Structure and non-essential function of glycerol kinase in <i>Plasmodium</i> <i>falciparum</i> blood stages

Malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). Microarray analysis identified glycerol kinase (GK) as the second most highly upregulated gene in Plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. Phosphorylation of glycerol by GK is the rate-limiting step in glycerol utilization. Deletion of this gene from P. falciparum had no effect on asexual parasite growth, but surprisingly also had no effect on gametocyte development or exflagellation, suggesting that these life cycle stages do not utilize host-derived glycerol as a carbon source. Kinetic studies of purified PfGK showed that the enzyme is not regulated by fructose 1,6 bisphosphate. The high-resolution crystal structure of P. falciparum GK, the first of a eukaryotic GK, reveals two domains embracing a capacious ligand-binding groove. In the complexes of PfGK with glycerol and ADP, we observed closed and open forms of the active site respectively. The 27° domain opening is larger than in orthologous systems and exposes an extensive surface with potential for exploitation in selective inhibitor design should the enzyme prove to be essential in vivo either in the human or in the mosquito

Molding photonic boxes into fluorescent emitters by direct laser writing

We acknowledge financial support from the European Research Council Starting Grant ABLASE (ERC StG 640012), the Scottish Funding Council (via SUPA) and the European Union Marie Curie Career Integration Grant (PCIG12-GA-2012-334407). M.K. acknowledges funding from the EPSRC DTG (EP/M506631/1). S.H. gratefully acknowledges support from the Engineering and Physical Sciences Research Council through the ’Hybrid Polaritonics’ Program Grant (Project EP/M025330/1).Direct laser writing of photonic boxes into active layers of biologically produced recombinant fluorescent protein in optical microcavities is demonstrated. Irradiation with laser light above the photobleaching threshold induces photonic confinement potentials on the order of 40 meV. The technique provides high spatial selectivity and enables room-temperature lasing in protein rings, and circular and elliptical pillars with customized beam shapes.Publisher PDFPeer reviewe

Operational Excellence in a continuous production line at ROCKWOOL Group

The constant search for improvement in production and efficiency in an industrial context is one of the main challenges faced by companies. This Master Thesis focuses in the development of a simulation model using AnyLogic software, aiming to represent a segment of a production line and facilitating its detailed analysis. The primary goal is to identify and address the factors limiting production excellence, employing Theory of Constraints to guide the model’s application. The research is conducted at ROCKWOOL Group, specifically examining the production line 09 at the Neuburg factory, within ROCKWOOL Germany context. This setting serves as a real-world example to apply and illustrate the Operational Excellence, Simulation Modeling and Theory of Constraints concepts. The development of a simulation model that represents the operation of the production line, combined with the input of data from the POP and RockFact systems over a specified period, enabled the analysis of the operational performance of one of the most frequently produced products. This analysis identified the critical points along the production line. The findings of this thesis illustrate methods to enhance production perfor- mance within a framework of continuous improvement. These methods have potential applications across the process industry, suggesting ways to generalize and adopt improved production strategies.Outgoin

Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background&#xd;&#xa;The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.&#xd;&#xa;&#xd;&#xa;Methodology/Principal Findings&#xd;&#xa;When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength &#x2013; a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.&#xd;&#xa;&#xd;&#xa;Conclusions/Significance&#xd;&#xa;Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

Förster Resonance Energy Transfer (FRET) Correlates of Altered Subunit Stoichiometry in Cys-Loop Receptors, Exemplified by Nicotinic α4β2

We provide a theory for employing Förster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose α4 and β2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (α4)3(β2)2 vs. (α4)2(β2)3. Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (α4)3(β2)2 exceeds that of (α4)2(β2)3. The simulations account for published data on transfected Neuro2a cells in which α4β2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (α4)3(β2)2 stoichiometry to mostly (α4)2(β2)3. The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (α4)2(β2)3 and (α4)3(β2)2 populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors

La casa de Alicia en el país de las maravillas: la casa del SER en su contexto

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