Can BioSAXS detect ultrastructural changes of antifungal compounds in Candida albicans?

The opportunistic yeast $\textit {Candida albicans}$ is the most common cause of candidiasis. With only four classes of antifungal drugs on the market, resistance is becoming a problem in the treatment of fungal infections, especially in immunocompromised patients. The development of novel antifungal drugs with different modes of action is urgent. In 2016, we developed a groundbreaking new medium-throughput method to distinguish the effects of antibacterial agents. Using small-angle X-ray scattering for biological samples (BioSAXS), it is now possible to screen hundreds of new antibacterial compounds and select those with the highest probability for a novel mode of action. However, yeast (eukaryotic) cells are highly structured compared to bacteria. The fundamental question to answer was if the ultrastructural changes induced by the action of an antifungal drug can be detected even when most structures in the cell stay unchanged. In this exploratory work, BioSAXS was used to measure the ultrastructural changes of $\textit {C. albicans}$ that were directly or indirectly induced by antifungal compounds. For this, the well-characterized antifungal drug Flucytosine was used. BioSAXS measurements were performed on the synchrotron P12 BioSAXS beamline, EMBL (DESY, Hamburg) on treated and untreated yeast $\textit {C. albicans}$. BioSAXS curves were analysed using principal component analysis (PCA). The PCA showed that Flucytosine-treated and untreated yeast were separated. Based on that success further measurements were performed on five antifungal peptides {1. Cecropin A-melittin hybrid [CA (1–7) M (2–9)], KWKLFKKIGAVLKVL; 2. Lasioglossin LL-III, VNWKKILGKIIKVVK; 3. Mastoparan M, INLKAIAALAKKLL; 4. Bmkn2, FIGAIARLLSKIFGKR; and 5. optP7, KRRVRWIIW}. The ultrastructural changes of $\textit {C. albicans}$ indicate that the peptides may have different modes of action compared to Flucytosine as well as to each other, except for the Cecropin A-melittin hybrid [CA (1–7) M (2–9)] and optP7, showing very similar effects on $\textit {C. albicans}$. This very first study demonstrates that BioSAXS shows promise to be used for antifungal drug development. However, this first study has limitations and further experiments are necessary to establish this application

Sol–Gel Transition in Nanodiamond Aqueous Dispersions by Small-Angle Scattering

This paper reports the results of the comparative structural characterization of detonation nanodiamond particles and their aggregates in hydrosols and hydrogels by small-angle scattering (SAS) techniques. The data from different neutron and X-ray (synchrotron radiation) diffractometers cover a wide range of momentum transfer and show multilevel structure organizations at the size scale from 1 to 1000 nm and higher. For this purpose, in addition to the conventional SAS techniques the methods of very small-angle and ultrasmall-angle neutron scattering were applied. The fraction of nanodiamond particles in the aggregates is determined. A complex two-step mechanism of nanodiamond cluster association into a network during the sol–gel transition is revealed. It is assumed that a reason for the reversibility of this process is a different compactness of the corresponding structural levels defined by different fractal organizations

C60\mathrm{C_{60}} fullerene enhances cisplatin anticancer activity and overcomes tumor cell drug resistance

We formulated and analyzed a novel nanoformulation of the anticancer drug cisplatin (Cis) with C$_{60}$ fullerene (C$_{60}$+Cis complex) and showed its higher toxicity toward tumor cell lines in vitro when compared to Cis alone. The highest toxicity of the complex was observed in HL-60/adr and HL-60/vinc chemotherapy-resistant human leukemia cell sublines (resistant to Adriamycin and Vinculin, respectively). We discovered that the action of the C$_{60}$+Cis complex is associated with overcoming the drug resistance of the tumor cell lines through observing an increased number of apoptotic cells in the Annexin V/PI assay. Moreover, in vivo assays with Lewis lung carcinoma (LLC) C$_{57}$BL/6J male mice showed that the C$_{60}$+Cis complex increases tumor growth inhibition, when compared to Cis or C$_{60}$ fullerenes alone. Simultaneously, we conducted a molecular docking study and performed an Ames test. Molecular docking specifies the capability of a C$_{60}$ fullerene to form van der Waals interactions with potential binding sites on P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP-1), and multidrug resistance protein 2 (MRP-2) molecules. The observed phenomenon revealed a possible mechanism to bypass tumor cell drug resistance by the C$_{60}$+Cis complex. Additionally, the results of the Ames test show that the formation of such a complex diminishes the Cis mutagenic activity and may reduce the probability of secondary neoplasm formation. In conclusion, the C$_{60}$+Cis complex effectively induced tumor cell death in vitro and inhibited tumor growth in vivo, overcoming drug resistance likely by the potential of the C$_{60}$ fullerene to interact with P-gp, MRP-1, and MRP-2 molecules. Thus, the C$_{60}$+Cis complex might be a potential novel chemotherapy modification

SAXS data for suspended melanosomes from C57BL/6J (B6) and DBA/2J (D2) mice.

<p>Melanosomes were exposed to photons at <i>E</i> = 12.8 keV for 10 s. (A) Comparison of scattering intensities for both phenotypes, in the range of 0.05 to 4.5 nm⁻¹. (B) Analysis of B6 melanosome structure by fitting a model of independent scatterers to the <i>q</i>-interval 0.07 nm⁻¹<<i>q</i><0.27 nm⁻¹. The resulting fitting parameters in the power law are <i>p</i>₁ = 4.034±0.001 (small <i>q</i>-values), <i>p</i>₂ = 3.887±0.118 (high <i>q</i>-values) und <i>R</i>_B6 = 22.57±5.57 nm (radius of gyration of melanosomal subunits). (C) Analysis of D2 melanosome structure by fitting a model of dependent scatterers to data within the <i>q</i>-interval 0.05 nm⁻¹<<i>q</i><0.27 nm⁻¹. The determined parameters are <i>p</i>₁ = 2.56 (modified power law at small <i>q</i>-values), <i>p</i>₂ = 3.60 (power law at high <i>q</i>-values) and <i>R</i>_D2 = 31.32±1.71 nm (radius of gyration of melanosomal subunits). In (B) and (C) the terms that contribute to the fit model used are labeled A1, A2 and A3. The analyzed data points are shown in green.</p

SEM images of freeze-dried C57BL/6J (B6) and DBA/2J (D2) melanosomes.

<p>The left column contains images recorded in the in-lens detector configuration (IL) and the right column shows micrographs acquired in the out-lens configuration (OL). A direct comparison shows that B6 melanosomes (A and B) have a rather smooth and featureless surface, whereas the D2 organelles (C and D) have an irregular surface. The samples were subjected to the identical preparation protocol. All scale bars represent 500 nm.</p

Correlation of maps generated by optical phase-contrast microscopy, cryo microscopy and darkfield cryo-STXM.

<p>Melanosomes of the genotypes C57BL/6J (B6) and DBA/2J (D2) are shown in tiles (A–C) and (D–F), respectively. In both cases, the sample is embedded in an amorphous ice matrix. The optical micrographs, recorded at 100 K, are superimposed with a semi-transparent (B and E) and an opaque (C and F) STXM map. Note that despite the fact that melanosome density is comparable in the two samples, the STXM images feature significant differences in signal intensity as well as the spatial distribution of that signal, revealing structural differences between the melanosomes of the two genotypes. The area of the scanned regions is 20.4×20.4 µm².</p

X-ray scattering data for cryogenically prepared melanosomes from C57BL/6J (B6) and DBA/2J (D2) mice.

<p>(A) Normalized intensity histograms derived from darkfield STXM maps of B6 and D2 samples like those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090884#pone-0090884-g002" target="_blank">Fig. 2</a>. The intensity of scatter for the D2 melanosomes is higher and has a broader distribution, than that for the B6 sample. The photon energy was 7.9 keV. (B) Sum of 13 background-corrected scattering events, i.e. <i>hits</i>, for a B6 sample. The scattering pattern is anisotropic. (C) Sum of 13 background-corrected scattering events for a D2 sample. The scattering pattern is isotropic. In (B) and (C), intensity is color-coded on a logarithmic scale. The horizontal bars in (B) and (C), which are devoid of signal, correspond to insensitive regions of the PILATUS, and separate the three detector modules. The artificial look of the regions in the centers is due to the stacking of two semi-transparent beamstops.</p

Nano-scale morphology of melanosomes revealed by small-angle X-ray scattering

Melanosomes are highly specialized organelles that produce and store the pigment melanin, thereby fulfilling essential functions within their host organism. Besides having obvious cosmetic consequences – determining the color of skin, hair and the iris – they contribute to photochemical protection from ultraviolet radiation, as well as to vision (by defining how much light enters the eye). Though melanosomes can be beneficial for health, abnormalities in their structure can lead to adverse effects. Knowledge of their ultrastructure will be crucial to gaining insight into the mechanisms that ultimately lead to melanosome-related diseases. However, due to their small size and electron-dense content, physiologically intact melanosomes are recalcitrant to study by common imaging techniques such as light and transmission electron microscopy. In contrast, X-ray-based methodologies offer both high spatial resolution and powerful penetrating capabilities, and thus are well suited to study the ultrastructure of electron-dense organelles in their natural, hydrated form. Here, we report on the application of small-angle X-ray scattering – a method effective in determining the three-dimensional structures of biomolecules – to whole, hydrated murine melanosomes. The use of complementary information from the scattering signal of a large ensemble of suspended organelles and from single, vitrified specimens revealed a melanosomal sub-structure whose surface and bulk properties differ in two commonly used inbred strains of laboratory mice. Whereas melanosomes in C57BL/6J mice have a well-defined surface and are densely packed with 40-nm units, their counterparts in DBA/2J mice feature a rough surface, are more granular and consist of 60-nm building blocks. The fact that these strains have different coat colors and distinct susceptibilities to pigment-related eye disease suggest that these differences in size and packing are of biological significance