the Fontan operation may have a blind pouch formed by the pulmonary stump or rudimentary ventricle according to the anatomy before surgery. Thrombosis in an intracardiac blind pouch of patients with a univentricular heart is a hazardous complication. Because only a few reports have described this complication, the authors evaluated the clinical significance of thrombosis in an intracardiac blind pouch of a univentricular heart. They performed a retrospective review of medical records from August 1986 to December 2007. Four patients were confirmed as having thrombosis in a pulmonary artery stump and one patient as having thrombosis in a rudimentary ventricle shown by cardiac computed tomography (CT). This represents 1.85% (5/271) of patients with ongoing regular follow-up evaluation after the Fontan operation. The median age at diagnosis was 14.2 years. Two of the five patients were taking aspirin and one patient was taking warfarin when they were identified for the development of thrombosis. None of the patients demonstrated thrombosis in the Fontan tract or venous side of the circulation. Brain magnetic resonance imaging (MRI) showed that three patients had cerebral infarction and one patient had suggestive old ischemia. Three patients with thrombus in the pulmonary stump underwent pulmonary artery stump thrombectomy and pulmonary valve obliteration. One patient with thrombus in the rudimentary ventricle underwent ventricular septal defect (VSD) closure with thrombectomy. Thrombus in a blind pouch could cause systemic thromboembolism despite little blood communication. Therefore, surgical modification of the pulmonary stump and VSD closure of the rudimentary ventricle are required to reduce the risk of later thrombus formation. Clinicians should not overlook the possibility of thrombus in a ligated pulmonary artery stump or a rudimentary ventricle after the Fontan operation, which may increase the risk of embolic stroke for patients with single-ventricle physiology