Genetic Characterization of Centromere-Mediated Uniparental Genome Elimination in Arabidopsis

The production of haploids through crossing was first discovered from instances of interspecies crosses in Nicotiana spp. in 1924. Since then, haploid induction crosses has been used to improve plant breeding programs and commercially utilized in a number of crop industries. The generation of doubled haploid instantaneously creates a pure homozygous line, therefore eliminating the need for several generations of inbreeding. There are several pathways to induce haploids in plants: of these methods, centromere-mediated genome elimination pathway engenders the highest haploid induction rate (HIR) with up to 45% in Arabidopsis compared to 15% through phospholipase-mediated haploid induction in maize. Centromere-mediated genome elimination operates through the manipulation of CENH3, a histone H3 variant that is associated with the formation of centromere on chromosomes. Although centromere-mediated genome elimination is highly efficient in inducing haploids, the best haploid inducer in the Arabidopsis system is stunted in its growth and is partially male sterile. In the work presented here, we were able to induce haploids in Arabidopsis using lines that were vigorous and can be crossed as either a male or female. This was achieved using four mutant allele combinations based on two recessive cenh3 alleles: cenh3-1, a null allele and cenh3-2, a missense allele. Our results demonstrated that we could induce haploids while balancing the trade-off between the efficacy to induce haploids and haploid inducer vigour. As CENH3 can be found across all plant species, centromere-mediated genome elimination pathway can be employed by other plants as well. On top of that, the concept that haploids can be induced without the introduction of transgene is attractive for crop industries as it can eliminate the need to go through regulatory bodies for plant breeding programs or crop improvement efforts. Meanwhile, detailed molecular characterization of events that govern haploid induction via centromere-mediated genome elimination is still largely not known. Here, we also provide a framework and potential protocol that would eventually allow expression profiling of early Arabidopsis embryos undergoing centromere-mediated genome elimination

Rhapso : automatic stitching of mass segments from fourier transform ion cyclotron resonance mass spectra

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the resolution and mass accuracy needed to analyze complex mixtures such as crude oil. When mixtures contain many different components, a competitive effect within the ICR cell takes place that hampers the detection of a potentially large fraction of the components. Recently, a new data collection technique, which consists of acquiring several spectra of small mass ranges and assembling a complete spectrum afterward, enabled the observation of a record number of peaks with greater accuracy compared to broadband methods. There is a need for statistical methods to combine and preprocess segmented acquisition data. A particular challenge of quadrupole isolation is that near the window edges there is a drop in intensity, hampering the stitching of consecutive windows. We developed an algorithm called Rhapso to stitch peak lists corresponding to multiple different m/z regions from crude oil samples. Rhapso corrects potential edge effects to enable the use of smaller windows and reduce the required overlap between windows, corrects mass shifts between windows, and generates a single peak list for the full spectrum. Relative to a stitching performed manually, Rhapso increased the data processing speed and avoided potential human errors, simplifying the subsequent chemical analysis of the sample. Relative to a broadband spectrum, the stitched output showed an over 2-fold increase in assigned peaks and reduced mass error by a factor of 2. Rhapso is expected to enable routine use of this spectral stitching method for ultracomplex samples, giving a more detailed characterization of existing samples and enabling the characterization of samples that were previously too complex to analyze

Oct-4 gene expression in the preattachment porcine conceptus and murine embryo with regulation of pluripotency through protein transduction

Scope and Method of Study: To investigate the presence of the Oct-4 transcript in the murine embryo and the preattachment porcine conceptus over time, we quantified the expression of Oct-4, a candidate master regulator gene for the maintenance of pluripotent cells. We also examined the cloning and subsequent protein purification of positive and negative regulators of the Oct-4 gene with the potential for protein transduction therapy. Our first objective was to test the hypothesis that porcine trophoblastic elongation and placental differentiation is associated with the Oct-4 gene expression. Porcine embryos were collected following days 10 to 17 of development. The quantitative analysis used a Real Time, one-step RT-PCR amplification. Our second objective was to test the hypothesis that individual murine embryos exhibit an up-regulation of Oct-4 in the 4-cell, 8-cell, and blastocyst whole embryos and that the individual pluripotent cells would exhibit an increase in single cell expression. RNA of individual murine embryos was reverse transcribed and Real Time PCR performed. Our final objective was to produce an Oct-4 gene enhancer TAT fusion protein, an Oct-4 gene repressor TAT fusion protein and an Oct-4 TAT fusion protein to be used in subsequent studies. We used site-directed PCR mutagenesis to clone the Bmp8b, GCNF and Oct-4 genes in prokaryote expression vectors. Through protein expression in E. coli and FPLC purification, fusion proteins were verified and tested on living cells.Findings and Conclusions: Using the comparative CT method, the porcine Oct-4 expression was greatest on days 10 and 12 of pregnancy and was approximately 2, 8, and 11-fold greater compared to expression on days 13, 15 and 17, respectively. The down regulation of Oct-4 gene expression between days 12 and 17 is temporally associated with porcine extended trophoblastic elongation and placental differentiation. The murine blastocysts and 8-cell embryos produced 85-fold and 7-fold more Oct-4, respectively, than the four cell embryos. A significant up-regulation of the Oct-4 transcript per totipotent blastocyst cell was demonstrated. The three fusion proteins were produced and verified. The protein transduction initial trials on living cells were unsuccessful in consistently regulating transcription

Disparities in lung transplantation in children

Lung transplantation is a recognized therapy for end-stage respiratory failure in children and young people. It is only available in selected countries and is limited by access to suitable organs. Data on disparities in access and outcomes for children undergoing lung transplantation are limited. It is clear from data from studies in adults, and from studies in other solid organ transplants in children, that systemic inequities exist in this field. While data relating specifically to pediatric lung transplantation are relatively sparse, professionals should be aware of the risk that healthcare systems may result in disparities in access and outcomes following lung transplantation in children.</p

Adaptive Data-based Predictive Control for Short Take-off and Landing (STOL) Aircraft

Data-based Predictive Control is an emerging control method that stems from Model Predictive Control (MPC). MPC computes current control action based on a prediction of the system output a number of time steps into the future and is generally derived from a known model of the system. Data-based predictive control has the advantage of deriving predictive models and controller gains from input-output data. Thus, a controller can be designed from the outputs of complex simulation code or a physical system where no explicit model exists. If the output data happens to be corrupted by periodic disturbances, the designed controller will also have the built-in ability to reject these disturbances without the need to know them. When data-based predictive control is implemented online, it becomes a version of adaptive control. The characteristics of adaptive data-based predictive control are particularly appropriate for the control of nonlinear and time-varying systems, such as Short Take-off and Landing (STOL) aircraft. STOL is a capability of interest to NASA because conceptual Cruise Efficient Short Take-off and Landing (CESTOL) transport aircraft offer the ability to reduce congestion in the terminal area by utilizing existing shorter runways at airports, as well as to lower community noise by flying steep approach and climb-out patterns that reduce the noise footprint of the aircraft. In this study, adaptive data-based predictive control is implemented as an integrated flight-propulsion controller for the outer-loop control of a CESTOL-type aircraft. Results show that the controller successfully tracks velocity while attempting to maintain a constant flight path angle, using longitudinal command, thrust and flap setting as the control inputs

Hippocampal neuronal cells that accumulate α-synuclein fragments are more vulnerable to Aβ oligomer toxicity via mGluR5--implications for dementia with Lewy bodies.

BackgroundIn dementia with Lewy bodies (DLB) abnormal interactions between α-synuclein (α-syn) and beta amyloid (Aβ) result in selective degeneration of neurons in the neocortex, limbic system and striatum. However, factors rendering these neurons selectively vulnerable have not been fully investigated. The metabotropic glutamate receptor 5 (mGluR5) has been shown to be up regulated in DLB and might play a role as a mediator of the neurotoxic effects of Aβ and α-syn in vulnerable neuronal populations. In this context, the main objective of the present study was to investigate the role of mGluR5 as a mediator of the neurotoxic effects of α-syn and Aβ in the hippocampus.ResultsWe generated double transgenic mice over-expressing amyloid precursor protein (APP) and α-syn under the mThy1 cassette and investigated the relationship between α-syn cleavage, Aβ, mGluR5 and neurodegeneration in the hippocampus. We found that compared to the single tg mice, the α-syn/APP tg mice displayed greater accumulation of α-syn and mGluR5 in the CA3 region of the hippocampus compared to the CA1 and other regions. This was accompanied by loss of CA3 (but not CA1) neurons in the single and α-syn/APP tg mice and greater loss of MAP 2 and synaptophysin in the CA3 in the α-syn/APP tg. mGluR5 gene transfer using a lentiviral vector into the hippocampus CA1 region resulted in greater α-syn accumulation and neurodegeneration in the single and α-syn/APP tg mice. In contrast, silencing mGluR5 with a lenti-shRNA protected neurons in the CA3 region of tg mice. In vitro, greater toxicity was observed in primary hippocampal neuronal cultures treated with Aβ oligomers and over-expressing α-syn; this effect was attenuated by down-regulating mGluR5 with an shRNA lentiviral vector. In α-syn-expressing neuronal cells lines, Aβ oligomers promoted increased intracellular calcium levels, calpain activation and α-syn cleavage resulting in caspase-3-dependent cell death. Treatment with pharmacological mGluR5 inhibitors such as 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) attenuated the toxic effects of Aβ in α-syn-expressing neuronal cells.ConclusionsTogether, these results support the possibility that vulnerability of hippocampal neurons to α-syn and Aβ might be mediated via mGluR5. Moreover, therapeutical interventions targeting mGluR5 might have a role in DLB

Fluorescence microscopy of inkjet prints

Inkjet printing technology has been developing rapidly during recent years, pressing the ink and paper manufacturers to develop a better understanding of the mechanism of fixation of inkjet dye into the substrate. The aim of the work described in this thesis was to investigate the three-dimensional distribution of inkjet dye in paper and the interaction between dye and paper using advanced fluorescence microscopy techniques, Confocal Laser Scanning Microscopy (CLSM), and Two-photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM). It has been shown that CLSM is a valuable, non-destructive, rapid technique for threedimensional imaging of printed samples and evaluation of print quality. The intrinsic fluorescence of both the inkjet dye and the paper substrate can be used to determine the spread and penetration of ink droplets in different inkjet papers. The optical sectioning capability of CLSM enables the position of the ink layer relative to the paper surface and the penetration depth of the ink to be quantified. It was observed that while in the microporous type of inkjet paper the penetration depends on the quantity of ink in the printed sample, in the swellable type of inkjet paper the penetration is almost the same for different amounts of ink. 2P-FLIM has been employed to spatially map, in three-dimensions, fluorescence lifetimes by measuring the lifetime at each pixel in the image. Fluorescent molecules in both the ink and paper were analysed. Because the fluorescence lifetime is affected by the local molecular environment, the fluorescence lifetime maps provide information on the interaction between inkjet dye and paper. Analysis of fluorescence lifetime maps reveals the interaction between dye molecules and silica or alumina particles in the paper, variations of the molecular environment within a single ink dot and that interaction between dye and paper is affected by pH

Visceral Leishmaniasis in Traveler to Guyana Caused by Leishmania siamensis, London, UK.

The parasite Leishmania siamensis is a zoonotic agent of leishmaniasis; infection in animals has been documented in Europe and the United States. Reported authochthonous human infections have been limited to Thailand. We report a case of human visceral Leishmania siamensis infection acquired in Guyana, suggesting colonization in South America