Establishing Minimal Conditions Sufficient for the Development of Titan-like Cells in <em>Cryptococcus neoformans</em>/<em>gattii</em> Species Complex

Opportunistic pathogens of the anamorphic genus Cryptococcus are unique considering their virulence factors that in the context of pathogenesis allowed them to achieve evolutionary success. Morphological transformation into giant (Titan) cells is one of the factors contributing to cryptococcosis. Recently established in vitro protocols demonstrate that 5 or 10% fetal bovine serum (FBS) combined with 5% CO2, 37 °C, and sufficiently low cell density, triggers cellular enlargement (Serum protocols). However, the FBS components that promote this morphological transition remain incompletely characterized. In search of minimal conditions necessary for stimulating the formation of Titan cells, we performed a study where we eliminated serum from the protocol (Serum-free protocol) and instead systematically adjusted the amount of glucose, source of nitrogen (ammonium sulfate), and the pH. We found that exposing cells to PBS with adjusted pH to 7.3, and supplemented with 0.05% glucose, 0.025% ammonium sulfate, 0.004% K2HPO4, 0.0035% MgSO4, in the presence of 5% CO2 at 37 °C triggers Titan-like cell formation to the same degree as the previously established protocol that utilized 10% FBS as the sole nutrient source. Titan-like cells obtained according to this Serum-free protocol were characterized by cell body size over ten microns, a single enlarged vacuole, thick cell wall, extensive polysaccharide capsule, and changes in the level of cell ploidy, all currently known hallmarks of Titan cells found in vivo. Strikingly, we found that in both, Serum and Serum-free protocols, an optimal pH for Titan-like cell development is ~7.3 whereas relatively acidic pH (5.5) prevents this morphological transition and promotes robust proliferation, while alkaline pH (~8.0) has a profound growth inhibitory effect. Our study demonstrates a critical role of pH response to the formation of Titan cells and indicates that conditions that allow restricted proliferation in the presence of 5% CO2 are sufficient for this morphological transition to form enlarged cells in Cryptococcus neoformans and Cryptococcus gattii species complex

Ganoderma spp.: A Promising Adjuvant Treatment for Breast Cancer

For the past several decades, cancer patients in the U.S. have chosen the use of natural products as an alternative or complimentary medicine approach to treat or improve their quality of life via reduction or prevention of the side effects during or after cancer treatment. The genus Ganoderma includes about 80 species of mushrooms, of which several have been used for centuries in traditional Asian medicine for their medicinal properties, including anticancer and immunoregulatory effects. Numerous bioactive compounds seem to be responsible for their healing effects. Among the approximately 400 compounds produced by Ganoderma spp., triterpenes, peptidoglycans and polysaccharides are the major physiologically-active constituents. Ganoderma anticancer effects are attributed to its efficacy in reducing cancer cell survival and growth, as well as by its chemosensitizing role. In vitro and in vivo studies have been conducted in various cancer cells and animal models; however, in this review, we focus on Ganoderma’s efficacy on breast cancers. Evidence shows that some species of Ganoderma have great potential as a natural therapeutic for breast cancer. Nevertheless, further studies are needed to investigate their potential in the clinical setting and to translate our basic scientific findings into therapeutic interventions for cancer patients