Liquid Crystalline Phase Formation in Suspensions of Solid Trimyristin Nanoparticles

The presence of liquid crystalline phases in suspensions of solid lipid nanoparticles can increase the risk of their gelling upon administration through fine needles. Here we study the formation of liquid crystalline phases in aqueous suspensions of platelet-like shaped solid lipid nanoparticles. A native lecithin-stabilized trimyristin (20 wt %) suspension was investigated at different dilution levels by small-angle X-ray scattering (SAXS) and visual inspection of their birefringence between two crossed polarizers. For trimyristin concentrations φ_MMM < 6 wt %, the dispersed platelets are well separated from each other whereas they start to self-assemble into stacked lamellae for 6 wt % ≤ φ_MMM < 12 wt %. For φ_MMM ≥ 12 wt %, the SAXS patterns become increasingly anisotropic, which is a signature of an evolving formation of a preferred orientation of the platelets on a microscopic scale. Simultaneously, the suspensions become birefringent, which proves the existence of an anisotropic liquid crystalline phase formed in the still low viscous liquid suspensions. Spatially resolved SAXS scans and polarization microscopy indicate rather small domains in the (sub)­micrometer size range in the nematic liquid crystalline phase and the presence of birefringent droplets (tactoids). The observed critical concentrations for the formation of stacks and the liquid crystalline phase are significantly higher as for equivalent suspensions prepared from triglycerides with longer chains. This can be explained with the lower aspect ratio of trimyristin platelets. Special emphasis is put on the isotropic–liquid crystalline phase transition as a function of the ionic strength of the dispersion medium and φ_MMM. Higher salt concentrations allow shifting of the phase transition to higher trimyristin concentrations. This can be attributed to a partial screening of the repulsive forces between the platelets, which allows higher packing densities within the platelet stacks and of remaining isolated platelets

Localization of formazan granules on filamentous <i>E. coli</i> cells.

<p>A, Percentage of filaments with different numbers of granules on the cell poles (n = 225). The inset shows a typical cell with three granules. They are bright spots in the dark-field image. B, The distribution of granules (open dots) on the filaments relative to the geometrical midpoint of cells. The green and blue lines mark the position of 2 and 4 cell-lengths (CLs) from the cell poles, respectively. C, The number of granules per 100 cells with the same color scheme as in B: OP, old pole; 2CL-O, the region within 2 CLs of the old pole; 4CL-O, the region between 2 CLsand 4 CLs of the old pole; 4CL-N, the region between 2 CLs and 4 CLs of the new pole; 2CL-N, the region within 2 CLs of the new pole; NP, new pole. D, A diffusion to capture model of granule formation. The old poles of the cells are depicted on the left-hand side. Arrows represent the diffusion of reduced formazan. The three cells shown on top are newborn, fully elongated, and dividing cells. The broken line represents a septum. A filamentous cell of 8 CLs is shown below.</p

Transmission electron microscopy images of <i>E. coli</i> cells containing formazan granules.

<p>Scale bars equal 400 nm. A, Longitudinal section of a control cell growing in medium without tetrazolium. B, Longitudinal section of a cell growing in the presence of TTC. The formazan granule is located at the right pole of the cell. C, Expanded view of the boxed area in panel. The cytoplasmic membrane is indicated by a closed arrow. B. The small lateral granule is indicated by an open arrow. D, Expanded view of the pole area of the control cell. E, Expanded view of the pole of a cell growing in TTC. The concentric contour lines on the cutting surface of the granule are indicated by open arrowheads. F, A further expanded picture of a cell pole, showing the concentric contour lines (open arrowhead) and the cytoplasmic membrane (closed arrow). G, An ultrathin section in which three control cells were cut at different positions, the bottom one being at the tip of the pole. H, Ultrathin sections of poles of cells growing in a medium containing tetrazolium for 12 h. (upper panels: longitudinal sections; lower panels: transverse sections). I, Sections of the poles of cells growing in the presence of tetrazolium for 18 h (upper panels: longitudinal sections; lower panels: transverse sections).</p

Computer simulation of the formazan diffusion in cell periplasm. A,

<p>Average free energy of a granule as a function of its center of mass (ΔE_COM) along the longitudinal cell axis. The energy of the granule at the poles was taken as zero. The curves correspond to different numbers of particles. B, Frequency of granule location as a function of position on the cell and aggregate size (<i>N</i>). The heat map of particles added at fast rate (<i>N_add</i>  = 10,000 MC steps) is shown above. The map of granules added at slow rate (<i>N_add</i>  = 50,000 MC steps) is shown below. The top color scheme shows the frequency value. C, Probability of granule localizing to different positions on cell at slow addition rate. Line colors are the same as in A. D, Snapshots of three critical steps of aggregate formation in a simulation movie. I, initial randomly diffusing particles; II, a seed forms stochastically; III, diffusion to and growth at the energetically favored cell pole.</p

Formazan granule formation in different <i>E. coli</i> mutants.

<p>Scale bars equal 20 µm. Granules were false-colored in dark magenta. The top two lines show the parental strain (MC1000) and its derivatives: the <i>dsb</i>D deletion, the <i>dsb</i>A deletion, and the <i>dsbA</i>/<i>dsbD</i> double mutant strains. The third line shows the parental strain (MC1061) and the Ccm null mutant derived from it. Photos of the corresponding cell cultures after growth in the presence of TTC are shown at the top-right corner of each DIC image. The percentage of cells containing different numbers of granules for each strain is compared in the histogram at the bottom of the figure.</p

Mesoscopic Structures of Triglyceride Nanosuspensions Studied by Small-Angle X‑ray and Neutron Scattering and Computer Simulations

Aqueous suspensions of platelet-like shaped tripalmitin nanocrystals are studied here at high tripalmitin concentrations (10 wt % tripalmitin) for the first time by a combination of small-angle X-ray and neutron scattering (SAXS and SANS). The suspensions are stabilized by different lecithins, namely, DLPC, DOPC, and the lecithin blend S100. At such high concentrations the platelets start to self-assemble in stacks, which causes interference maxima at low <i>Q</i>-values in the SAXS and SANS patterns, respectively. It is found that the stack-related interference maxima are more pronounced for the suspension stabilized with DOPC and in particular DLPC, compared to suspensions stabilized by S100. By use of the X-ray and neutron powder pattern simulation analysis (XNPPSA), the SAXS and SANS patterns of the native tripalmitin suspensions could only be reproduced simultaneously when assuming the presence of both isolated nanocrystals and stacks of nanocrystals of different size in the simulation model of the dispersions. By a fit of the simulated SAXS and SANS patterns to the experimental data, a distribution of the stack sizes and their volume fractions is determined. The volume fraction of stacklike platelet assemblies is found to rise from 70% for S100-stabilized suspensions to almost 100% for the DLPC-stabilized suspensions. The distribution of the platelet thicknesses could be determined with molecular resolution from a combined analysis of the SAXS and SANS patterns of the corresponding diluted tripalmitin (3 wt %) suspensions. In accordance with microcalorimetric data, it could be concluded that the platelets in the suspensions stabilized with DOPC, and in particular DLPC, are significantly thinner than those stabilized with S100. The DLPC-stabilized suspensions exhibit a significantly narrower platelet thickness distribution compared to DOPC- and S100-stabilized suspensions. The smaller thicknesses for the DLPC- and DOPC-stabilized platelets explain their higher tendency to self-assemble in stacks. The finding that the nanoparticles of the suspension stabilized by the saturated lecithin DLPC crystallize in the stable β-tripalmitin modification with its characteristic platelet-like shape is surprising and can be explained by the fact that the main phase transformation temperature for DLPC is, as for unsaturated lecithins like DOPC and S100, well below the crystallization temperature of the supercooled tripalmitin emulsion droplets

Timelapse images showing transfer of GFP between cells

Co-cultures of cross-feeding genotypes of E.coli and A.baylyi were observed after 9 hours of growth in liquid media. Images of this co-culture were taken every 2 minutes to visualize the transfer of GFP between cells connected by nanotubes. The zip file contains unprocessed images in tiff format of three different channels- bright-field, green and red. Each folder contains 18 images, the file name indicates the time point

Complexity and Variability of Gut Commensal Microbiota in Polyphagous Lepidopteran Larvae

<div><h3>Background</h3><p>The gut of most insects harbours nonpathogenic microorganisms. Recent work suggests that gut microbiota not only provide nutrients, but also involve in the development and maintenance of the host immune system. However, the complexity, dynamics and types of interactions between the insect hosts and their gut microbiota are far from being well understood.</p> <h3>Methods/Principal Findings</h3><p>To determine the composition of the gut microbiota of two lepidopteran pests, <em>Spodoptera littoralis</em> and <em>Helicoverpa armigera</em>, we applied cultivation-independent techniques based on 16S rRNA gene sequencing and microarray. The two insect species were very similar regarding high abundant bacterial families. Different bacteria colonize different niches within the gut. A core community, consisting of Enterococci, Lactobacilli, Clostridia, <em>etc</em>. was revealed in the insect larvae. These bacteria are constantly present in the digestion tract at relatively high frequency despite that developmental stage and diet had a great impact on shaping the bacterial communities. Some low-abundant species might become dominant upon loading external disturbances; the core community, however, did not change significantly. Clearly the insect gut selects for particular bacterial phylotypes.</p> <h3>Conclusions</h3><p>Because of their importance as agricultural pests, phytophagous Lepidopterans are widely used as experimental models in ecological and physiological studies. Our results demonstrated that a core microbial community exists in the insect gut, which may contribute to the host physiology. Host physiology and food, nevertheless, significantly influence some fringe bacterial species in the gut. The gut microbiota might also serve as a reservoir of microorganisms for ever-changing environments. Understanding these interactions might pave the way for developing novel pest control strategies.</p> </div

Different gut bacterial community structures in <i>S. littoralis</i> larvae of different instars feeding on artificial diet.

<p>A, The bacterial community compositions detected by cloning and sequencing from insects that are 2-days (n = 33), 6-days (n = 104), 10-days (n = 232), and 14-days (n = 490). The arrow represents the life span of an <i>S. littoralis</i> larva. The developmental stages, hatch, pupation, and larval instars are represented by bars. The inset shows the relative abundance of bacteria detected on the epithelium of 10-day old larvae (n = 94). B, The rarefaction curves of the richness indices Chao1 and ACE, and the diversity indices Shannnon and Simpson based on sequences retrieved from larvae. Indices were calculated using 95% confidence level and 0.03 distance cutoff for OUT clustering.</p

Bacterial localization in the gut of S. littoralis larvae with Fluorescent In Situ Hybridization.

<p>Scale bar equals 10 µm. A, Detection of <i>Clostridium</i> sp. In the midgut. The three images shown are TIC image, fluorescent image of universal probe (EUB, red) and of specific probe (SPE, green). B to G are merged images of TIC, EUB and SPE. The bacteria detected only with universal probe are red, and the bacterial with both probes are green. B, a large aggregate of <i>Clostridium</i> sp. deep in the gut lumen. C, Detection of <i>E. mundtii</i>. D, Detection of <i>E. casseliflavus</i>. E, <i>P. acnes</i> in the midgut. F, <i>E. coli</i> detected in the midgut; G, <i>K. pneumonia</i> detected in the midgut. Bacteria detected only by universal probe are highlighted with white arrows; Bacteria stained by sequence-specific probes are pointed by open arrows. Insect tissue is indicated by arrow heads.</p