INTRODUCTION

Oral candidiasis,a worldwide medical challenge for fungal superficialinfection,isresponsible forthe high morbidity especially in children,denture wearers and the immunocompromised population,such as human immunodeficiency virus(HIV)infected patients and head/neck cancer patients received radiation or chemo therapy.1–4Candida albicans(C.albicans)is the most pronounced conditional fungal pathogen colonized in oral cavity.5The filamentous growth of C.albicans is considered as the most essential virulence factor for the adhesion and invasion.6,7C.albicans can also produce many virulent molecules companied with the hyphal development,such as the cell-surface adhesin and secreted aspartyl proteases(Sap).8,9Agglutinin-like sequence(ALS)genes encoded cell-surface glycoproteins have important roles in the adherence to host surfaces,10such as agglutinin-like sequence 1(Als1),which is capable of the inducing adherence to endothelial and epithelial cells.11,12Although the major protein of the hyphal cell wall hyphal wall protein-1(Hwp1)also functions as a cell-surface adhesion with the ability to mimic mammalian transglutaminase substrates for the formation of covalent crosslinking between C.albicans and epithelial cells.13The family of Sap of C.albicans is responsible for the adhesion,cell-surface integrity,and tissue damage.7,14,15SAP6 is the predominant protease gene expressed in the patients with oral candidiasis and the expression occurs concomitantly at the place of tissue damage.16

The epithelium is thought to be the first mechanical barrier against tissue invading by C.albicans.When the epithelial cells are infected by the C.albicans hyphae,they activate the activating protein-1(AP-1),c-Fos,and mitogen-activated protein kinase 1(MKP1)to sense the C.albicans hyphal damage and produce the epithelial cytokine(such as interleukin(IL)-1α,IL-1β,IL-6,and IL-17),and then recruit immune cells(such as macrophages).17,18 However,it remains unclear that which cell components of C.albicans hyphae are important for mediating the damage of epithelial cells.Recently,the first fungal cytolytic peptide toxin“Candidalysin”(encoded by ECE1)was identified in C.albicans.19 The ECE1 deleted mutant can form normal hyphae similar to the wild type strain but not cause the epithelial cell damage,suggesting that candidalysin is a critical factor for the potential of C.albicans hyphae to cause invasive mucosal infections and tissue damage without the impact upon filamentous growth.The morphological identity between ECE1 deletion and wild type strains combined the opposite capabilities on epithelial cell damage highlight the idea that there are “missing links”between hyphal growth and host cell damage.This type of “missing link”genes will provide further insight into the transformation process from commensal to pathogenic state of C.albicans,and perhaps additional therapeutic targets.

To combat with C.albicans infections,several types of antifungal drugs are developed,such as azoles targeted at ergosterol(key elementin cellmembrane)biosynthesis,20 polyenes binding to ergosterol to form poles in cell membrane,21 and echinocandins targeted at cell wall biosynthesis.22–24Fluconazole(FLC),a clinical first-line fungistatic antifungal azole,can bind to Erg11 to inhibit the ergosterol biosynthesis and cause the accumulation of toxic sterols,indicating the importance of ergosterol in C.albicans.25–27ERG3 and ERG11 are the most important genes in ergosterol biosynthesis pathway and they have key roles in azole drug resistance.28–30However,their contributions to oral epithelial infections are not under investigated.Here we identified that the ERG3 and ERG11 genes were also belonged to the “missing link”type of genes for the first time since their deletions were incapable of causing oral mucosal infection similar to ECE1 gene,but they can also form hyphae.Meanwhile,fluconazole can relieve the epithelial infection even at non-growth inhibitory dosage both in vitro and in vivo,indicating its dual-functional abilities to not only eliminate the C.albicans but also inhibit the interaction between fungal pathogens and host cells by reducing the infective virulence.

RESULT

ERG3 and ERG11 genes are critical for epithelial cell damage in vitro

The expression of both ERG3 and ERG11 genes were significantly upregulated when C.albicans strains co-cultured with epithelial cell,indicating the positive relationship between ERG3 and ERG11 and the epithelial pathogenesis(Figure S1a,b).Then we subjected wild type,erg3Δ/Δ and erg11Δ/Δ to epithelial cell culture to probe the functions of ERG3 and ERG11 genes during epithelium infection in vitro.The erg3Δ/Δ and erg11Δ/Δ strains both can form typical hyphae identical with wild type(Fig.1a),but they were incapable of inducing epithelial cell damage(Fig.1b)after co-cultured with epithelial cell for 24h compared to wild type,indicating that erg3Δ/Δ and erg11Δ/Δ only formed non-virulent hyphae.Meanwhile,the erg3Δ/Δ and erg11Δ/Δ strains significantly reduced the adhesion to the epithelial cells compared to wild type(Fig.1c).Interestingly,both erg3Δ/Δ and erg11Δ/Δ strains were capable of extensive epithelial invasion and penetrating through multiple epithelial cells same as the wild type after 24 h cocultured with epithelial cells,in line with the morphological similarity of hyphae between the mutants and wild type strain(Fig.1d,e).Although the invasion was not affected,both erg3Δ/Δ and erg11Δ/Δ strains significantly reduced cell damage and inflammatory through the decrease of the reactive oxygen species(ROS)(Fig.1f)and cytokine(IL-1α)production(Fig.1g)in epithelial cells compared to the wild type strain.To identify the reason for the non-virulent hyphae of erg3Δ/Δ and erg11Δ/Δ,we measured theexpressionsofsome importantvirulence factors.The expressionsof ALS1 and SAP6 tested in thisstudy were significantly downregulated in erg3Δ/Δ strain as well as the obvious downregulation of SAP6 and HWP1 in erg11Δ/Δ strain(Fig.1h),respectively,consistent with their incapability of causing the cell damage and induction of ROS and cytokine,indicating the critical roles of ERG3 and ERG11 for epithelial infection in vitro.

FLC protect the epithelial cells from the damage of C.albicans in vitro

As azole drugs can target at the ergosterol biosynthesis pathway,22we employed FLC as an inhibitor to confirm the critical roles of ERG3 and ERG11 of C.albicans during the epithelial cell infection in vitro.The growth of C.albicans was inhibited by FLC at the concentration of 1µg·mL−1(Figure S2a),so we tried low doses of FLC,at which the growth was not inhibited(Figure S2b),to evaluate its inhibitory ability on the interactions between C.albicans and epithelial cell.Interestingly,C.albicans wild type stains was significantly reduced the epithelial cell damage at both 0.25 and 0.125µg·mL−1(Fig.2a).FLC also significantly suppressed the inductions of ROS and IL-1α productions(Fig.2b,c).Meanwhile,the non-growth inhibitory concentration could remarkably reduce the adherence rate to the epithelial cells(Fig.2d).In combination,these results indicate that FLC can not only work as fungal eliminative drug,but also be served as virulence inhibitor when at non-growth dosage due to the critical functions of ergosterol biosynthesis pathway in epithelial cell infection.

Epithelial cell adhesion assay

We next assessed the role of ERG3 and ERG11 in murine oropharyngeal candidiasis model.The ERG3 and ERG11 null mutants were considered as non-virulent hyphae,whereas the wild type served as normal-virulent hyphae control.As expected,the mice infected with wild type strains exhibited typical hyperplastic white plaques on the lingual surface,whereas the erg3Δ/Δ and erg11Δ/Δ strains failed to form the lesions(Fig.3a).Quantification of histology sections including micro-abscesses,the extensive hyphal invasion of the tongue epithelium and tissue damage indicated the typical tongue epithelial infectious disease symptoms when the mice infected with the wild type.In contrast,mice infected with erg3Δ/Δ and erg11Δ/Δ strains showed no obvious invasive fungal hyphae and no inflammatory infiltrates or tissue damage(Fig.3b–d).The incapability of erg3Δ/Δ and erg11Δ/Δ strains to cause mucosal infection in mice was not due to the intolerance to phagocyte challenge as both the null mutants and wild type strains were cleared by macrophage as the same(Fig.3e),but may mainly related the decrease of virulence factors in erg3Δ/Δ and erg11Δ/Δ strains(Fig.1h).Therefore,ERG3 and ERG11 genes in C.albicans were critical for mucosal infection in vivo.

FLC can cure the epithelial infection caused by C.albicans at low dosage in vivo

The powerful efficacy of FLC in epithelial cell infection in vitro highlighted the expectation of its potential effects in vivo at low dosage.As expected,after the mice infected by C.albicans,the FLC-treated group demonstrated absent white patches and low fungal burdens on the tongues at both 0.25 and 0.125 µg·mL−1 compared to the no drug treatment group(Fig.4a,b).These dosages of FLC also significantly reduced the epithelium infection area,inflammatory infiltrates,and local epithelial damage(Fig.4c,d),indicating the curative efficacy of FLC even at non-growth inhibitory dosage and suggesting the ability of FLC to block the interactions between fungal pathogens and host.The macrophage clearance rate of C.albicans was not effected by FLC(Fig.4e)in line with the wild type strain,indicated the inhibition of FLC on C.albicans virulence.

DISCUSSION

Ergosterol,the mostimportantcomponentin fungalcell membrane,functions many of the same as cholesterol in animal cells to regulate the fluidity and biogenesis of plasma membrane.31,32Here we identified the functionsofergosterol biosynthesis pathway contributed to the oral epithelial infection caused by C.albicans for the first time.The ergosterol biosynthesis dysfunction mutants erg3Δ/Δ and erg11Δ/Δ failed to damage the oral epithelial cells in vitro and importantly they lost the function to cause the mucosal infection in vivo.Miyazaki et al.33observed that the ERG3 null mutants showed defective hyphal formation when induced by human serum in vitro and in systemic infected mice.However,when the erg3Δ/Δ strain co-cultured with the epithelial cells in vitro,it can form the typical hyphae and show the same invasion rate similar to the wild type strain in our study,indicated that the attachment between C.albicans and epithelial cells may regulate the morphological development of C.albicans.As the typical hyphae of erg3Δ/Δ mutant were observed in vitro,there were no extensive fungal invasion in murine oropharyngeal candidiasis model in line with the observation in systemic C.albicans infection mice,indicated the defective virulence of ERG3 deletion.Becker et al.34found that ERG3 and ERG11 were required for virulence in murine model of systemic infection.Our results corroborated the finding that ERG3 and ERG11 were critical for the virulence in murine oropharyngeal candidiasis model.Furthermore,we dominated that the reason for that may result from the decrease of key virulence factors such as Als1,Hwp1 and Sap6 in ERG3 and ERG11 null mutants instead of their immune intolerance to macrophage.The production of ROS from immune cells,such as macrophage,is an important immune weapon to wipe out C.albicans during the early infectious stage.We found that the ERG3 and ERG11 gene from C.albicans contributed to the ROS production in oral epithelial cells,which may contribute to the C.albicans-infected epithelial cell damage.The dysfunction of ERG11 gene was reported as more sensitive to ROS generated from neutrophils,35,36which may also one of the reasons for the failure of erg11Δ/Δ mucosal infection in vivo in our study.In combination of the previous findings in C.albicans systemic infection murine model37,38and our results in murine oropharyngeal candidiasis model,ergosterol biosynthesis pathway is proved to be essential for C.albicans pathogenesis both in invasive and superficial fungal infection.

The epithelial cells are physically the first defensive surface barrier against C.albicans caused invasion and tissue damage.The transition between C.albicans yeast and hyphal forms has been proved as the most essential virulence factor by numerous investigations both in vitro and in vivo.39,40However,the recent identified fungal peptide toxin Ece1 functions as the key element to damage the epithelial cell instead of the hyphae of C.albicans as the deletion of ECE1 also formed the morphological identical hyphae compared to the wild type with the same invasive ability.19This type of genes whose deletion will lost epithelial cell damage without the effect on hyphal formation is important to understand the pathogenesis of C.albicans during the superficial infection and can be served as new therapeutic potential targets for the treatment of mucosal candidiasis.Our results from the contribution of ERG3 and ERG11 in epithelial cell infection model and murine oropharyngeal candidiasis model confirmed that these two genes were another “missing-link”genes between epithelial damage and hyphal development.The erg3Δ/Δ and erg11Δ/Δ in this study combined previous ece1Δ/Δ suggest that this type of “missing-link”genes is likely related to the virulence factors as ERG3 and ERG11 deletion decreased the virulence factors in our study while Ece1 acted as toxin to epithelium itself.In view of C.albicans infection in vivo,some of its immune evasion-related genes whose deletion will not affect the hyphal development may also lost the infectious ability due to the clearance of immune system in vivo(Y.Zhou et al.unpublished data.2017).Therefore,the “missing-link”genes may typically include virulence and immune evasion correlative genes or pathways,which will be new type of targets for antifungal drug discovery beyond the killing targets.These targets may reduce the fungal drug resistance as the inhibition to these genes will not kill the fungi but cause their incapability in infection.

其间，盛京医院护理工作的相关亮点先后闻名业界。在2005年全面实施垂直分配护理绩效津贴机制后，2007年，医院自主研发了工作量统计软件，依据护理工作量、技术含量、风险责任、技术职称、能级对应等多方面因素，绩效津贴打破了以往分配制度的平均主义，被进一步科学合理分配。

Owing to the important role of ergosterol in fungal membranes,azole drugs that inhibit ergosterol biosynthesis are widely used for the treatment of fungal infections.41Usually,azole drugs,such as FLC,are clinically used at killing dosage to inhibit the growth of C.albicans.Here we identified the multi-functions of FLC,which can not only eliminate the fungi but also inhibit the infective virulence of C.albicans both in vitro and in vivo.Especially,when FLC served at non-growth inhibitory dosage,it still reduced the epithelial cell damage in vitro and mucosal infection in vivo indicated its capability to block the pathogenesis of C.albicans and suggested that non-growth inhibitory dosage of FLC can also relieve the mucosal infection and be also served as synergistic potentiator to other antifungal compounds for C.albicans infection treatment.

MATERIALS AND METHODS

Ethics statement

基于石墨烯材料的敏感膜对pH的响应机理大致有以下两种情况，一是石墨烯边角的酚羟基[4]与pH溶液接触时，会发生质子交换从而产生电位差，这为石墨烯修饰电极在pH传感器中的应用提供了可能。同时石墨烯表面的少量含氧基团可与水及氢氧根离子形成氢键，因此，晶体外延型的1～2层石墨烯可灵敏地感知表面的离子浓度，从而成为性能良好的pH传感器[5-6]。但是鉴于石墨烯材料的灵敏特性，此类传感器中石墨烯材料对氢离子的选择性以及其他离子的干扰情况还有待进一步研究。

在开展机插秧工作的过程中，市县（区）农机推广部门通过召开育秧、机插秧现场演示会和培训会的形式对技术人员和农民进行广泛宣传、培训，取得了一定的成效，但是由于育秧繁琐、土地规模化程度低等因素的影响，机插秧技术推广进展缓慢。截至2017年底，全市共有插秧机115台，完成水稻机插面积1万亩，机插水平仅为4.7%。

All mouse experiments described in this study were conducted in strict accordance with the guidelines of Ethics Committee of West China Hospital of Sichuan University and the protocols were full approved by this Agency(license number WCHSIRB-D-2016-131).All efforts were made to minimize suffering and ensure the highest ethical and humane standards.

For scanning electron microscopy(SEM)analysis,TR146 cells were grown to confluence on glass coverslips.After the co-cultured with C.albicans(1×105cells per mL)for 24h,cell media was removed.Post washing with sterile PBS for three times,samples were fixed overnight at 4°C with 4%paraformaldehyde.Next,samples were dehydrated through a graded ethanol series,and sputter-coated with gold.Samples were then examined and images recorded using a scanning electron microscopy(SEM;FEI,Hillsboro,OR,USA).

Fluconazole(98.5%)was commercially obtained from Sigma-Aldrich(China)and dissolved with dimethylsulfoxide(DMSO,Merck-China).It was then stored at−20°C until use.Concanavalin A-Alexa-Fluor 647(ConA,Thermo Fisher)were dissolved in sterile phosphate buffer solution(PBS)(10 μg·mL−1,stored at −20 °C)and Calcofluor White(CFW,Sigma-Aldrich)(stored at room temperature).

综上所述，桑树育苗技术和种植技术直接影响了桑树的生长情况，保证桑树的良好质量，为桑蚕养殖创造更好的条件，所以，在育苗和种植过程中，需要保证使用正规方式。

Strains and media

All the C.albicans strains used in this study were listed in Table S1.C.albicans strains were maintained on YPD plates(1%yeast extract,2%peptone,2%glucose,2%agar)and then single colony was picked out and subjected into liquid YPD medium at 35°C overnight.C.albicans cells were harvested by centrifugation at 6000 r·min−1,4 °C for 5min,followed the wash in PBS for three time.The final C.albicans suspension was counted by a hemacytometer and then adjusted to the desired concentration in culture medium(Dulbecco’s modified Eagle’s medium(DMEM,HYclone)medium without fetal calf serum).

教养方式体现家庭中父母的文化水平、人生观、世界观、价值观。家庭的教养方式有：父母对孩子过度保护，使孩子丧失基本的判断力和自理能力；父母对孩子过分干涉，使孩子在成长的过程中没有自由；孩子出现错误，父母对孩子过分地辱骂、体罚、羞辱，使孩子产生恐惧心理和怨恨心理，缺乏安全感；父母拒绝孩子学习生活中的正常要求，使孩子的学习生活没有保障；父母适度地宽容，有耐心，有底线，有规则，这是家庭教养方式中较好的方式。在咨询笔者的案例中，一名抑郁学生有自杀意向，人际关系差，通过咨询治疗，有所缓解，笔者与其家长沟通，希望家长配合，但是家长表示孩子人际关系差不要紧。

Cell lines

Experiments were carried out by using buccal epithelial squamous cell carcinoma line TR146 and R human immortalized macrophage line RAW 264.7(ATCC,TIB-71™).TR146 was commercially obtained from JENNIO Biological Technology(Guangzhou,China),whereas macrophage RAW cell line was obtained from the American Type Culture Collection(ATCC).These cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium(DMEM,HYclone)supplemented with 10%fetal bovine serum(FBS,Gibco)and 1%penicillin–streptomycin at 37 °C.

Antifungal susceptibility test and growth measurement

针对ELV充电站设施选址评价问题，提出了一种基于组合赋权法的充电站选址评价模型。在充分考虑经济、社会等4个因素的基础上，从中选取了建设与运维成本、交通便利度等8项指标构建了评价指标体系。通过算例实证发现，科学合理的ELV充电站选址评价研究可以降低企业的建设成本，有效满足客户配送服务需求和城市规划布局要求。算例结果验证了所提出方法的有效性，为物流企业更好地进行ELV充电站选址评价提供了决策支撑。

Cytokine levels produced from cell culture supernatants were determined using Quantikine®ELISA kit(R&D Systems,USA)and a Varioskan Flash machine(Thermo Scientific)according to the manufacture’s introduction.All of the experiments were performed in triplicates.

(5)Because he is too busy to have a good rest,he feels tired of his life.

Relative quantification of differentially expressed genes by real time PCR

C.albicanscultures were harvested by centrifugation at 6000 r·min-1at 4 °C for 5min.The pellets were flash frozen in liquid nitrogen and stored at−80°C until RNA preparation.RNA isolation was carried out according to the E.Z.N.A.®Yeast RNA Kit(OMEGA Bio-tek.)instructions.Then 1µg RNA was subjected to the One Step RNA PCR kit(Takara Inc.)to prepare the cDNA according to themanufacturer’sinstructions.TheRT-PCR werethen proceeded by using the SYBR®PremixEx TaqTM kit(Takara Inc.)with following two-step strategy:(1)94°C for 30 s;(2)40 PCR cycles(94°C for 30s,a gene-specific annealing temperature for 30 s).All primer sequences are listed in Table S2.Real time PCRs of triplicate samples were performed using CFX 96 TouchTM(Bio-Rad,Hercules,CA,USA).The gene expression level relative to the calibrator was expressed as 2−ΔΔCT.

Cytokine production qualification assay

Fluconazole susceptibility measurements were carried out in flat bottom,96-well microtiter plates(Greiner,Germany),using a broth microdilution protocol modified from the Clinical and Laboratory Standards Institute M-27A methods(National Committee for Clinical Laboratory Standards 2002).Overnight cultures were picked to prepare the strain suspension with medium RPMI 1640 at the concentration of 1 ×104CFU·mL−1.Overall,2μL of thefluconazole was added then followed by an additional 80μL of the strain suspension.The test plates were incubated at 35°C for 16 h.Minimal inhibition concentrations(MICs)were determined by measuring and comparing the optical densities of the blank control and test wells.Representative aliquot of well-mixed and diluted 100µL of cultures treated by 0.25 and 0.125μg·mL−1fluconazole was spotted on YEPD media to monitor cells recovery.All experiments were done in triplicate.

Cell damage assay

在改革进程中，全国人大常委会是否有权授权进行监察体制改革试点，存在较大争议。监察体制是政治体制的重要组成部分，监察体制改革超越了当时宪法规定，人大常委会采取授权试点的方式，一定程度上解决了改革合宪性问题，为缓解改革与法治之间的紧张关系，提供了一种变通模式。但有学者认为，授权监察体制改革试点属于全国人大的职权，没有全国人大授权，作为全国人大常设机构的常委会，无权授权监察体制改革试点[15]。而“授权试点”是改革过程中的一项宪法工程，应当具有法律依据。这表明，《立法法》第13条能否为“授权试点”提供规范依据，如何使其具有更坚固的法律基础，是个值得研究的问题。

TR146 cells were grown to confluence on 24-well plates for 48h in DMEM medium.Lactatedehydrogenase(LDH)arraywere conducted to determine the TR146 cell damage after the cells co-cultured with C.albicans or FLC.Briefly,the culture supernatants were collected after incubation for 24h and subjected to the LDH activity test by using a Roche cytotoxicity detection kitplus according to the manufacturer’s instructions.All of the experiments were performed at least in triplicates.

ERG3 and ERG11 genes are essential for mucosal pathogenesis in vivo

Statistical significance was decided by Student’s t-test with Welch’s correction,one-way ANOVA with Dunnett’s or Tukey’s multiple comparison test,or two-way ANOVA with Tukey’s multiple comparison test using GraphPad Prism software.For data plotted on a logarithmic scale the geometric mean is indicated,and data were log-transformed before statistical analysis.

Epithelial invasion assay

TR146 cells were grown to confluence on glass coverslips for 48 h and then infected with C.albicans yeast cells(1×105cells per mL)for 24 h in a humidified incubator(37°C,5%CO2).After the wash for three times with PBS,the cells were fixed overnight(4°C in 4%paraformaldehyde)and stained with Concanavalin A-Alexa-Fluor 647(Thermo Fisher)in PBS(10μg·mL−1)for 45min at room temperature in the dark with gentle shaking to stain the fungal cell wall.After rinsing with PBS,TR146 cells were permeabilized by 0.1%Triton X-100 in PBS for 15min and fungal cells(invading and non-invading)were stained with Calcofluor White.After rinsing with water,coverslips were visualized using laser scanning confocal microscopy(FV1000,Olympus).The percentage of invading C.albicans cells was determined by dividing the number of(partially)internalized cells by the total number of adherent cells.At least 100 fungal cells were counted on each coverslip and all experiments were performed in duplicates on at least three separate occasions.

Macrophage clearance assay

RAW cells were grown to confluence on 96-well plates for 24 h in DMEM medium.C.albicans yeast cells(1×104cells per mL)were added into 100 μL DMEM with serum,incubated for 3h(37 °C,5%CO2).RAW cells were lysed by soaking with sterile double distilled water in 37°C for 1h.The suspensions were diluted and spread on YPD plates to derive quantitative fungal counts.

Scanning electron microscopy

Chemicals

目前，单一控制算法已经无法满足对复杂的工业机器人运动控制，往往采用模糊控制等算法与PID控制或自适应控制相结合方法，这样工业机器人运动控制的效果能实现多种控制方法融合的优点，具有很强的鲁棒性。

Murine oropharyngeal candidiasis model was performed according to the previous description.19,42Briefly,female BALB/c mice were injected subcutaneously with 3mg per mouse(in 200μL PBS with 0.5%Tween 80)of cortisone acetate on days before and post infection.The second day after injected,mice were in a coma for at least 75 min with an intra-peritoneal injection of 5%chloral hydrate 10 mL·kg−1.Then a swab soaked in a 1 × 107CFU·mL−1of C.albicans yeast in sterile saline was placed under the tongue.After 2 days,mice were executed.The tongue was cut out and divided longitudinally in two.After weighed,one half was homogenized and cultured to quantify candida counts on CHROMagarTM Candidaplate,whereastheotheronewas processed for histopathology analysis.To monitor the efficacy offluconazole,different dosages of fluconazole were added into the drinking water after the mice were infected.

Immunohistochemistry of murine tissue

C.albicans-infected murine tongues were fixed in 10%(v/v)formaldehyde before being embedded and processed in paraffin wax using standard protocols.For each tongue,5-μm sections were prepared using a Leica microtome and silane-coated slides.Sections were dewaxed using xylene.Then C.albicans and infiltrating inflammatory cells were visualized by staining using Periodic Acid-Schiff(PAS)stain and hematein eosin(HE)stain.Sections were then examined by light microscopy.Histological quantification of infection was undertaken by measuring the area of infected epithelium and expressed as a percentage relative to the entire epithelial area.

ROS assays

TR146 cells were grown to confluence on 96-well plates for 24h in DMEM medium.The ROS production was determined using a Reactive Oxygen Species Assay Kit(Beyotime,China)according to the manufacturer’s instructions.Briefly,cells were loaded with 10 μmol·L-1H2DCF-DA in serum-free DMEM for 20min in a humidified incubator(37°C,5%CO2)in the dark.After washing with serum-free DMEM for three times,100mL DMEM medium containing Rosup(100 mg·mL−1)was served as positive controls and equal volumes of C.albicans strains with or withoutfluconazole atthe indicated concentrations were added.Chemiluminescence was measured at 15 min intervals at 37°C with a Varioskan Flash machine(Thermo Scientific).Data are expressed as relative luciferase per well TR146 cells over time.

Murine oropharyngeal candidiasis model

Statistics

TR146 cells were grown to confluence on 24-well plates for 48h in DMEM medium.After washed by PBS for three times,1mL serumfree DMEM of C.albicans yeast cells(2×105cells per mL)were added and then followed the incubation for 60 min(37°C,5%CO2).The non-adherent C.albicans cells were aspirated and washed with PBS for three times.The cells were then collected and soaked with sterile double distilled water at 37°C for 1h until the epithelial cells were lysed.The suspensions were diluted and spread on YPD plates to derive quantitative candida counts at 35°C overnight.

ACKNOWLEDGEMENTS

We greatly thank Prof.Dominique Sanglard for providing the C.albicans mutants.This work was supported by the National Key Research and Development Program of China 2016YFC1102700(X.Z.);National Natural Science Foundation of China Grant 81600858(B.R.),81372889(L.C.),and 81430011(X.Z.);the Recruitment Program for Young Professionals(M.F.);the Youth Grant of the Science and Technology Department of Sichuan Province,China 2017JQ0028(L.C.).

ADDITIONAL INFORMATION

第三，我们还有承前启后、继往开来的社会主义先进文化。社会主义先进文化是对中华民族优秀传统文化和红色革命文化的继承和发展，是以马克思主义为指导创造的新型文化成果。社会主义先进文化的核心是中国特色社会主义的共同理想、以爱国主义为核心的民族精神和以改革创新为核心的时代精神，以及社会主义荣辱观。在短短几十年的社会主义实践中，我们创造了属于自己的中国道路、中国模式、中国奇迹，这说明社会主义先进文化是一种有生命力的文化，是一种体现了人类文明发展进步方向的文化。

三是中小河流洪水频发，山洪灾害严重。全年因局部强降雨引发中小河流洪水和山洪、滑坡、泥石流等灾害造成的人员死亡占洪涝死亡总人数七成。7月份四川都江堰100年一遇强降雨引发特大型高位山体滑坡。8月中旬辽宁暴雨导致浑河上游发生超历史纪录特大洪水。

The online version ofthis article (https://doi.org/10.1038/s41368-018-0013-2)contains supplementary material,which is available to authorized users.

Competing interests:The authors declare no competing interests.

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