Effect of Virulence Factors on the Photodynamic Inactivation of Cryptococcus neoformans

Opportunistic fungal pathogens may cause an array of superficial infections or serious invasive infections, especially in immunocompromised patients. Cryptococcus neoformans is a pathogen causing cryptococcosis in HIV/AIDS patients, but treatment is limited due to the relative lack of potent antifungal agents. Photodynamic inactivation (PDI) uses the combination of non-toxic dyes called photosensitizers and harmless visible light, which produces singlet oxygen and other reactive oxygen species that produce cell inactivation and death. We report the use of five structurally unrelated photosensitizers (methylene blue, Rose Bengal, selenium derivative of a Nile blue dye, a cationic fullerene and a conjugate between poly-L-lysine and chlorin(e6)) combined with appropriate wavelengths of light to inactivate C. neoformans. Mutants lacking capsule and laccase, and culture conditions that favoured melanin production were used to probe the mechanisms of PDI and the effect of virulence factors. The presence of cell wall, laccase and melanin tended to protect against PDI, but the choice of the appropriate photosensitizers and dosimetry was able to overcome this resistance.Fundação de Amparo à Pesquisa do Estado de São Paulo (2010/13313–9

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution

Inhomogeneity in optical properties of rat brain: a study for LLLT dosimetry

Over the last few years, low-level light therapy (LLLT) has shown an incredible suitability for a wide\ud range of applications for central nervous system (CNS) related diseases. In this therapeutic modality light\ud dosimetry is extremely critical so the study of light propagation through the CNS organs is of great\ud importance. To better understand how light intensity is delivered to the most relevant neural sites we\ud evaluated optical transmission through slices of rat brain point by point. We experimented red\ud (λ = 660 nm) and near infrared (λ = 808 nm) diode laser light analyzing the light penetration and\ud distribution in the whole brain. A fresh Wistar rat (Rattus novergicus) brain was cut in sagittal slices and\ud illuminated with a broad light beam. A high-resolution digital camera was employed to acquire data of\ud transmitted light. Spatial profiles of the light transmitted through the sample were obtained from the\ud images. Peaks and valleys in the profiles show sites where light was less or more attenuated. The peak\ud intensities provide information about total attenuation and the peak widths are correlated to the scattering\ud coefficient at that individual portion of the sample. The outcomes of this study provide remarkable\ud information for LLLT dose-dependent studies involving CNS and highlight the importance of LLLT\ud dosimetry in CNS organs for large range of applications in animal and human diseases.CNP

Irradiation and fluorescence parameters for each photosensitizer.

<p>Irradiation and fluorescence parameters for each photosensitizer.</p

Chemical structures of the photosensitizers used in this study.

<p>(A) Methylene blue; (B) Rose Bengal; (C) Selenium Nile blue derivatrive (EtNBSe); (D) Tris-cationic fullerene (BB6); poly-L-lysine chlorin (e6) conjugate (pL-ce6).</p

Confocal microscopy image of <i>C. neoformas</i> KN99α.

<p>Cells were treated with APDI mediated by pL-ce6 (10 µM) and then incubated with FITC-annexin V and PI. Green represents fluorescence of externalized phosphatidylserine that is correlated to the initial steps of apoptosis, and red corresponds to fluorescence of PI (advanced apoptosis/necrosis). We present three pictures of the same field: Transmittance in column A, green and red fluorescence in columns B and C respectively. The first line of figures is the stained samples before APDI (0J/cm²), the second line is following an irradiation of 10J/cm² and the last one was irradiated with fluence of 40J/cm². Scale bars 8µm.</p

Molecular features that could affect APDI effectiveness.

a<p>As the conjugate has 37 primary amino groups, the number of charges depends of pH and microenviroment.</p>b<p>Average extinction coefficient over range 400–700-nm.</p

Effect of laccase enzyme on photodynamic inactivation of <i>C. neoformans</i> ATCC 208820 (laccase positive strain, black squares), and ATCC 208819 (laccase negative strain, open circles).

<p>(A) methylene blue, (B) rose bengal, (C) EtNBSe, (D) BB6, (E) pL-ce6 were used as photosensitizers at 10 µM in PBS for 30 min followed by a wash and illumination with the wavelengths specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054387#pone-0054387-t002" target="_blank">Table 2</a>. Data are means and bars are the standard deviation. * P<0.05; ** P<0.01; *** P<0.001 for survival of 208820 vs 208820.</p