3,4,6-Tri-O-acetyl-2-deoxy-2-[18F]fluoroglucopyranosyl phenylthiosulfonate: a thiol-reactive agent for the chemoselective 18F-glycosylation of peptides

3,4,5-Tri-O-acetyl-2-[18F]fluoro-2-deoxy-d-glucopyranosyl 1-phenylthiosulfonate (Ac3-[18F]FGlc-PTS) was developed as a thiol-reactive labeling reagent for the site-specific 18F-glycosylation of peptides. Taking advantage of highly accessible 1,3,4,6-tetra-O-acetyl-2-deoxy-2-[18F]fluoroglucopyranose, a three-step radiochemical pathway was investigated and optimized, providing Ac3-[18F]FGlc-PTS in a radiochemical yield of about 33% in 90 min (decay-corrected and based on starting [18F]fluoride). Ac3-[18F]FGlc-PTS was reacted with the model pentapeptide CAKAY, confirming chemoselectivity and excellent conjugation yields of >90% under mild reaction conditions. The optimized method was adopted to the 18F-glycosylation of the alphavbeta3-affine peptide c(RGDfC), achieving high conjugation yields (95%, decay-corrected). The alphavbeta3 binding affinity of the glycosylated c(RGDfC) remained uninfluenced as determined by competition binding studies versus 125I-echistatin using both isolated alphavbeta3 and human umbilical vein endothelial cells (Ki = 68 +/- 10 nM (alphavbeta3) versus Ki = 77 +/- 4 nM (HUVEC)). The whole radiosynthetic procedure, including the preparation of the 18F-glycosylating reagent Ac3-[18F]FGlc-PTS, peptide ligation, and final HPLC purification, provided a decay-uncorrected radiochemical yield of 13% after a total synthesis time of 130 min. Ac3-[18F]FGlc-PTS represents a novel 18F-labeling reagent for the mild chemoselective 18F-glycosylation of peptides indicating its potential for the design and development of 18F-labeled bioactive S-glycopeptides suitable to study their pharmacokinetics in vivo by positron emission tomography (PET).     

PLMaddon: a power-law module for the Matlab SBToolbox

PLMaddon is a General Public License (GPL) software module designed to expand the current version of the SBToolbox (a Matlab toolbox for systems biology; www.sbtoolbox.org) with a set of functions for the analysis of power-law models, a specific class of kinetic models, set in ordinary differential equations (ODE) and in which the kinetic orders can have positive/negative non-integer values. The module includes functions to generate power-law Taylor expansions of other ODE models (e.g. Michaelis-Menten type models), as well as algorithms to estimate steady-states. The robustness and sensitivity of the models can also be analysed and visualized by computing the power-law's logarithmic gains and sensitivities.     

Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion

The main function of the photosynthetic process is to capture solar energy and to store it in the form of chemical 'fuels'. Increasingly, the photosynthetic machinery is being used for the production of biofuels such as bio-ethanol, biodiesel and bio-H₂. Fuel production efficiency is directly dependent on the solar photon capture and conversion efficiency of the system. Green algae (e.g. Chlamydomonas reinhardtii ) have evolved genetic strategies to assemble large light-harvesting antenna complexes (LHC) to maximize light capture under low-light conditions, with the downside that under high solar irradiance, most of the absorbed photons are wasted as fluorescence and heat to protect against photodamage. This limits the production process efficiency of mass culture. We applied RNAi technology to down-regulate the entire LHC gene family simultaneously to reduce energy losses by fluorescence and heat. The mutant Stm3LR3 had significantly reduced levels of LHCI and LHCII mRNAs and proteins while chlorophyll and pigment synthesis was functional. The grana were markedly less tightly stacked, consistent with the role of LHCII. Stm3LR3 also exhibited reduced levels of fluorescence, a higher photosynthetic quantum yield and a reduced sensitivity to photoinhibition, resulting in an increased efficiency of cell cultivation under elevated light conditions. Collectively, these properties offer three advantages in terms of algal bioreactor efficiency under natural high-light levels: (i) reduced fluorescence and LHC-dependent heat losses and thus increased photosynthetic efficiencies under high-light conditions; (ii) improved light penetration properties; and (iii) potentially reduced risk of oxidative photodamage of PSII.     

The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells

Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.     

Widespread Expression of Erythropoietin Receptor in Brain and Its Induction by Injury

Erythropoietin (EPO) exerts potent neuroprotective, neuroregenerative and procognitive functions. However, unequivocal demonstration of erythropoietin receptor (EPOR) expression in brain cells has remained difficult since previously available anti-EPOR antibodies (EPOR-AB) were unspecific. We report here a new, highly specific, polyclonal rabbit EPOR-AB directed against different epitopes in the cytoplasmic tail of human and murine EPOR and its characterization by mass spectrometric analysis of immuno-precipitated endogenous EPOR, Western blotting, immunostaining and flow cytometry. Among others, we applied genetic strategies including overexpression, Lentivirus-mediated conditional knockout of EpoR and tagged proteins, both on cultured cells and tissue sections, as well as intracortical implantation of EPOR-transduced cells to verify specificity. We show examples of EPOR expression in neurons, oligodendroglia, astrocytes and microglia. Employing this new EPOR-AB with double-labeling strategies, we demonstrate membrane expression of EPOR as well as its localization in intracellular compartments such as the Golgi apparatus. Moreover, we show injury-induced expression of EPOR. In mice, a stereotactically applied stab wound to the motor cortex leads to distinct EpoR expression by reactive GFAP-expressing cells in the lesion vicinity. In a patient suffering from epilepsy, neurons and oligodendrocytes of the hippocampus strongly express EPOR. To conclude, this new analytical tool will allow neuroscientists to pinpoint EPOR expression in cells of the nervous system and to better understand its role in healthy conditions, including brain development, as well as under pathological circumstances, such as upregulation upon distress and injury.