Using mass spectrometry imaging to visualize age-related subcellular disruption.

Metabolic homeostasis balances the production and consumption of energetic molecules to maintain active, healthy cells. Cellular stress, which disrupts metabolism and leads to the loss of cellular homeostasis, is important in age-related diseases. We focus here on the role of organelle dysfunction in age-related diseases, including the roles of energy deficiencies, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, changes in metabolic flux in aging (e.g., Ca2+ and nicotinamide adenine dinucleotide), and alterations in the endoplasmic reticulum-mitochondria contact sites that regulate the trafficking of metabolites. Tools for single-cell resolution of metabolite pools and metabolic flux in animal models of aging and age-related diseases are urgently needed. High-resolution mass spectrometry imaging (MSI) provides a revolutionary approach for capturing the metabolic states of individual cells and cellular interactions without the dissociation of tissues. mass spectrometry imaging can be a powerful tool to elucidate the role of stress-induced cellular dysfunction in aging.

Design and synthesis of new drugs inhibitors of Candida albicans hyphae and biofilm formation by upregulating the expression of TUP1 transcription repressor gene.

Candida albicans is a common human fungal pathogen that causes disease ranging from superficial to lethal infections. C. albicans grows as budding yeast which can transform into hyphae in response to various environmental or biological stimuli. Although both forms have been associated with virulence, the hyphae form is responsible for the formation of multi-drug resistance biofilm. Here, new compounds were designed to selectively inhibit C. albicans hyphae formation without affecting human cells to afford sufficient safety. The newly designed 5-[3-substitued-4-(4-substituedbenzyloxy)-benzylidene]-2-thioxo-thiazolidin-4-one derivatives, named SR, showed very specific and effective inhibition activity against C. albicans hyphae formation. SR compounds caused hyphae inhibition activity at concentrations 10-40 fold lower than the concentration required to inhibit Candida yeast and bacterial growths. The anti-hyphae inhibition activities of SR compounds were via activation of the hyphae transcription repressor gene, TUP1. Correlation studies between the expression of TUP1 gene and the activity of SR compounds confirmed that the anti-C. albicans activities of SR compounds were via inhibition of hyphae formation. The newly designed SR compounds showed 10-40% haemolytic activity on human erythrocytes when compared to 100% haemolysis by 0.1% triton employed as positive control. Furthermore, theoretical prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET) of SR compounds confirmed their safety, efficient metabolism and possible oral bioavailability. With the minimal toxicity and significant activity of the newly-designed SR compounds, a future optimization of pharmaceutical formulation may develop a promising inhibitor of hyphal formation not only for C. albicans but also for other TUP1- dependent dimorphic fungal infections.

Inhibition of EGFR Signaling Protects from Mucormycosis.

Mucormycosis is a life-threatening, invasive fungal infection that is caused by various species belonging to the order Mucorales. Rhizopus species are the most common cause of the disease, responsible for approximately 70% of all cases of mucormycosis. During pulmonary mucormycosis, inhaled Rhizopus spores must adhere to and invade airway epithelial cells in order to establish infection. The molecular mechanisms that govern this interaction are poorly understood. We performed an unbiased survey of the host transcriptional response during early stages of Rhizopus arrhizus var. delemar (R. delemar) infection in a murine model of pulmonary mucormycosis using transcriptome sequencing (RNA-seq). Network analysis revealed activation of the host's epidermal growth factor receptor (EGFR) signaling. Consistent with the RNA-seq results, EGFR became phosphorylated upon in vitro infection of human alveolar epithelial cells with several members of the Mucorales, and this phosphorylated, activated form of EGFR colocalized with R. delemar spores. Inhibition of EGFR signaling with cetuximab or gefitinib, specific FDA-approved inhibitors of EGFR, significantly reduced the ability of R. delemar to invade and damage airway epithelial cells. Furthermore, gefitinib treatment significantly prolonged survival of mice with pulmonary mucormycosis, reduced tissue fungal burden, and attenuated the activation of EGFR in response to pulmonary mucormycosis. These results indicate EGFR represents a novel host target to block invasion of alveolar epithelial cells by R. delemar, and inhibition of EGFR signaling provides a novel approach for treating mucormycosis by repurposing an FDA-approved drug.IMPORTANCE Mucormycosis is an increasingly common, highly lethal fungal infection with very limited treatment options. Using a combination of in vivo animal models, transcriptomics, cell biology, and pharmacological approaches, we have demonstrated that Mucorales fungi activate EGFR signaling to induce fungal uptake into airway epithelial cells. Inhibition of EGFR signaling with existing FDA-approved drugs significantly increased survival following R. arrhizus var. delemar infection in mice. This study enhances our understanding of how Mucorales fungi invade host cells during the establishment of pulmonary mucormycosis and provides a proof-of-concept for the repurposing of FDA-approved drugs that target EGFR function.

Calli Essential Oils Synergize with Lawsone against Multidrug Resistant Pathogens.

The fast development of multi-drug resistant (MDR) organisms increasingly threatens global health and well-being. Plant natural products have been known for centuries as alternative medicines that can possess pharmacological characteristics, including antimicrobial activities. The antimicrobial activities of essential oil (Calli oil) extracted from the Calligonum comosum plant by hydro-steam distillation was tested either alone or when combined with lawsone, a henna plant naphthoquinone, against MDR microbes. Lawsone showed significant antimicrobial activities against MDR pathogens in the range of 200-300 µg/mL. Furthermore, Calli oil showed significant antimicrobial activities against MDR bacteria in the range of 180-200 µg/mL, Candida at 220-240 µg/mL and spore-forming Rhizopus fungus at 250 µg/mL. Calli oil's inhibition effect on Rhizopus, the major cause of the lethal infection mucormycosis, stands for 72 h, followed by an extended irreversible white sporulation effect. The combination of Calli oil with lawsone enhanced the antimicrobial activities of each individual alone by at least three-fold, while incorporation of both natural products in a liposome reduced their toxicity by four- to eight-fold, while maintaining the augmented efficacy of the combination treatment. We map the antimicrobial activity of Calli oil to its major component, a benzaldehyde derivative. The findings from this study demonstrate that formulations containing essential oils have the potential in the future to overcome antimicrobial resistance.