Intestinal long non-coding RNAs in response to simulated microgravity stress in Caenorhabditis elegans.

Long non-coding RNAs (lncRNAs) are important in regulating the response to environmental stresses in organisms. In this study, we used Caenorhabditis elegans as an animal model to determine the functions of intestinal lncRNAs in regulating response to simulated microgravity stress. Among the intestinal lncRNAs, linc-2, linc-46, linc-61, and linc-78 were increased by simulated microgravity treatment, and linc-13, linc-14, linc-50, and linc-125 were decreased by simulated microgravity treatment. Among these 8 intestinal lncRNAs, RNAi knockdown of linc-2 or linc-61 induced a susceptibility to toxicity of simulated microgravity, whereas RNAi knockdown of linc-13, linc-14, or linc-50 induced a resistance to toxicity of simulated microgravity. In simulated microgravity treated nematodes, linc-50 potentially binds to three transcriptional factors (DAF-16, SKN-1, and HLH-30). RNAi knockdown of daf-16, skn-1, or hlh-30 could suppress resistance of linc-50(RNAi) nematodes to the toxicity of simulated microgravity. Therefore, our results provide an important basis for intestinal lncRNAs, such as the linc-50, in regulating the response to simulated microgravity in nematodes.

Comparison of transgenerational reproductive toxicity induced by pristine and amino modified nanoplastics in Caenorhabditis elegans.

Certain modifications can aggravate the toxicity of nanoplastics. However, the influence of surface amino modification on transgenerational impairment induced by nanoplastics remains largely unclear. Pristine nanopolystyrene (NPS) and amino modified NPS (NPS-NH2) were used to determine their transgenerational toxicity in Caenorhabditis elegans. Exposure to 100 µg/L pristine NPS in parents (P0) cause a decrease in reproductive capacity in the F1-F3 generations and the damage on gonad development in the F1-F2 generations. In contrast, exposure to 10 µg/L NPS-NH2 caused toxicity on reproductive capacity and gonad development in the F1 generation. The toxic effects of NPS-NH2 on reproductive capacity and gonad development in the F1-F3 generations were more severe than those of pristine NPS. Moreover, amino modification could increase transgenerational toxicity of NPS in inducing apoptosis of germline and in affecting expressions of ced-1, ced-4, and ced-9. Our data demonstrate that surface modification of NPS with amino groups enhances transgenerational reproductive toxicity of NPS in C. elegans.

The Effect of Honokiol on Ergosterol Biosynthesis and Vacuole Function in Candida albicans.

Ergosterol, an essential constituent of membrane lipids of yeast, is distributed in both the cell membrane and intracellular endomembrane components such as vacuoles. Honokiol, a major polyphenol isolated from Magnolia officinalis, has been shown to inhibit the growth of Candida albicans. Here, we assessed the effect of honokiol on ergosterol biosynthesis and vacuole function in C. albicans. Honokiol could decrease the ergosterol content and upregulate the expression of genes related with the ergosterol biosynthesis pathway. The exogenous supply of ergosterol attenuated the toxicity of honokiol against C. albicans. Honokiol treatment could induce cytosolic acidification by blocking the activity of the plasma membrane Pma1p H+-ATPase. Furthermore, honokiol caused abnormalities in vacuole morphology and function. Concomitant ergosterol feeding to some extent restored the vacuolar morphology and the function of acidification in cells treated by honokiol. Honokiol also disrupted the intracellular calcium homeostasis. Amiodarone attenuated the antifungal effects of honokiol against C. albicans, probably due to the activation of the calcineurin signaling pathway which is involved in honokiol tolerance. In conclusion, this study demonstrated that honokiol could inhibit ergosterol biosynthesis and decrease Pma 1p H+-ATPase activity, which resulted in the abnormal pH in vacuole and cytosol.

microRNAs involved in the control of toxicity on locomotion behavior induced by simulated microgravity stress in Caenorhabditis elegans.

microRNAs (miRNAs) post-transcriptionally regulate the expression of targeted genes. We here systematically identify miRNAs in response to simulated microgravity based on both expressions and functional analysis in Caenorhabditis elegans. After simulated microgravity treatment, we observed that 19 miRNAs (16 down-regulated and 3 up-regulated) were dysregulated. Among these dysregulated miRNAs, let-7, mir-54, mir-67, mir-85, mir-252, mir-354, mir-789, mir-2208, and mir-5592 were required for the toxicity induction of simulated microgravity in suppressing locomotion behavior. In nematodes, alteration in expressions of let-7, mir-67, mir-85, mir-252, mir-354, mir-789, mir-2208, and mir-5592 mediated a protective response to simulated microgravity, whereas alteration in mir-54 expression mediated the toxicity induction of simulated microgravity. Moreover, among these candidate miRNAs, let-7 regulated the toxicity of simulated microgravity by targeting and suppressing SKN-1/Nrf protein. In the intestine, a signaling cascade of SKN-1/Nrf-GST-4/GST-5/GST-7 required for the control of oxidative stress was identified to act downstream of let-7 to regulate the toxicity of simulated microgravity. Our data demonstrated the crucial function of miRNAs in regulating the toxicity of simulated microgravity stress in organisms. Moreover, our results further provided an important molecular basis for epigenetic control of toxicity of simulated microgravity.

Regulation of Innate Immune Response to Fungal Infection in Caenorhabditis elegans by SHN-1/SHANK.

In Caenorhabditis elegans, SHN-1 is the homologue of SHANK, a scaffolding protein. In this study, we determined the molecular basis for SHN-1/SHANK in the regulation of innate immune response to fungal infection. Mutation of shn-1 increased the susceptibility to Candida albicans infection and suppressed the innate immune response. After C. albicans infection for 6, 12, or 24 h, both transcriptional expression of shn-1 and SHN-1::GFP expression were increased, implying that the activated SHN-1 may mediate a protection mechanism for C. elegans against the adverse effects from fungal infection. SHN-1 acted in both the neurons and the intestine to regulate the innate immune response to fungal infection. In the neurons, GLR-1, an AMPA ionotropic glutamate receptor, was identified as the downstream target in the regulation of innate immune response to fungal infection. GLR-1 further positively affected the function of SER-7-mediated serotonin signaling and antagonized the function of DAT-1-mediated dopamine signaling in the regulation of innate immune response to fungal infection. Our study suggests the novel function of SHN-1/SHANK in the regulation of innate immune response to fungal infection. Moreover, our results also denote the crucial role of neurotransmitter signals in mediating the function of SHN-1/SHANK in regulating innate immune response to fungal infection.

Lipid metabolic sensors of MDT-15 and SBP-1 regulated the response to simulated microgravity in the intestine of Caenorhabditis elegans.

Caenorhabditis elegans is a useful animal model to determine the underlying mechanism for the response to simulated microgravity. In this study, we employed C. elegans as an animal model to investigate the role of lipid metabolic sensors in regulating the response to simulated microgravity. Among the lipid metabolic sensors, simulated microgravity treatment could increase the expressions of sbp-1 and mdt-15. RNAi knockdown of sbp-1 or mdt-15 induced a susceptibility to toxicity of simulated microgravity, suggesting the alteration in SBP-1 and MDT-1 mediated a protective response to simulated microgravity. Tissue-specific activity analysis demonstrated that both MDT-15 and SBP-1 could act in the intestine to regulate the response to simulated microgravity. Genetic interaction analysis further indicated that intestinal MDT-15 acted upstream of SBP-1 to regulate the response to simulated microgravity. During the control of response to simulated microgravity, fatty acyl CoA desaturase FAT-6 was identified as the downstream target of intestinal SBP-1. Therefore, the identified signaling cascade of MDT-15-SBP-1-FAT-6 suggested the important function of lipid metabolic sensors in mediating a novel intestinal signaling pathway to regulate the response to simulated microgravity in nematodes.

Opposite effects of vitamin C and vitamin E on the antifungal activity of honokiol.

The aim of the present study was to evaluate the effects of two well-known natural antioxidants vitamin C (VC) and vitamin E (VE) on the antifungal activity of honokiol against Candida albicans. The broth microdilution method was employed to test the antifungal activities of honokiol with or without antioxidants in the medium against C. albicans strain. Intracellular reactive oxygen species (ROS) and lipid peroxidation were determined by fluorescence staining assay. Mitochondrial dysfunction was assessed by detecting the mitochondrial DNA and the mitochondrial membrane potential. We observed that VC could significantly potentiate the antifungal activities of honokiol while VE reduced the effectiveness of honokiol against C. albicans. In addition, VC accelerated honokiol-induced mitochondrial dysfunction and inhibited glycolysis leading to a decrease in cellular ATP. However, VE could protect against mitochondrial membrane lipid peroxidation and rescue mitochondrial function after honokiol treatment. Our research provides new insight into the understanding of the action mechanism of honokiol and VC combination against C. albicans.

Roles of the Hsp90-Calcineurin Pathway in the Antifungal Activity of Honokiol.

Honokiol, a bioactive compound isolated from the cone and bark of Magnolia officinalis, has been shown to have various activities including inhibition of the growth of Candida albicans. We investigated the roles of the Hsp90-calcineurin pathway in the antifungal activity of honokiol. The pharmacologic tool was employed to evaluate the effects of Hsp90 and calcineurin in the antifungal activity of honokiol. We also evaluated the protective effects of the calcineurin inhibitor cyclosporin A (CsA) on honokiol-induced mitochondrial dysfunction by the fluorescence staining method. The Hsp90 inhibitor potentiated the antifungal activity of honokiol. A C. albicans strain with the calcineurin gene deleted displayed enhanced sensitivity to honokiol. However, co-treatment with calcineurin inhibitor CsA attenuated the cytotoxic activity of honokiol due to the protective effect on mitochondria. Our results provide insight into the action mechanism of honokiol.

Caffeic acid phenethyl ester synergistically enhances the antifungal activity of fluconazole against resistant Candida albicans.

BACKGROUND: Owing to the increased morbidity and mortality associated with invasive fungal infections, treatments with a combination of antifungal agents are often considered. Caffeic acid phenethyl ester (CAPE), a major active component of propolis, possesses many biological activities, including antibacterial, antiviral, antioxidant, anti-inflammatory, and anticancer effects. PURPOSE: This study aimed to evaluate the interaction between CAPE and fluconazole (FLC) against Candida albicans. METHODS: Microdilution checkerboard and time-kill assays were employed to evaluate the in vitro interaction between CAPE and FLC. The data obtained from the checkerboard tests were analyzed by the fractional inhibitory concentration index (FICI).The antifungal activity of the CAPE and FLC combination was evaluated in vivo in a Caenorhabditis elegans model of infection. RESULTS: We observed that when used in combination, CAPE acted synergistically with FLC against FLC-resistant clinical isolates of C. albicans. In addition, the CAPE-FLC combination significantly extended the longevity and reduced fungal burden in C. elegans when compared with treatment with FLC or CAPE alone. CONCLUSION: These results indicate that the use of CAPE and FLC in combination has considerable therapeutic potential against resistant C. albicans.

Honokiol induces superoxide production by targeting mitochondrial respiratory chain complex I in Candida albicans.

BACKGROUND: Honokiol, a compound extracted from Magnolia officinalis, has antifungal activities by inducing mitochondrial dysfunction and triggering apoptosis in Candida albicans. However, the mechanism of honokiol-induced oxidative stress is poorly understood. The present investigation was designed to determine the specific mitochondrial reactive oxygen species (ROS)-generation component. METHODS/RESULTS: We found that honokiol induced mitochondrial ROS accumulation, mainly superoxide anions (O2•-) measured by fluorescent staining method. The mitochondrial respiratory chain complex I (C I) inhibitor rotenone completely blocked O2•- production and provided the protection from the killing action of honokiol. Moreover, respiratory activity and the C I enzyme activity was significantly reduced after honokiol treatment. The differential gene-expression profile also showed that genes involved in oxidoreductase activity, electron transport, and oxidative phosphorylation were upregulated. CONCLUSIONS: The present work shows that honokiol may bind to mitochondrial respiratory chain C I, leading to mitochondrial dysfunction, accompanied by increased cellular superoxide anion and oxidative stress. GENERAL SIGNIFICANCE: This work not only provides insights on the mechanism by which honokiol interferes with fungal cell, demonstrating previously unknown effects on mitochondrial physiology, but also raises a note of caution on the use of M. officinalis as a Chinese medicine due to the toxic for mitochondria and suggests the possibility of using honokiol as chemosensitizer.

Honokiol induces reactive oxygen species-mediated apoptosis in Candida albicans through mitochondrial dysfunction.

OBJECTIVE: To investigate the effects of honokiol on induction of reactive oxygen species (ROS), antioxidant defense systems, mitochondrial dysfunction, and apoptosis in Candida albicans. METHODS: To measure ROS accumulation, 2',7'-dichlorofluorescein diacetate fluorescence was used. Lipid peroxidation was assessed using both fluorescence staining and a thiobarbituric acid reactive substances (TBARS) assay. Protein oxidation was determined using dinitrophenylhydrazine derivatization. Antioxidant enzymatic activities were measured using commercially available detection kits. Superoxide dismutase (SOD) genes expression was measured using real time RT-PCR. To assess its antifungal abilities and effectiveness on ROS accumulation, honokiol and the SOD inhibitor N,N'-diethyldithiocarbamate (DDC) were used simultaneously. Mitochondrial dysfunction was assessed by measuring the mitochondrial membrane potential (mtΔψ). Honokiol-induced apoptosis was assessed using an Annexin V-FITC apoptosis detection kit. RESULTS: ROS, lipid peroxidation, and protein oxidation occurred in a dose-dependent manner in C. albicans after honokiol treatment. Honokiol caused an increase in antioxidant enzymatic activity. In addition, honokiol treatment induced SOD genes expression in C. albicans cells. Moreover, addition of DDC resulted in increased endogenous ROS levels and potentiated the antifungal activity of honokiol. Mitochondrial dysfunction was confirmed by measured changes to mtΔψ. The level of apoptosis increased in a dose-dependent manner after honokiol treatment. CONCLUSIONS: Collectively, these results indicate that honokiol acts as a pro-oxidant in C. albicans. Furthermore, the SOD inhibitor DDC can be used to potentiate the activity of honokiol against C. albicans.

microRNAs Involved in the Control of Innate Immunity in Candida Infected Caenorhabditis elegans.

The role of microRNAs (miRNAs) in regulating innate immune response to Candida albicans infection in Caenorhabditis elegans is still largely unclear. Using small RNA SOLiD deep sequencing technique, we profiled the miRNAs that were dysregulated by C. albicans infection. We identified 16 miRNAs that were up-regulated and 4 miRNAs that were down-regulated in nematodes infected with C. albicans. Bioinformatics analysis implied that these dysregulated miRNAs may be involved in the control of many important biological processes. Using available mutants, we observed that mir-251 and mir-252 loss-of-function mutants were resistant to C. albicans infection, whereas mir-360 mutants were hypersensitive to C. albicans infection. The expression pattern of antimicrobial genes suggested that mir-251, mir-252, and mir-360 played crucial roles in regulating the innate immune response to C. albicans infection. Fungal burden might be closely associated with altered lifespan and innate immune response in mir-251, mir-252, and mir-360 mutants. Moreover, mir-251 and mir-252 might function downstream of p38 mitogen activated protein kinase (MAPK) or IGF-1/insulin-like pathway to regulate the innate immune response to C. albicans infection. Our results provide an important molecular basis for further elucidating how miRNA-mRNA networks may control the innate immune response to C. albicans infection.

Synergy Between Polyvinylpyrrolidone-Coated Silver Nanoparticles and Azole Antifungal Against Drug-Resistant Candida albicans.

In the clinical practice, resistance of Candida albicans to antifungal agents has frequently emerged. Silver-nanoparticles (Ag-NPs) have been demonstrated to have the antifungal property. We investigated the potential for synergy between polyvinylpyrrolidone (PVP)-coated Ag-NPs and azole antifungal, such as fluconazole or voriconazole, against drug-resistant C. albicans strain CA10. When antifungal agent was examined alone, fluconazole and voriconazole did not kill drug-resistant C. albicans, and PVP-coated Ag-NPs had only the moderate killing ability. In contrast, the combinational treatment of PVP-coated Ag-NPs with fluconazole or voriconazole was effective in being against the drug-resistant C. albicans. After the combinational treatment, we detected the disruption of cell membrane integrity, the tendency of PVP-coated Ag-NPs to adhere to cell membrane, and the inhibition of budding process. Moreover, after the combinational treatment, the defects in ergosterol signaling and efflux pump functions were detected. Our results suggest that the combinational use of engineered nanomaterials (ENMs), such as PVP-coated Ag-NPs, with the conventional antifungal may be a viable strategy to combat drug-resistant fungal infection.

Thymol has antifungal activity against Candida albicans during infection and maintains the innate immune response required for function of the p38 MAPK signaling pathway in Caenorhabditis elegans.

The Caenorhabditis elegans model can be used to study Candida albicans virulence and host immunity, as well as to identify plant-derived natural products to use against C. albicans. Thymol is a hydrophobic phenol compound from the aromatic plant thyme. In this study, the in vitro data demonstrated concentration-dependent thymol inhibition of both C. albicans growth and biofilm formation during different developmental phases. With the aid of the C. elegans system, we performed in vivo assays, and our results further showed the ability of thymol to increase C. elegans life span during infection, inhibit C. albicans colony formation in the C. elegans intestine, and increase the expression levels of host antimicrobial genes. Moreover, among the genes that encode the p38 MAPK signaling pathway, mutation of the pmk-1 or sek-1 gene decreased the beneficial effects of thymol's antifungal activity against C. albicans and thymol's maintenance of the innate immune response in nematodes. Western blot data showed the level of phosphorylation of pmk-1 was dramatically decreased against C. albicans. In nematodes, treatment with thymol recovered the dysregulation of pmk-1 and sek-1 gene expressions, the phosphorylation level of PMK-1 caused by C. albicans infection. Therefore, thymol may act, at least in part, through the function of the p38 MAPK signaling pathway to protect against C. albicans infection and maintain the host innate immune response to C. albicans. Our results indicate that the p38 MAPK signaling pathway plays a crucial role in regulating the beneficial effects observed after nematodes infected with C. albicans were treated with thymol.

Contribution of heavy metals to toxicity of coal combustion related fine particulate matter (PM2.5) in Caenorhabditis elegans with wild-type or susceptible genetic background.

Contribution of chemical components in coal combustion related fine particulate matter (PM2.5) to its toxicity is largely unclear. We focused on heavy metals in PM2.5 to investigate their contribution to toxicity formation in Caenorhabditis elegans. Among 8 heavy metals examined (Fe, Zn, Pb, As, Cd, Cr, Cu, and Ni), Pb, Cr, and Cu potentially contributed to PM2.5 toxicity in wild-type nematodes. Combinational exposure to any two of these three heavy metals caused higher toxicity than exposure to Pb, Cr, or Cu alone. Toxicity from the combinational exposure to Pb, Cr, and Cu at the examined concentrations was higher than exposure to PM2.5 (100 mg/L). Moreover, mutation of sod-2 or sod-3 gene encoding Mn-SOD increased susceptibility in nematodes exposed to Fe, Zn, or Ni, although Fe, Zn, or Ni at the examined concentration did not lead to toxicity in wild-type nematodes. Our results highlight the potential contribution of heavy metals to PM2.5 toxicity in environmental organisms.

Multi-walled carbon nanotubes enhanced fungal colonization and suppressed innate immune response to fungal infection in nematodes.

The underlying molecular mechanisms for multi-walled carbon nanotube (MWCNT)-induced in vivo toxicity on innate immunity are still largely unclear. Considering the potential of Caenorhabditis elegans for the study of innate immune response of animals, we employed this in vivo assay system to investigate the effects of MWCNTs on innate immune response of animals and the underlying mechanisms. Pre-exposure to MWCNTs at concentrations more than 100 µg L-1 enhanced the adverse effect of fungal infection in reducing lifespan. With regard to the underlying cellular mechanisms, we found that MWCNT pre-exposure enhanced colony formation of Candida albicans in the body of nematodes, and suppressed innate immune response of nematodes by decreasing expression levels of some antimicrobial genes. With regard to the underlying molecular mechanisms, we found that MWCNTs decreased expression levels of pmk-1, sek-1, and nsy-1 genes encoding the p38 mitogen activated protein kinase (MAPK) signaling pathway, and inhibited translational expression of PMK-1::GFP in the intestine and phosphorylation of PMK-1. Epistasis assays showed that MWCNTs required the involvement of the p38 MAPK signaling pathway mediated by a NSY-1-SEK-1-PMK-1 cascade to enhance the toxicity of fungal infection, increase fungal colony formation, and suppress innate immune response. Thus, our results suggest that MWCNTs may possess immunoinhibitory effects by affecting the functions of the p38 MAPK signaling pathway. Our study also provides meaningful insights into the role of innate immune system of hosts against the toxicity of environmental toxicants.

Bioactive ent-Pimarane and ent-Abietane Diterpenoids from the Whole Plants of Chloranthus henryi.

Two new ent-pimarane (1 and 2), eight new ent-abietane (3-10) diterpenoids, and eight known analogues (11-18) were isolated from the whole plants of Chloranthus henryi. The absolute configuration of 1 was determined on the basis of single-crystal X-ray diffraction data. Compound 8 represents a class of rare naturally occurring C-14 norabietanes, and compounds 9 and 10 feature rare 13,14-seco-abietane skeletons. Compounds 5, 12, 13, and 15 inhibited the yeast-to-hyphae transition of Candida albicans with IC50 values between 97.3 and 738.7 µM.

Sesquiterpenes from Carpesium macrocephalum inhibit Candida albicans biofilm formation and dimorphism.

One new xanthanolide, 4-(2-methybutyryl)-4H-tomentosin (1) was isolated from the whole plant of Carpesium macrocephalum together with nine known sesquiterpenes (2-10), including four eudesmane sesquiterpenes (2, 4, 5, and 10), one guaianolide (3), two xanthanolides (6 and 9) and two carabranolides (7 and 8). Their structures were elucidated on the basis of detailed spectroscopic analyses. All isolates were evaluated for their antifungal activities against the growth, biofilm formation and yeast-hyphal transition in Candida albicans. All compounds lacked the antifungal activity (MIC50>256 µg/ml) except compound 6 with the MIC50 value of 128 µg/ml. However, compounds 3, 5 and 10 strongly inhibited biofilm formation with IC50 values ranging from 15.4 to 38.0 µg/mL, and compounds 1, 3, 4, 6 and 7 inhibited the yeast-to-hyphae morphogenetic transition with the IC50 values between 31.6 and 118.4 µg/mL. The above results indicated that sesquiterpenes from C. macrocephalum may have therapeutic potential for candidiasis as virulence inhibitors.

Effects of magnolol and honokiol on adhesion, yeast-hyphal transition, and formation of biofilm by Candida albicans.

BACKGROUND: The first step in infection by Candida albicans is adhesion to host cells or implanted medical devices and this followed by hyphal growth and biofilm formation. Yeast-to-hyphal transition has long been identified as a key factor in fungal virulence. Following biofilm formation, C. albicans is usually less sensitive or insensitive to antifungals. Therefore, development of new antifungals with inhibitory action on adhesion, yeast-hyphal transition and biofilm formation by C. albicans is very necessary. METHODS: The effects of magnolol and honokiol on hypha growth were investigated using different induction media. Their inhibitory effects were determined using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide assay, and biofilm thickness and viability were observed by a confocal scanning laser microscope. Mammalian cells were used in adhesion assays. Genes related to hyphae development and cell adhesions were analyzed by real-time reverse transcription-polymerase chain reaction. The exogenous cyclic adenosine monophosphate was used to determine the mechanisms of action of magnolol and honokiol. Caenorhabditis elegans was used as an in vivo model to estimate the antifungal activities of magnolol and honokiol. RESULTS AND CONCLUSIONS: Magnolol and honokiol inhibited adhesion, the transition from yeast to hypha, and biofilm formation by C. albicans through the Ras1-cAMP-Efg1 pathway. Moreover, magnolol and honokiol prolonged the survival of nematodes infected by C. albicans. Magnolol and honokiol have potential inhibitory effects against biofilm formation by C. albicans. GENERAL SIGNIFICANCE: This study provides useful information towards the development of new strategies to reduce the incidence of C. albicans biofilm-associated infection.