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.

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.

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.

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.

Potentiation of azole antifungals by 2-adamantanamine.

Azoles are among the most successful classes of antifungals. They act by inhibiting α-14 lanosterol demethylase in the ergosterol biosynthesis pathway. Oropharyngeal candidiasis (OPC) occurs in about 90% of HIV-infected individuals, and 4 to 5% are refractory to current therapies, including azoles, due to the formation of resistant biofilms produced in the course of OPC. We reasoned that compounds affecting a different target may potentiate azoles to produce increased killing and an antibiofilm therapeutic. 2-Adamantanamine (AC17) was identified in a screen for compounds potentiating the action of miconazole against biofilms of Candida albicans. AC17, a close structural analog to the antiviral amantadine, did not affect the viability of C. albicans but caused the normally fungistatic azoles to become fungicidal. Transcriptome analysis of cells treated with AC17 revealed that the ergosterol and filamentation pathways were affected. Indeed, cells exposed to AC17 had decreased ergosterol contents and were unable to invade agar. In vivo, the combination of AC17 and fluconazole produced a significant reduction in fungal tissue burden in a guinea pig model of cutaneous candidiasis, while each treatment alone did not have a significant effect. The combination of fluconazole and AC17 also showed improved efficacy (P value of 0.018) compared to fluconazole alone when fungal lesions were evaluated. AC17 is a promising lead in the search for more effective antifungal therapeutics.

Phaeosphaerins A-F, cytotoxic perylenequinones from an endolichenic fungus, Phaeosphaeria sp.

Six novel phototoxins, phaeosphaerins A-F, together with six known perylenequinones were isolated from an endolichenic fungus Phaeosphaeria sp. Their structures were determined unequivocally on the basis of comprehensive analysis of MS and NMR data as well as electronic circular dichroism calculations. These toxins kill cancer cells in vitro with accumulation in lysosomes, and the killing effects were potently intensified in the presence of light.

Plagiochin E, an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway.

BACKGROUND: Plagiochin E (PLE) is an antifungal active macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. To elucidate the mechanism of action, previous studies revealed that the antifungal effect of PLE was associated with the accumulation of ROS, an important regulator of apoptosis in Candida albicans. The present study was designed to find whether PLE caused apoptosis in C. albicans. METHODS: We assayed the cell cycle by flow cytometry using PI staining, observed the ultrastructure by transmission electron microscopy, studied the nuclear fragmentation by DAPI staining, and investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V staining. The effect of PLE on expression of CDC28, CLB2, and CLB4 was determined by RT-PCR. Besides, the activity of metacaspase was detected by FITC-VAD-FMK staining, and the release of cytochrome c from mitochondria was also determined. Furthermore, the effect of antioxidant L-cysteine on PLE-induced apoptosis in C. albicans was also investigated. RESULTS: Cells treated with PLE showed typical markers of apoptosis: G(2)/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. The expression of CDC28, CLB2, and CLB4 was down-regulated by PLE, which may contribute to PLE-induced G(2)/M cell cycle arrest. Besides, PLE promoted the cytochrome c release and activated the metacaspase, which resulted in the yeast apoptosis. The addition of L-cysteine prevented PLE-induced nuclear fragmentation, phosphatidylserine exposure, and metacaspase activation, indicating the ROS was an important mediator of PLE-induced apoptosis. CONCLUSIONS: PLE induced apoptosis in C. albicans through a metacaspase-dependent apoptotic pathway. GENERAL SIGNIFICANCE: In this study, we reported for the first time that PLE induced apoptosis in C. albicans through activating the metacaspase. These results would conduce to elucidate its underlying antifungal mechanism.

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.

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.

Plagiochin E, an antifungal bis(bibenzyl), exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species accumulation in Candida albicans.

BACKGROUND: Plagiochin E (PLE) is an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. Its antifungal mechanism is unknown. To elucidate the mechanism of action, its effect on mitochondria function in Candida albicans was studied. METHODS: We assayed the mitochondrial membrane potential (mtDeltapsi) using rhodamine 123, measured ATP level in mitochondria by HPLC, and detected the activities of mitochondrial F(0)F(1)-ATPase and dehydrogenases. Besides, the mitochondrial dysfunction-induced reactive oxygen species (ROS) production was determined by a fluorometric assay, and the effects of antioxidant L-cysteine on PLE-induced ROS production and the antifungal effect of PLE on C. albicans were also investigated. RESULTS: Exposure to PLE resulted in an elevation of mtDeltapsi, and a decrease of ATP level in mitochondria. The ATP depletion owed to PLE-induced enhancement of mitochondrial F(0)F(1)-ATPase and inhibition of the mitochondrial dehydrogenases. These dysfunctions of mitochondria caused ROS accumulation in C. albicans, and this increase in the level of ROS production and PLE-induced decrease in cell viability were prevented by addition of L-cysteine, indicating that ROS was an important mediator of the antifungal action of PLE. CONCLUSIONS: PLE exerts its antifungal activity through mitochondrial dysfunction-induced ROS accumulation in C. albicans. GENERAL SIGNIFICANCE: The effect of PLE on the mitochondria function in C. albicans was assayed for the first time. These results would conduce to elucidate its underlying antifungal mechanism.

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.

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.

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.

In vitro activities of retigeric acid B alone and in combination with azole antifungal agents against Candida albicans.

The vitro antifungal activity of retigeric acid B (RAB), a pentacyclic triterpenoid from the lichen species Lobaria kurokawae, was evaluated alone and in combination with fluconazole, ketoconazole, and itraconazole against Candida albicans using checkerboard microdilution and time-killing tests. The MICs for RAB against 10 different C. albicans isolates ranged from 8 to 16 microg/ml. A synergistic action of RAB and azole was observed in azole-resistant strains, whereas synergistic or indifferent effects were observed in azole-sensitive strains when interpreted by a separate approach of the fractional inhibitory concentration index and DeltaE model (the difference between the predicted and measured fungal growth percentages). In time-killing tests, we used both colony counts and a colorimetric assay to evaluate the combinational antifungal effects of RAB and azoles, which further confirmed their synergistic interactions. These findings suggest that the natural product RAB may play a certain role in increasing the susceptibilities of azole-resistant C. albicans strains.

Dracotanosides A-D, spermidine glycosides from Dracocephalum tanguticum: structure and amide rotational barrier.

Four new spermidine glycosides, dracotanosides A-D (1-4), have been isolated from Dracocephalum tanguticum. These molecules represent the first spermidine glycosides from this plant genus. The structures, including absolute configurations, were determined by spectroscopic and chemical methods. The amide bond rotational barrier of aglycone 1a was calculated by density functional theory (DFT) computation.

The inhibitory effect of a macrocyclic bisbibenzyl riccardin D on the biofilms of Candida albicans.

Biofilm formation plays a key role in the life cycles and subsistence of many microorganisms. The human fungal pathogen Candida albicans has a high propensity to develop biofilms and resulted resistant to traditional antifungal agents. Biofilms are composed of a mixture of cell types, including yeast, pseudohyphal and hyphal cells, and hyphae are a prominent feature of biofilms. Riccardin D is a macrocyclic bisbibenzyl isolated from the liverwort Dumortiera hirsute in our laboratory. In the present investigation, the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assay and live/dead cell staining were employed for evaluating the effects of riccardin D on C. albicans biofilms. The results demonstrated that riccardin D can interfere with the biofilm formation. To investigate whether this effect was due to the inhibition of hyphae formation, morphological observation and real-time reverse transcriptase polymerase chain reaction (RT-PCR) were employed for evaluating the effects of riccardin D on the hyphae formation and the expression of hyphae specific genes. The results showed that the hyphae formation was strongly inhibited and the mRNA expression levels of hyphae specific genes were downregulated after riccardin D treatment. We concluded that riccardin D interfered with the biofilm formation of C. albicans through downregulating the expression of hyphae specific genes and inhibiting the formation of hyphae.

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.

Effect of plagiochin E, an antifungal macrocyclic bis(bibenzyl), on cell wall chitin synthesis in Candida albicans.

AIM: To investigate the effect of plagiochin E (PLE), an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L, on cell wall chitin synthesis in Candida albicans. METHODS: The effect of PLE on chitin synthesis in Candida albicans was investigated at the cellular and molecular levels. First, the ultrastructural changes were observed under transmission electron microscopy (TEM). Second, the effects of PLE on chitin synthetase (Chs) activities in vitro were assayed using 6-O-dansyl-N-acetylglucosamine as a fluorescent substrate, and its effect on chitin synthesis in situ was assayed by spheroplast regeneration. Finally, real-time RT-PCR was performed to assay its effect on the expression of Chs genes (CHS). RESULTS: Observation under TEM showed that the structure of the cell wall in Candida albicans was seriously damaged, which suggested that the antifungal activity of PLE was associated with its effect on the cell wall. Enzymatic assays and spheroplast regeneration showed that PLE inhibited chitin synthesis in vitro and in situ. The results of the PCR showed that PLE significantly downregulated the expression of CHS1, and upregulated the expression of CHS2 and CHS3. Because different Chs is regulated at different stages of transcription and post-translation, the downregulation of CHS1 would decrease the level of Chs1 and inhibit its activity, and the inhibitory effects of PLE on Chs2 and Chs3 would be at the post-translational level or by the inhibition on the enzyme-active center. CONCLUSION: These results indicate that the antifungal activity of PLE would be attributed to its inhibitory effect on cell wall chitin synthesis in Candida 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.