Dr. Indrani Bose's Seminar, “Using RNA Interference to Understand the Virulence and Biology of the Yeast Cryptococcus Neoformans

The interference of RNA and ME in the Bradford assay was compensated by using the blank, because the difference between the addition of the protein and the non-protein blank

Cryptococcus neoformans (Cn) virulence depends on the active transport of vesicles that contain melanin and capsule precursors, proteinases, and other macromolecules. We previously found that the Cn intersectin protein Cin1 regulates intracellular trafficking critical for growth and virulence and that Cin1-S isoform confers a marked survival advantage in the CNS of a murine model of cryptococcosis. In addition, we found that the expression of extracellular RNAs (exRNAs) including small RNA (sRNA), mRNA, and long noncoding RNA (lncRNA) was significantly differentiated among cin1, CIN1-S, and wild type stains. Further investigation of these observations could promote our understanding of Cn propensity for the host CNS and the virulence

When he started the presentation he gave a quick introduction then outlined what he did. Instead of going into every procedure in his experiment he focused on the largest, and possibly the most interesting, portion which was the 16S RNA assay. As he described this relatively complex topic he used

Yeast, a unicellular fungus, is a eukaryotic organism that reproduces through mitosis and meiosis, allowing it to be either a haploid or a diploid cell. There are about 1000 species of yeast and yeast-like organisms in the world today. It is commonly found on the surfaces of plants, gastrointestinal tracts and body surfaces of insects and warm-blooded animals, soils from all regions of the world, and aquatic environments. Yeast is used in bread-making and wine-making, since, by breaking down glucose in a low oxygen

H. capsulatum is a dimorphic fungal pathogen that thrives in its mycelial phase in soil containing high concentrations of bird or bat guano, and every year hundreds of thousands of individuals who live in areas of endemicity are infected1,2. Though found worldwide, histoplasmosis is primarily a disease of North and Central America that is generally asymptomatic in healthy persons but may be fatal in immunosuppressed individuals2. Infection occurs by inhalation of small mycelial fragments or microconidia into the terminal bronchioles and alveoli of the lung. Inhaled conidia are phagocytosed by macrophages and the change in temperature from 250C to 370C induces the transformation into the yeast phase, which is responsible for the pathogenesis of histoplasmosis3–5.

There is still much to be discovered about this disease. Researchers will be studying the fungi and its effects on ecosystems for many years.

spores that are inhaled. The spores make their way to the lungs and adhere to

rhodochrous’s success in white-nose syndrome prevention began as a research project on the conservation of edible fruit for maximum sustainability in agriculture. Researchers knew that the Rhodoccus genus of bacterium were particularly useful in preventing fungi to spread. To induce R. rhodochrous’s enzyme production, researchers allowed the bacterium to thrive in a perfect environment where the enzyme production would be at it’s peak, enabling the anti-fungal properties (Inman and LaTina 2014). The 6 volatile organic compounds discovered in total interact with P. destructans by inhibiting the growth of the hyphae, the filamentous structure that grows in fungi (Cornelison et. al). Inhibiting the source of growth for P. destructans slows down the destructive and painful spread of the disease, making it possible for bats to survive hibernation and for the fungi to disappear altogether. Despite the VOCs not killing P. destructans, it will eventually die on it’s own without any hyphae growth. [HOW DOES THE CURRENT TREATMENT OF BATS FOR THIS DISEASE WORK?] [HOW HAS IT BEEN SUCCESSFUL?] [WHY WAS IT SUCCESSFUL?] [SHORT DISCUSSION] A concern for both scientists and conservationists alike is the rapid spread of white-nose syndrome across the United States’ northeast region. Proactively using VOCs in bat caves that are affected with white-nose syndrome has not occurred yet, but doing so will defend those caves from the fungi

A steady increase in the types and number of organ transplants was seen after the 1970’s largely due to the success of an immunosuppressant isolated from Tolypocladium inflatum. One of the many inconspicuous creatures found in soil is the fungus Tolypocladium inflatum, which is a major contributor in the field of immunopharmacology. Belonging to the phylum Ascomycota (Bushley et al. 2013), T.inflatum is an asexual reproductive stage of Cordyceps subsessilis, a very rare stage of the fungus (Hodge et al. 1996). Interestingly, this was discovered in Cornell University when students collected a beetle infected with the fungi. Later, Professor Hodge

elegans was modified [76]. Freshly grown C. albicans of the indicated genotype were picked from a single colony and used to inoculate 1 mL of YPD broth, which was allowed to grow overnight with agitation at 30˚C. The following day, 10 mL of yeast were spread into a square lawn in a 4 cm tissue culture plate (BD) containing 4 mL of BHI agar and kanamycin (45 mg/mL). For experiments that compared heat-killed and live C. albicans, cells were subjected to the exact same preparatory conditions. A single colony of yeast was grown in 1 mL BHI at 30uC overnight and then inoculated into 50 mL YPD. After approximately 20 hours of incubation, cells were split into two aliquots, collected by centrifugation and washed twice with sterile PBS (pH 7.4). One aliquot was resuspended in 1 mL PBS, exposed to 75uC for 60 minutes and washed again with sterile PBS. The other aliquot was processed in parallel with the heat-killed sample. Cells were suspended in 25 mL PBS, incubated at room temperature for 60 minutes and washed again with sterile PBS. 10 mL of this sample were added to the killing assay plates containing 4 mL of BHI agar and kanamycin (45 mg/mL). The plates were then incubated for approximately 20 hours at 30˚C. These pathogenesis assay plates are then used to give infection to C. elegans at young adult stage for four

Since shRNA does not interact with the Drosha/DGCR8 microprocessor complex, this RNA must be designed with care in order to achieve correct downstream processing and gene suppression.1 Proper dosage of shRNAs is another factor to consider, as it has been found that they may saturate the endogenous pathway and disrupt natural miRNAs.4

Yeast growth is activated in 49° C–55°C temperatures, and yeast can be grown in either anaerobic or aerobic environments. Baker’s yeast grown in anaerobic environments produces alcohol. In this experiment yeast will be grown an aerobic environment. It is known that yeast grows well when fed glucose and maltose, but this experiment will test food that is sources of different sugars. Salt, which

For instance, insoluble and amorphous eumelanin pigment assemblies of the human fungal pathogen Cryptococcus neoformans are associated with microbial virulence and therapeutic drug resistance; I have employed multi-dimensional magnetic resonance spectroscopy to deconstruct the heterogeneous (supra) molecular frameworks of these chemically recalcitrant organic pigment assemblies synthesized from obligatory catecholamine precursors, shedding light on the action of these poorly understood fungal virulence factors. This research work has involved extensive collaborative studies with Professor Arturo Casadevall’s immunology and microbiology laboratory at Johns Hopkins University.

albicans can gain an advantage over the normal bacterial flora. Two common substances are steroids and birth control pills. These both act to alter the host’s body chemistry in a way that is favorable to the over growth of C. albicans. If the host is immunocompromised to begin with as in the case of AIDS patients or organ transplant receivers that are on immunosupresive drugs C. albicans infections are very prominent. A common symptom among AIDS patients is oral thrush, where there is a huge over population of C. albicans on the back of the hosts tongue, it appears as white speckles.

For the last three decades, fungal infections have become a major problem worldwide, especially among the immunocompromised individuals (Pfaller MA, Epidemiology of invasive candidiasis, 2007). Despite that Candida is the leading cause of the opportunistic fungal infections, there is a limited number of antimycotics available for therapy (Naglik JR, 2003) (Cannon RD, 2009). Perea et al. (Perea S, 2002) divided antifungal agents commonly used for candidiasis treatment in five major groups basing on their mode of action; group I: inhibition of RNA and/or DNA synthesis (fluorinated pyrimidine analogs 5-FC); group II: alteration of the membrane function (polyenes: nystatin, natamycin, amphotericin B AMB); group III: alteration of cell wall biosynthesis by inhibition of β(1,3)-glucan synthase (echinocandins: caspofungin,