Secondary Metabolites from Food-Derived Yeasts Inhibit Virulence of Candida albicans.

A sparse number of available antifungal drugs, therapeutic side effects, and drug resistance are major challenges in current antifungal therapy to treat Candida albicans-associated infections. Here, we describe two food-derived yeasts, Saccharomyces cerevisiae and Issatchenkia occidentalis, that inhibit virulence traits of C. albicans, including hyphal morphogenesis, biofilm formation, and adhesion to intestinal epithelial cells. These yeasts also protect the model host Caenorhabditis elegans from C. albicans infection. We demonstrate that the protective activity is primarily retained in the secretome of the beneficial yeasts, and the protection they provide as a physical barrier is negligible. S. cerevisiae aro8 aro9 mutant analysis demonstrate that phenylethanol and tryptophol are necessary for protection, and experiments with commercially procured compounds indicate that they are sufficient to inhibit C. albicans virulence. We propose food-derived yeasts as an alternative or combination therapy to conventional antifungal therapy for C. albicans infection. IMPORTANCE The gut microbiome, primarily established by food, is complex and contributes to the health of the host. Molecular mechanisms that regulate microbial interactions and host health remain unclear. Here, we show that the pathogen C. albicans interacts with food-derived beneficial yeasts in the gut of the microscopic worm, C. elegans, forming a simple microbiome. C. albicans can colonize the worm gut, compromising the worm's health, and exposure to the food-derived yeasts ameliorates this effect protecting the nematode host. We identify small molecules from food-derived yeasts that are necessary and sufficient to inhibit multiple virulence traits of C. albicans and protect the nematode host. The nematode gut faithfully recapitulates a mammalian intestine. This could be an effective alternative or combination therapy for C. albicans infection.

Probiotic Yeasts Inhibit Virulence of Non-albicans Candida Species.

Systemic infections of Candida species pose a significant threat to public health. Toxicity associated with current therapies and emergence of resistant strains present major therapeutic challenges. Here, we report exploitation of the probiotic properties of two novel, food-derived yeasts, Saccharomyces cerevisiae (strain KTP) and Issatchenkia occidentalis (strain ApC), as an alternative approach to combat widespread opportunistic fungal infections. Both yeasts inhibit virulence traits such as adhesion, filamentation, and biofilm formation of several non-albicans Candida species, including Candida tropicalis, Candida krusei, Candida glabrata, and Candida parapsilosis as well as the recently identified multidrug-resistant species Candida auris They inhibit adhesion to abiotic surfaces as well as cultured colon epithelial cells. Furthermore, probiotic treatment blocks the formation of biofilms of individual non-albicans Candida strains as well as mixed-culture biofilms of each non-albicans Candida strain in combination with Candida albicans The probiotic yeasts attenuated non-albicans Candida infections in a live animal. In vivo studies using Caenorhabditis elegans suggest that exposure to probiotic yeasts protects nematodes from infection with non-albicans Candida strains compared to worms that were not exposed to the probiotic yeasts. Furthermore, application of probiotic yeasts postinfection with non-albicans Candida alleviated pathogenic colonization of the nematode gut. The probiotic properties of these novel yeasts are better than or comparable to those of the commercially available probiotic yeast Saccharomyces boulardii, which was used as a reference strain throughout this study. These results indicate that yeasts derived from food sources could serve as an effective alternative to antifungal therapy against emerging pathogenic Candida species.IMPORTANCE Non-albicans Candida-associated infections have emerged as a major risk factor in the hospitalized and immunecompromised patients. Besides, antifungal-associated complications occur more frequently with these non-albicans Candida species than with C. albicans Therefore, as an alternative approach to combat these widespread non-albicans Candida-associated infections, here we showed the probiotic effect of two yeasts, Saccharomyces cerevisiae (strain KTP) and Issatchenkia occidentalis (ApC), in preventing adhesion and biofilm formation of five non-albicans Candida strains, Candida tropicalis, Candida krusei, Candida glabrata, Candida parapsilosis, and Candida auris The result would influence the current trend of the conversion of conventional antimicrobial therapy into beneficial probiotic microbe-associated antimicrobial treatment.

Prolyl endopeptidase-degraded low immunoreactive wheat flour attenuates immune responses in Caco-2 intestinal cells and gluten-sensitized BALB/c mice.

Targeted degrading Aspergillus niger-derived prolyl endopeptidase (AN-PEP) is promising in gluten hydrolysis because it specifically cleaves the proline-rich sites in gluten. The current study aims to understand the safety aspects of AN-PEP hydrolysed low immunoreactive wheat flours by testing immune responses using cell line and animal models. In the AN-PEP hydrolysed wheat flour (AN-PEP HWF) gliadin extract, there was no increase in the levels of zonulin-1 (Zo-1) and pro-inflammatory cytokines (IL-6 and IL-8) but a significant increase was noted in the control wheat flour (CWF) gliadin-treated Caco-2 cells. The Zo-1 localization in Caco-2 cells was significantly noted in the reacted positive fluorescence cells that were treated with the control wheat flour. Further, a safety evaluation of HWF was carried out in gluten-sensitized BALB/c mice. Mouse anti-gliadin (IgG, IgA and IgE) antibodies were significantly generated in the CWF treated animals rather than the AN-PEP HWF groups. The serum pro-inflammatory (IL-1ß, IL-4, IL-6, IL-15, TNF-α and IFN-γ) markers were observed in significant levels in CWF challenged mice and a similar trend was observed in ex-vivo splenocyte cells. A small intestine histopathological sectioning revealed that there are no abnormalities or structural changes in AN-PEP HWF challenged mice.

Laser-ablation-synthesized nanoparticles and animal cell lines studies.

Nanoparticles (NPs) synthesized by laser ablation in distilled water were used to study their biological effect on normal and cancer cells. Parameters such as cell morphology, cell proliferation and viability were examined for treated cell lines, and the effect was represented in terms of cells cytotoxicity using standard procedures. The study reveals the higher cytotoxic effect of nanoparticles on cancerous cells of breast, melanoma and colon origin compared to normal fibroblast cells NIH- 3T3. Furthermore, DNA fragmentation assay results demonstrated the apoptosis mediated cell death in nanoparticle-treated cancer cells. The distinct role of nanoparticles in normal and cancer cells of different origin showed that nanoparticles were specific to cause cytotoxicity in particular cancer cells type. NPs exhibit cytotoxic effects in cancer cells by inducing apoptosis. These studies provide fundamental evidence for the easy, simple and safe mode of nanoparticles synthesis and their application in cancer cells death.

Antigen-Specific Gut Inflammation and Systemic Immune Responses Induced by Prolonging Wheat Gluten Sensitization in BALB/c Murine Model.

Gluten-related diseases such as wheat allergy, celiac disease, and gluten intolerance are widespread around the globe to genetically predisposed individuals. The present study aims to develop a wheat-gluten induced BALB/c murine model for addressing wheat-gluten related disorders by sensitizing the wheat gluten through the route of intraperitoneal and oral challenge in prolonged days. During the sensitization, the sera were collected for specific antigliadin antibodies response and proinflammatory markers quantification. Ex vivo primary cells and organs were collected for subsequent analysis of inflammatory profile. Prolonging sensitization of gluten can moderate the antigen-specific inflammatory markers such as IL-1ß, IL-4, IL-15, IL-6, IFN-γ and TNF-α levels in mice sera. However, ex vivo primary cells of splenocytes (SPLs) and intestinal epithelial lymphocytes (IELs) significantly increased the IL-6, IL-15, IL-1ß, and IL-4 levels in G+ (gliadin and gluten) treated cells. Histopathology staining of jejunum sections indicates enterocyte degeneration in the apical part of villi and damage of tight junctions in G+ (gliadin and gluten) sensitized murine model. Immunohistochemistry of embedded jejunum sections showed significant expression of positive cells of IL-15, tTG and IL-4 in G+ sensitized murine model. In contrast, all markers of gluten-related disorders are expressed exclusively such as tTG, ZO-1, IL-15, IL-6, IL-4, and intestinal inflammation was mediated by iNOS, COX-2, TLR-4 and NF-kBp50 signaling mechanism in G+ sensitized mice.

Encapsulation of sesamol in phosphatidyl choline micelles: Enhanced bioavailability and anti-inflammatory activity.

Sesamol, the phenolic degradation product of sesamolin, although recognised for its anti-inflammatory effects, has low bioavailability. In this manuscript, we attempted to improve its bioavailability by encapsulation in mixed phosphatidylcholine micelles. Sesamol could be solubilised and entrapped in phosphatidylcholine mixed micelles (PCS) with 96.8% efficiency (particle size 3.0±0.06nm). Fluorescence spectra of PCS revealed lower relative fluorescence intensity (RFI 112) compared to 'free' sesamol (FS) (RFI 271). The bioaccessibility, transport across a monolayer of cells and cellular uptake of PCS was 8.58%, 1.5-fold and 1.2-fold better, respectively, compared to FS. The anti-inflammatory effects of FS and PCS were compared using LPS treated RAW 264.7 cell line and lipoxygenase inhibition. PCS effected downregulation of iNOS protein expression (27%), NO production (20%), ROS (32%) and lipoxygenase inhibition (IC50=31.24µM) compared to FS.

Uncovering molecular events associated with the chemosuppressive effects of flaxseed: a microarray analysis of the laying hen model of ovarian cancer.

BACKGROUND: The laying hen model of spontaneous epithelial ovarian cancer (EOC) is unique in that it is the only model that enables observations of early events in disease progression and is therefore also uniquely suited for chemoprevention trials. Previous studies on the effect of dietary flaxseed in laying hens have revealed the potential for both amelioration and prevention of ovarian cancer. The objective of this study was to assess the effect of flaxseed on genes and pathways that are dysregulated in tumors. We have used a bioinformatics approach to identify these genes, followed by qPCR validation, immunohistochemical localization, and in situ hybridization to visualize expression in normal ovaries and tumors from animals fed a control diet or a diet containing 10% flaxseed. RESULTS: Bioinformatic analysis of ovarian tumors in hens led to the identification of a group of highly up-regulated genes that are involved in the embryonic process of branching morphogenesis. Expression of these genes coincides with expression of E-cadherin in the tumor epithelium. Levels of expression of these genes in tumors from flax-fed animals are reduced 40-60%. E-cadherin and miR200 are both up-regulated in tumors from control-fed hens, whereas their expression is decreased 60-75% in tumors from flax-fed hens. This does not appear to be due to an increase in ZEB1 as mRNA levels are increased five-fold in tumors, with no significant difference between control-fed and flax-fed hens. CONCLUSIONS: We suggest that nutritional intervention with flaxseed targets the pathways regulating branching morphogenesis and thereby alters the progression of ovarian cancer.

Anti-inflammatory effects of fish oil in ovaries of laying hens target prostaglandin pathways.

BACKGROUND: An effective way to control cancer is by prevention. Ovarian cancer is the most lethal gynecological malignancy. Progress in the treatment and prevention of ovarian cancer has been hampered due to the lack of an appropriate animal model and absence of effective chemo-prevention strategies. The domestic hens spontaneously develop ovarian adenocarcinomas that share similar histological appearance and symptoms such as ascites and metastasis with humans. There is a link between chronic inflammation and cancer. Prostaglandin E2 (PGE2) is the most pro-inflammatory ecoisanoid and one of the downstream products of two isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. PGE2 exerts its effects on target cells by coupling to four subtypes of receptors which have been classified as EP1-4. Fish oil is a source of omega-3 fatty acids (OM-3FAs) which may be effective in prevention of ovarian cancer. Our objective was to assess the potential impact of fish oil on expression of COX enzymes, PGE2 concentration, apoptosis and proliferation in ovaries of laying hens. METHODS: 48 white Leghorn hens were fed 50, 100, 175, 375 and 700 mg/kg fish oil for 21 days. The OM3-FAs and omega-6 fatty acids contents of egg yolks were determined by Gas Chromatography. Proliferation, apoptosis, COX-1, COX-2 and prostaglandin receptor subtype 4 (EP4) protein and mRNA expression and PGE2 concentration in ovaries were measured by PCNA, TUNEL, Western blot, quantitative real-time qPCR and ELISA, respectively. RESULTS: Consumption of fish oil increased the incorporation of OM-3FAs into yolks and decreased both COX-1 and COX-2 protein and mRNA expression. In correlation with COXs down-regulation, fish oil significantly reduced the concentrations of PGE2 in ovaries. EP4 protein and mRNA expression in ovaries of hens was not affected by fish oil treatment. A lower dose of fish oil increased the egg laying frequency. 175 and 700 mg/kg fish oil reduced proliferation and 700 mg/kg increased apoptosis in hen ovaries. CONCLUSIONS: Our findings suggest that the lower doses of fish oil reduce inflammatory PG and may be an effective approach in preventing ovarian carcinogenesis. These findings may provide the basis for clinical trials utilizing fish oil as a dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer.

Snail and slug mediate radioresistance and chemoresistance by antagonizing p53-mediated apoptosis and acquiring a stem-like phenotype in ovarian cancer cells.

The transcriptional repressors Snail and Slug contribute to cancer progression by mediating epithelial-mesenchymal transition (EMT), which results in tumor cell invasion and metastases. We extend this current understanding to demonstrate their involvement in the development of resistance to radiation and paclitaxel. The process is orchestrated through the acquisition of a novel subset of gene targets that is repressed under conditions of stress, effectively inactivating p53-mediated apoptosis, while another subset of targets continues to mediate EMT. Repressive activities are complemented by a concurrent derepression of specific genes resulting in the acquisition of stem cell-like characteristics. Such cells are bestowed with three critical capabilities, namely EMT, resistance to p53-mediated apoptosis, and a self-renewal program, that together define the functionality and survival of metastatic cancer stem cells. EMT provides a mechanism of escape to a new, less adverse niche; resistance to apoptosis ensures cell survival in conditions of stress in the primary tumor; whereas acquisition of "stemness" ensures generation of the critical tumor mass required for progression of micrometastases to macrometastases. Our findings, besides achieving considerable expansion of the inventory of direct genes targets, more importantly demonstrate that such elegant cooperative modulation of gene regulation mediated by Snail and Slug is critical for a cancer cell to acquire stem cell characteristics toward resisting radiotherapy- or chemotherapy-mediated cellular stress, and this may be a determinative aspect of aggressive cancer metastases.

Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer.

The cellular mechanisms underlying the increasing aggressiveness associated with ovarian cancer progression are poorly understood. Coupled with a lack of identification of specific markers that could aid early diagnoses, the disease becomes a major cause of cancer-related mortality in women. Here we present direct evidence that the aggressiveness of human ovarian cancer may be a result of transformation and dysfunction of stem cells in the ovary. A single tumorigenic clone was isolated among a mixed population of cells derived from the ascites of a patient with advanced ovarian cancer. During the course of the study, yet another clone underwent spontaneous transformation in culture, providing a model of disease progression. Both the transformed clones possess stem cell-like characteristics and differentiate to grow in an anchorage-independent manner in vitro as spheroids, although further maturation and tissue-specific differentiation was arrested. Significantly, tumors established from these clones in animal models are similar to those in the human disease in their histopathology and cell architecture. Furthermore, the tumorigenic clones, even on serial transplantation continue to establish tumors, thereby confirming their identity as tumor stem cells. These findings suggest that: (a) stem cell transformation can be the underlying cause of ovarian cancer and (b) continuing stochastic events of stem and progenitor cell transformation define the increasing aggression that is characteristically associated with the disease.