Differential survival and reproduction in colour forms of Philaenus spumarius give new insights to the study of its balanced polymorphism

1. Colour polymorphisms are common across animals and are often the result of complex selection regimes. Philaenus spumarius (Linnaeus) (Hemiptera, Aphrophoridae) shows a widely studied dorsal colour polymorphism with several described phenotypes whose variation in their occurrence and frequency, as well as their maintenance across time, have been reported. Several selective influences have been suggested to play a role, but the mechanisms underlying the maintenance of this polymorphism are still poorly understood. 2. To explore the adaptive significance of the colour polymorphism of P. spumarius, an experiment was conducted in captivity under semi‐natural conditions to measure survival, reproductive success, and duration of egg maturation. 3. It was found that there was higher longevity, a higher number of oviposition events, and a higher number of eggs laid by trilineatus phenotype females than by typicus and marginellus, supporting previous reports of an increase in trilineatus frequency during the season. The duration of egg maturation did not differ among phenotypes. 4. The higher longevity and fertility of the trilineatus phenotype may compensate, for example, the higher rate of attack by parasitoids and/or higher solar radiation reflectance in this phenotype, which have already been reported in previous studies, constituting a possible mechanism for the maintenance of this polymorphism.     

APSY-NMR for protein backbone assignment in high-throughput structural biology

A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h. The results of the automated procedure provided a basis for efficient interactive validation and extension to near-completion of the assignments by reference to the same 3D heteronuclear-resolved [¹H,¹H]-NOESY spectra that were subsequently used for the collection of conformational constraints. High-quality structures were obtained for all 30 proteins, using the J-UNIO protocol, which includes extensive automation of NMR structure determination.     

Sorting of Clathrin‐Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin‐Mediated Endocytosis

Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.      

Engineering cell surfaces via liposome fusion

In this study, we have rewired cell surfaces with ketone and oxyamine molecules based on liposome fusion for applications in cell-surface engineering. Lipid vesicles, functionalized with ketone and oxyamine molecules, display complementary chemistry and undergo recognition, docking, and subsequent fusion upon covalent oxime bond formation. Liposome fusion was characterized by several techniques including matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS), light scattering, fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). When cultured with cells, ketone- and oxyamine-containing liposomes undergo spontaneous membrane fusion to present the respective molecules from cell surfaces. Ketone-functionalized cell surfaces serve as sites for chemoselective ligation with oxyamine-conjugated molecules. We tailored and fluorescently labeled cell surfaces with an oxyamine-conjugated rhodamine dye. As an application of this cell-surface engineering strategy, ketone- and oxyamine-functionalized cells were patterned on oxyamine- and ketone-presenting surfaces, respectively. Cells adhered, spread, and proliferated in the patterned regions via interfacial oxime linkage. The number of ketone molecules on the cell surface was also quantified by flow cytometry.     

Physicochemical Characterization of NPC 1161C,A Novel Antimalarial 8-Aminoquinoline,in Solution and Solid State

NPC 1161C is a novel antimalarial drug of interest because of its superior curative and prophylactic activity, and favorable toxicity profile against in vivo and in vitro models of malaria, pneumocystis carinii pneumonia, and leishmaniasis. The preformulation studies performed included determination of pK_as, aqueous and pH solubility, cosolvent solubility, log P, pH stability, thermal analysis, and preliminary hygroscopicity studies. The mean pK_a1, pK_a2, and pK_a3 were determined to be 10.12, 4.07, and 1.88, respectively. The aqueous solubility was found to be 2.4 × 10⁻⁴ M having a saturated solution pH of 4.3–5.0 and a low intrinsic solubility of 1.6 × 10⁻⁶ M. A mathematical model of the pH-solubility profile was derived from pH 2.2 to 8.0. An exponential decrease in solubility was observed with increasing pH. The excess solid phase in equilibrium with the solution in aqueous buffers was determined to be the free-base form of the drug. A significant increase in solubility was observed with all the cosolvents studied, in both unbuffered and buffered systems. Mean log P of the salt and the free base were estimated to be 2.18 and 3.70, respectively. The compound had poor stability at pH 7.0 at 37°C, with a t ₉₀ of 3.58 days. Thermal analysis of the drug using DSC and TGA revealed that the drug is present as a semi-crystalline powder, which transformed into the amorphous state after melting. The drug was also found to sublime at higher temperatures. Determination of physicochemical properties of NPC 1161C provided useful information for the development of a dosage form and preclinical evaluation.     

Therapeutic siRNA silencing in inflammatory monocytes in mice

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism

The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases. Kinetic stability analyses (T(50) and t(1/2) ) and refolding experiments show similar thermal stability and refolding efficiency for both forms. These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism.