LC-MS characterization and cell-binding properties of chelate modified somatropin

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is recently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. In some of these cancers, over-expression of the human growth hormone receptor (hGHR) is described. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. As site-specific labelling is necessary to avoid active region interactions (1-16, 41-68, 103-119 and 167-175), characterization of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and a combination of both enzymes. The resulting peptides were then monitored using HPLC-MSn, allowing the investigation of the exact amino acid modifications. The use of a mixture of trypsin and chymotrypsin gave an enhanced information efficiency. Moreover, the intact protein, without enzymatic degradation, was analysed on a protein HPLC column using UV detection for quantification and ESI-MS/MS for characterization. Based upon the HPLC-MSn results of the digested somatropin, the chelating molecule is mainly bound to a specific lysine amino acid that is located away from the receptor binding site. Therefore, the cell-binding functionality of the characterized NOTA-somatropin is measured, using a HepG2 cell line

Genetic diversity and core subset selection in ex situ seed collections of the banana crop wild relative Musa balbisiana

Crop wild relatives (CWRs) play a key role in crop breeding by providing beneficial trait characteristics for improvement of related crops. CWRs are more efficiently used in breeding if the plant material is genetically characterized, but the diversity in CWR genetic resources has often poorly been assessed. Seven seed collections of Musa balbisiana, an important CWR of dessert and cooking bananas, originating from three natural populations, two feral populations and two ex situ field collections were retrieved and their genetic diversity was quantified using 18 microsatellite markers to select core subsets that conserve the maximum genetic diversity. The highest genetic diversity was observed in the seed collections from natural populations of Yunnan, a region that is part of M. balbisiana's centre of origin. The seeds from the ex situ field collections were less genetically diverse, but contained unique variation with regards to the diversity in all seed collections. Seeds from feral populations displayed low genetic diversity. Core subsets that maximized genetic distance incorporated almost no seeds from the ex situ field collections. In contrast, core subsets that maximized allelic richness contained seeds from the ex situ field collections. We recommend the conservation and additional collection of seeds from natural populations, preferentially originating from the species' region of origin, and from multiple individuals in one population. We also suggest that the number of seeds used for ex situ seed bank regeneration must be much higher for the seed collections from natural populations

Controllable Tuning Plasmonic Coupling with Nanoscale Oxidation.

The nanoparticle on mirror (NPoM) construct is ideal for the strong coupling of localized plasmons because of its simple fabrication and the nanometer-scale gaps it offers. Both of these are much harder to control in nanoparticle dimers. Even so, realizing controllable gap sizes in a NPoM remains difficult and continuous tunability is limited. Here, we use reactive metals as the mirror so that the spacing layer of resulting metal oxide can be easily and controllably created with specific thicknesses resulting in continuous tuning of the plasmonic coupling. Using Al as a case study, we contrast different approaches for oxidation including electrochemical oxidation, thermal annealing, oxygen plasma treatments, and photo-oxidation by laser irradiation. The thickness of the oxidation layer is calibrated with depth-mode X-ray photoemission spectroscopy (XPS). These all consistently show that increasing the thickness of the oxidation layer blue-shifts the plasmonic resonance peak while the transverse mode remains constant, which is well matched by simulations. Our approach provides a facile and reproducible method for scalable, local and controllable fabrication of NPoMs with tailored plasmonic coupling, suited for many applications of sensing, photochemistry, photoemission, and photovoltaics.EPSRC grant EP/G060649/1, EP/I012060/1, ERC grant LINASS 320503.This is the final version of the article. It first appeared from the American Chemical Society via http://dx.doi.org/10.1021/acsnano.5b0128

The importance of feeding status and desiccation rate in successful anhydrobiosis of Panagrolaimus detritophagus

We examined the effect of nutritional status and desiccation rate on the ability of Panagrolaimus detritophagus to undergo anhydrobiosis, as well as to survive high temperatures in the dried state. Both nutrition and drying rate were found to be important, with starvation and slow drying providing better success at anhydrobiosis. The upper temperature for survival of dried animals in laboratory studies was 80 degrees C. Starved worms recovered from drying more successfully when the starvation period was followed by a smooth, gradual dry period prior to undergoing desiccation. Thus, the ability of these worms to enter and leave anhydrobiosis is dependent on critical stress signals

Paradigm of biased PAR1 (protease-activated receptor-1) activation and inhibition in endothelial cells dissected by phosphoproteomics

Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of PARs (protease-activated receptors). The predominant thrombin receptor is PAR1, and in endothelial cells (ECs), thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear whether thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N terminus have remained an unresolved issue. Here, we used a quantitative phosphoproteomics approach to show that classical and peptide activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in the presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs. Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrate that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-tethered ligand peptide induce similar phosphoregulation, and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating that it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology

Correction: Schelfhout, S.; et al. Tree Species Identity Shapes Earthworm Communities. Forests 2017, 8, 85

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