Analiza nakupovalnih centrov v Ljubljani s pomočjo atributivnega pristopa

Alginate microgels are widely used as delivery systems in food, cosmetics, and pharmaceutical industries for encapsulation and sustained release of hydrophilic compounds and cells. However, the encapsulation of lipophilic molecules inside these microgels remains a great challenge because of the complex oil-core matrix required. The present study describes an original two-step approach allowing the easy encapsulation of several oil microdroplets within alginate microgels. In the first step, stable oil microdroplets were formed by preparing an oil-in-water (O/W) Pickering emulsion. To stabilize this emulsion, we used two solid particles, namely the cotton cellulose nanocrystals (CNC) and calcium carbonate (CaCO₃). It was observed that the surface of the oil microdroplets formed was totally covered by a CNC layer, whereas CaCO₃ particles were adsorbed onto the cellulose layer. This solid CNC shell efficiently stabilized the oil microdroplets, preventing them from undesired coalescence. In the second step, oil microdroplets resulting from the Pickering emulsion were encapsulated within alginate microgels using microfluidics. Precisely, the outermost layer of oil microdroplets composed of CaCO₃ particles was used to initiate alginate gelation inside the microfluidic device, following the internal gelation mode. The released Ca²⁺ ions induced the gel formation through physical cross-linking with alginate molecules. This innovative and easy to carry out two-step approach was successfully developed to fabricate monodisperse alginate microgels of 85 μm in diameter containing around 12 oil microdroplets of 15 μm in diameter. These new oil-core alginate microgels represent an attractive system for encapsulation of lipophilic compounds such as vitamins, aroma compounds or anticancer drugs that could be applied in various domains including food, cosmetics, and medical applications

The life cycle of <i>S</i>. <i>boydii</i> under scanning electron microscopy.

<p>After release, conidia (<b>A</b>) germinate (<b>B</b>) and the hyphal part of germ tubes elongates (<b>C</b>) until a first branch emerges near the mother cell (<b>D</b>). Both hyphae grow and more branching sites appear on filaments at the subapical region of the articles (<b>E</b>) until the mother cell can no more be distinguished (<b>F</b>). Arrows indicate sites of first branching, and later branching are indicated by arrowheads. All cultures were performed in YPD broth with incubation at 37°C for 6h (<b>B</b>), 8h (<b>C</b>), 10 h (<b>D</b> and <b>E</b>) or 24 h (<b>F</b>). Bars: 1 μm in A, B and C; 0.5 μm in D; and 5 μm in E and F.</p

Surface charge modifications during germination of <i>S</i>. <i>boydii</i> by ferritin labeling and zeta potential measurements.

<p>TEM images of germ tubes labeled with cationized ferritin (<b>A</b>), germ tubes treated with neuraminidase prior cationized ferritin labeling (<b>B</b>) or germ tubes incubated with native ferritin (<b>C</b>). (<b>D</b>) Comparison of the surface electrostatic charge of resting and germinated conidia calculated from the electrophoretic mobility of 10 000 cells using Zetasizer Nano ZS (<i>P</i> = 0.0005). <b>H</b>: hyphal part of germ tube; <b>MC</b>: mother cell of germ tube. Bars: 1 μm.</p

Fluorescence labeling of <i>S</i>. <i>boydii</i> surface carbohydrates with FITC-conjugated lectins.

<p>Germ tubes after labeling with concanavalin A (<b>A</b> and <b>C</b>) or wheat germ agglutinin (<b>B</b> and <b>D</b>) lectins. The same fields are presented under fluorescence (<b>A</b> and <b>B</b>) and phase contrast microscopy (<b>C</b> and <b>D</b>) respectively. Arrows indicate mother cells.</p

Kinetics of germination of <i>S</i>. <i>boydii</i> in various conditions.

<p><b>(A</b>) Conidia isolated from 5-, 9- and 14-day-old cultures on yeast peptone dextrose (YPD) agar were incubated in YPD liquid medium over 8 h at 37°C. (<b>B</b>) Conidia isolated from 9-day-old cultures on Malt or YPD agar were incubated in Malt or YPD liquid media over 8 h at 37°C. (<b>C</b>) Conidia isolated from 9-day-old cultures on YPD agar were incubated in YPD liquid medium for 16 h at 20°C, 25°C or 37°C (200X).</p

High resolution imaging and chemical force spectroscopy analysis of <i>S</i>. <i>boydii</i> resting conidia and germ tubes.

<p>AFM amplitude images of a resting (<b>A</b>) or germinated (<b>B</b>) <i>S</i>. <i>boydii</i> conidium. (<b>C) Left</b>, scheme for chemical functionalization of AFM tips. Gold-coated tips were modified with CH₃-terminated alkanethiols or OH-terminated alkanethiols. (<b>C</b>) <b>Right</b>, histograms of hydrophobic adhesion forces measured on the surface of a resting conidium (1.8 ± 0.3 nN, in red) and the hyphal part of a germinated conidium (0.85 ± 0.15 nN, in blue).</p

Sequence analysis of the identified GPI-anchored proteins.

<p>^<i>a</i> RC: resting conidia; GT: germ tube.</p><p>^<i>b</i> S: serine; T: threonine.</p><p>^<i>c</i> Bold: best predicted ω site. Underlined: alternative ω site (second best). Italic: basic amino acids.</p><p>^<i>d</i> GRAVY index > 0: hydrophobic; GRAVY index < 0: hydrophilic.</p><p>Sequence analysis of the identified GPI-anchored proteins.</p

Gold labeling of cell wall mannan groups in <i>S</i>. <i>boydii</i> germ tubes.

<p>Germ tubes labeled with gold-conjugated concanavalin A (Con A; 5-nm gold particles) showing higher affinity of gold particles to the hyphal part (H) of germ tubes compared to the mother cell (MC) under transmission electron microscopy. Arrow indicates the limit of the outer cell wall layer of the mother cell. Bar: 0.5 μm.</p

UV-visible spectrophotometry of melanin extracts.

<p>Melanin extracts from control conidia or conidia recovered from cultures grown in the presence of DOPA-melanin (kojic acid or glyphosate) or DHN-melanin (pyroquilon, tricyclazol or carpropamid) inhibitors were examined under UV-visible spectrophotometry. A spectrum similar to synthetic melanin (0.05 mg/ml) was obtained only for extracts from control conidia or conidia produced with DOPA-melanin inhibitors.</p

Changes in melanin amount in conidia as the age of culture progresses.

<p>Melanin amount in the conidial wall changed with the age of culture as revealed by UV-visible spectrophotometry (<b>A</b>) or electron paramagnetic resonance (<b>B</b>). (<b>A</b>) The mean rank of the quantity of melanin extracted from conidia was significantly different among cultures of different age (<i>p</i><0.05). (<b>B</b>) EPR spectra of <i>P. boydii</i> conidia taken from cultures of different age (5, 9 and 14 days). The spectra were recorded at a microwave power of 30 db. Spectra are representative of three independent experiments.</p