Breathing Life into Polycations: Functionalization with pH-Responsive Endosomolytic Peptides and Polyethylene Glycol Enables siRNA Delivery

The lack of efficient delivery systems is still limiting the full therapeutic potential of siRNA. For the purpose of nucleic acid transfer, among other synthetic carrier systems, polycations have been applied. Favorable characteristics of suitable polymers include nucleic acid binding, compaction, protection, and biocompatibility. However the lack of nucleic acid transfer activity in transfection-based screening often abandons promising candidates. Here we present that functionalization may turn polycations with poor delivery activity into efficient carriers:  for example, polylysine, on its own lacking nucleic acid transfer activity, displayed high efficiency in siRNA delivery after modification with polyethylene glycol and a pH-responsive endosomolytic peptide. Hence these findings have implication for the selection process of polymeric carriers for siRNA

Molecular Recognition with 2,4-Diaminotriazine-Functionalized Colloids

New polymeric colloids functionalized with 2,4-diaminotriazine residues have been prepared. The functionalities provide a triple hydrogen bond motif with a donor–acceptor–donor (DAD) pattern. The colloids are based on cross-linked poly-4-methoxymethyl styrene and are polymerized by means of surfactant-free emulsion polymerization. The reaction pathway including five steps was successfully tracked and verified via 13C CP/MAS solid-state NMR. Characterization of the colloids was done by combined static and dynamic light scattering and indicates a compact spherical particle shape. In solvents with the appropriate polarity, intercolloidal hydrogen bonding was enabled, including colloidal aggregation. In highly dilute solutions of THF, this aggregation was recordable by means of time-resolved static light scattering experiments. If THF was saturated with uracil, then aggregation could be completely inhibited. Uracil bears a triple hydrogen bond motif of the form acceptor–donor–acceptor (ADA) and is a direct antagonist of 2,4-diaminotriazine. The charging of the colloids with uracil via hydrogen bond formation as a typical molecular recognition mechanism could be confirmed by IR spectroscopy

Multilamellar Vesicle Formation Probed by Rheo-NMR and Rheo-SALS under Large Amplitude Oscillatory Shear

The formation of multilamellar vesicles (MLVs) in the lyotropic lamellar phase of the system triethylene glycol mono n-decyl ether (C10E3)/water is investigated under large amplitude oscillatory shear (LAOS) using spatially resolved rheo-NMR spectroscopy and a combination of rheo-small angle light scattering (rheo-SALS) and conventional rheology. Recent advances in rheo-NMR hardware development facilitated the application of LAOS deformations in high-field NMR magnets. For the range of investigated strain amplitudes (10–50) and frequencies (1 and 2 rad s–1), MLV formation is observed in all NMR and most SALS experiments. It is found that the MLV size depends on the applied frequency in contrast to previous steady shear experiments where the shear rate is the controlling parameter. The onset of MLV formation, however, is found to vary with the shear amplitude. The LAOS measurements bear no indication of the intermediate structures resembling aligned multilamellar cylinders observed in steady shear experiments. Lissajous curves of stress vs strain reveal a transition from a viscoelastic solid material to a pseudoplastic material

Tuning the “Roadblock” Effect in Kinesin-Based Transport

Major efforts are underway to harness motor proteins for technical applications. Yet how to best attach cargo to microtubules that serve as kinesin-driven “molecular shuttles” without compromising transport performance remains challenging. Furthermore, microtubule-associated proteins (MAPs) can block motor protein-powered transport in neurons, which can lead to neurodegenerative diseases. Again it is unclear how different physical roadblock parameters interfere with the stepping motion of kinesins. Here, we employ a series of MAPs, tailored (strept)­avidins, and DNA as model roadblocks and determine how their geometrical, nanomechanical, and electrochemical properties can reduce kinesin-mediated transport. Our results provide insights into kinesin transport regulation and might facilitate the choice of appropriate cargo linkers for motor protein-driven transport devices

A Yeast Mutant Deleted of <i>GPH1</i> Bears Defects in Lipid Metabolism

<div><p>In a previous study we demonstrated up-regulation of the yeast <i>GPH1</i> gene under conditions of phosphatidylethanolamine (PE) depletion caused by deletion of the mitochondrial (M) phosphatidylserine decarboxylase 1 (<i>PSD1</i>) (Gsell et al., 2013, PLoS One. 8(10):e77380. doi: <a href="http://dx.doi.org/10.1371/journal.pone.0077380" target="_blank">10.1371/journal.pone.0077380</a>). Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC), triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a Δ<i>gph1</i> strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. <i>In vivo</i> labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP)-choline and the methylation route, is negatively affected by a Δ<i>gph1</i> mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism.</p></div

Phospholipid composition of cell-free homogenate, plasma membrane and mitochondria from cells grown on YPD or minimal medium (*).

<p>CF, cellular fraction; LPL, lysophospholipids; PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; CL, cardiolipin; DMPE, dimethylphosphatidylethanolamine; PA, phosphatidic acid. WT [pYES2], wild type bearing plasmid pYES2; WT [pYES_gph1], overexpression of <i>GPH1</i> on plasmid pYES in wild type background. Mean values of at least three measurements and standard deviations are shown.</p

Gelled Lyotropic Liquid Crystals

In our previous work we were able to prove that gelled bicontinuous microemulsions are a novel type of orthogonal self-assembled system. The study at hand aims at complementing our previous work by answering the question of whether gelled lyotropic liquid crystals are also orthogonal self-assembled systems. For this purpose we studied the same system, namely, water–<i>n</i>-decane/12-hydroxy­octa­decanoic acid (12-HOA)–<i>n</i>-decyl tetra­oxy­ethylene glycol ether (C₁₀E₄). The phase boundaries of the nongelled and the gelled lyotropic liquid crystals were determined visually and with ²H NMR spectroscopy. Oscillating shear measurements revealed that the absolute values of the storage and loss moduli of the gelled liquid crystalline (LC) phases do not differ very much from those of the binary organogel. While both the phase behavior and the rheological properties of the LC phases support the hypothesis that gelled lyotropic liquid crystals are orthogonal self-assembled systems, freeze–fracture electron microscopy (FFEM) seems to indicate an influence of the gel network on the structure of the L_α phase and vice versa

Tuning the “Roadblock” Effect in Kinesin-Based Transport

Major efforts are underway to harness motor proteins for technical applications. Yet how to best attach cargo to microtubules that serve as kinesin-driven “molecular shuttles” without compromising transport performance remains challenging. Furthermore, microtubule-associated proteins (MAPs) can block motor protein-powered transport in neurons, which can lead to neurodegenerative diseases. Again it is unclear how different physical roadblock parameters interfere with the stepping motion of kinesins. Here, we employ a series of MAPs, tailored (strept)­avidins, and DNA as model roadblocks and determine how their geometrical, nanomechanical, and electrochemical properties can reduce kinesin-mediated transport. Our results provide insights into kinesin transport regulation and might facilitate the choice of appropriate cargo linkers for motor protein-driven transport devices

Expression levels of <i>PSD1</i>, <i>PSD2</i>, <i>CHO2</i> and <i>OPI3</i> in the Δ<i>gph1</i> deletion mutant.

<p>Relative gene expression of <i>PSD1</i>, <i>PSD2</i>, <i>CHO2</i> and <i>OPI3</i> in the Δ<i>gph1</i> deletion mutant was measured by RT-qPCR. A <i>Δpsd1</i> mutant was used as a negative control. Wild type was set at 1. Data are mean values from three independent experiments with the respective deviation.</p