High efficiency regeneration and genetic stability analysis of somatic clones of Gynura bicolor DC.

Gynura bicolor DC. is a perennial vegetable and medicinal plant. It is an important source of anthocyanins. The effects of different growth regulators on callus induction and plant regeneration were evaluated. The best SFC index (8.6) of plant regeneration was obtained in combination of 2,4-D at 2.0 mg/l and BA at 0.5 mg/l, and the frequency of regenerating explants was 78.3%. The highest number of shoots per explant was 11. The genetic stability of the regenerants was analyzed by random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) molecular markers and flow cytometry. The results indicated that no somaclonal variation was detected among the regenerants. To our knowledge, this is the first report of somatic clone study in G. bicolor. The high efficient and reproducible protocol will be advanced for the further studies on secondary metabolic products, transformations and breeding of this potential medicinal plant.Key words: Flow cytometry analysis, genetic stability, Gynura bicolor, inter-simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD), regeneration

Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: Implications for the treatment of brain arteriovenous malformations

© 2016 AANS. Objective Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. methods An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. results Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25-and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. coNclusioNs Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for AVM treatment

Human preferences for sexually dimorphic faces may be evolutionarily novel

This article has been made available through the Brunel Open Access Publishing Fund.A large literature proposes that preferences for exaggerated sex typicality in human faces (masculinity/femininity) reflect a long evolutionary history of sexual and social selection. This proposal implies that dimorphism was important to judgments of attractiveness and personality in ancestral environments. It is difficult to evaluate, however, because most available data come from largescale, industrialized, urban populations. Here, we report the results for 12 populations with very diverse levels of economic development. Surprisingly, preferences for exaggerated sex-specific traits are only found in the novel, highly developed environments. Similarly, perceptions that masculine males look aggressive increase strongly with development, specifically, urbanization. These data challenge the hypothesis that facial dimorphism was an important ancestral signal of heritable mate value. One possibility is that highly developed environments provide novel opportunities to discern relationships between facial traits and behavior by exposing individuals to large numbers of unfamiliar faces, revealing patterns too subtle to detect with smaller samples

Screening for inborn errors of metabolism in high-risk children: a 3-year pilot study in Zhejiang Province, China

<p>Abstract</p> <p>Background</p> <p>Tandem mass spectrometry (MS/MS) has been available in China for 8 years. This technique makes it possible to screen for a wide range of previously unscreened inborn errors of metabolism (IEM) using a single test. This 3-year pilot study investigated the screening, diagnosis, treatment and outcomes of IEM in symptomatic infants and children.</p> <p>Methods</p> <p>All children encountered in the Newborn Screening Center of Zhejiang Province during a 3-year period with symptoms suspicious for IEM were screened for metabolic diseases. Dried blood spots were collected and analyzed by tandem mass spectrometry. The diagnoses were further confirmed by clinical symptoms and biochemical analysis. Neonatal intrahepatic cholestasis caused by citrin deficiency, ornithine transcarbamylase deficiency and primary carnitine deficiency were confirmed by DNA analysis.</p> <p>Results</p> <p>A total of 11,060 symptomatic patients (6,720 boys, 4,340 girls) with a median age of 28.8 months (range: 0.04-168.2 months) were screened. Among these, 62 were diagnosed with IEM, with a detection rate of 0.56%. Thirty-five were males and 27 females and the median age was 3.55 months (range 0.07-143.9 months). Of the 62 patients, 27 (43.5%) had aminoacidemias, 26 (41.9%) had organic acidemias and nine (14.5%) had fatty acid oxidation disorders.</p> <p>Conclusions</p> <p>Because most symptomatic patients are diagnosed at an older age, mental retardation and motor delay are difficult to reverse. Additionally, poor medication compliance reduces the efficacy of treatment. More extensive newborn screening is thus imperative for ensuring early diagnosis and enhancing the treatment efficacy of IEM.</p

A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation

BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12860-014-0041-5) contains supplementary material, which is available to authorized users

Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations

© 2018 Elsevier Ltd Background: Our goal is to develop a vascular targeting treatment for brain arteriovenous malformations (AVMs). Externalized phosphatidylserine has been established as a potential biomarker on the endothelium of irradiated AVM blood vessels. We hypothesize that phosphatidylserine could be selectively targeted after AVM radiosurgery with a ligand-directed vascular targeting agent to achieve localized thrombosis and rapid occlusion of pathological AVM vessels. Objective: The study aim was to establish an in vitro parallel-plate flow chamber to test the efficacy of a pro-thrombotic conjugate targeting phosphatidylserine. Methods: Conjugate was prepared by Lys-Lys cross-linking of thrombin with the phosphatidylserine-targeting ligand, annexin V. Cerebral microvascular endothelial cells were irradiated (5, 15, and 25 Gy) and after 1 or 3 days assembled in a parallel-plate flow chamber containing whole human blood and conjugate (1.25 or 2.5 μg/mL). Confocal microscopy was used to assess thrombus formation after flow via binding and aggregation of fluorescently-labelled platelets and fibrinogen. Results and conclusions: The annexin V-thrombin conjugate induced rapid thrombosis (fibrin deposition) on irradiated endothelial cells under shear stress in the parallel-plate flow device. Unconjugated, non-targeting thrombin did not induce fibrin deposition. A synergistic interaction between radiation and conjugate dose was observed. Thrombosis was greatest at the highest combined doses of radiation (25 Gy) and conjugate (2.5 μg/mL). The parallel-plate flow system provides a rapid method to pre-test pro-thrombotic vascular targeting agents. These findings validate the translation of the annexin V-thrombin conjugate to pre-clinical studies

P5CS expression study in a new family with ALDH18A1-associated hereditary spastic paraplegia SPG9

In 2015\u20132016, we and others reported ALDH18A1 mutations causing dominant (SPG9A) or recessive (SPG9B) spastic paraplegia. In vitro production of the ALDH18A1 product, \u3941-pyrroline-5-carboxylate synthetase (P5CS), appeared necessary for cracking SPG9 disease-causing mechanisms. We now describe a baculovirus\u2013insect cell system that yields mgs of pure human P5CS and that has proven highly valuable with two novel P5CS mutations reported here in new SPG9B patients. We conclude that both mutations are disease-causing, that SPG9B associates with partial P5CS deficiency and that it is clinically more severe than SPG9A, as reflected in onset age, disability, cognitive status, growth, and dysmorphic traits

From regional pulse vaccination to global disease eradication: insights from a mathematical model of Poliomyelitis

Mass-vaccination campaigns are an important strategy in the global fight against poliomyelitis and measles. The large-scale logistics required for these mass immunisation campaigns magnifies the need for research into the effectiveness and optimal deployment of pulse vaccination. In order to better understand this control strategy, we propose a mathematical model accounting for the disease dynamics in connected regions, incorporating seasonality, environmental reservoirs and independent periodic pulse vaccination schedules in each region. The effective reproduction number, $R_e$, is defined and proved to be a global threshold for persistence of the disease. Analytical and numerical calculations show the importance of synchronising the pulse vaccinations in connected regions and the timing of the pulses with respect to the pathogen circulation seasonality. Our results indicate that it may be crucial for mass-vaccination programs, such as national immunisation days, to be synchronised across different regions. In addition, simulations show that a migration imbalance can increase $R_e$ and alter how pulse vaccination should be optimally distributed among the patches, similar to results found with constant-rate vaccination. Furthermore, contrary to the case of constant-rate vaccination, the fraction of environmental transmission affects the value of $R_e$ when pulse vaccination is present.Comment: Added section 6.1, made other revisions, changed titl

Preparation and Measurement of Three-Qubit Entanglement in a Superconducting Circuit

Traditionally, quantum entanglement has played a central role in foundational discussions of quantum mechanics. The measurement of correlations between entangled particles can exhibit results at odds with classical behavior. These discrepancies increase exponentially with the number of entangled particles. When entanglement is extended from just two quantum bits (qubits) to three, the incompatibilities between classical and quantum correlation properties can change from a violation of inequalities involving statistical averages to sign differences in deterministic observations. With the ample confirmation of quantum mechanical predictions by experiments, entanglement has evolved from a philosophical conundrum to a key resource for quantum-based technologies, like quantum cryptography and computation. In particular, maximal entanglement of more than two qubits is crucial to the implementation of quantum error correction protocols. While entanglement of up to 3, 5, and 8 qubits has been demonstrated among spins, photons, and ions, respectively, entanglement in engineered solid-state systems has been limited to two qubits. Here, we demonstrate three-qubit entanglement in a superconducting circuit, creating Greenberger-Horne-Zeilinger (GHZ) states with fidelity of 88%, measured with quantum state tomography. Several entanglement witnesses show violation of bi-separable bounds by 830\pm80%. Our entangling sequence realizes the first step of basic quantum error correction, namely the encoding of a logical qubit into a manifold of GHZ-like states using a repetition code. The integration of encoding, decoding and error-correcting steps in a feedback loop will be the next milestone for quantum computing with integrated circuits.Comment: 7 pages, 4 figures, and Supplementary Information (4 figures)