Histone H3K4 methylation regulates deactivation of the spindle assembly checkpoint through direct binding of Mad2.

Histone H3 methylation on Lys4 (H3K4me) is associated with active gene transcription in all eukaryotes. In Saccharomyces cerevisiae, Set1 is the sole lysine methyltransferase required for mono-, di-, and trimethylation of this site. Although H3K4me3 is linked to gene expression, whether H3K4 methylation regulates other cellular processes, such as mitosis, is less clear. Here we show that both Set1 and H3K4 mutants display a benomyl resistance phenotype that requires components of the spindle assembly checkpoint (SAC), including Bub3 and Mad2. These proteins inhibit Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mutations in Cdc20 that block Mad2 interactions suppress the benomyl resistance of both set1 and H3K4 mutant cells. Furthermore, the HORMA domain in Mad2 directly binds H3, identifying a new histone H3 "reader" motif. Mad2 undergoes a conformational change important for execution of the SAC. We found that the closed (active) conformation of both yeast and human Mad2 is capable of binding methylated H3K4, but, in contrast, the open (inactive) Mad2 conformation limits interaction with methylated H3. Collectively, our data indicate that interactions between Mad2 and H3K4 regulate resolution of the SAC by limiting closed Mad2 availability for Cdc20 inhibition.

Suppression of arbuscular mycorrhizal fungi increased lead uptake in maize leaves and loss via surface runoff and interflow from polluted farmland.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous in farmland. But the knowledge on AMF impact on lead (Pb) migration in farmland is limited. A field experiment was conducted in the rainy season (May-October) for two years in a Pb-polluted farmland. Benomyl was used to specifically suppress the native AMF growth in the farmland. The effect of benomyl-induced AMF suppression on the Pb uptake in maize, and Pb loss via surface runoff and interflows (20 cm and 40 cm depth) from the farmland was investigated. The benomyl significantly inhibited the AMF growth, resulting in decreases in the colonization rate, spore number, and contents of total and easily extractable glomalin-related soil protein (GRSP); and promoted the Pb migration into maize shoots and mainly enriched in leaves. The particulate Pb accounted for 83.2%-90.6% of Pb loss via surface runoff, while the proportion of particulate Pb loss via interflow was decreased and the proportion of dissolved Pb loss increased with the increase of soil depth. The AMF suppression led to a decrease in dissolved Pb concentration and loss, but an increase in particulate Pb concentration and loss, and enhanced the total Pb loss via surface runoff and interflows. Moreover, significant or very significant negative correlations were observed between the AMF colonization rate in roots with the Pb uptake in leaves, and the content of easily extractable GRSP with the particulate Pb loss. These results indicated the native AMF contributed to immobilizing Pb in soil and inhibited its migration to crops and the surrounding environment.

The cytoskeleton influences the formation and distribution of eisosomes in Neurospora crassa.

Eisosomes are stable protein complexes at the plasma membrane, with punctate distributional patterns. Their formation and how their locations are determined remain unclear. The current study discovered that the formation and distribution of eisosomes are influenced by the cytoskeleton. Disassembly of either the F-actin or the microtubules leads to eisosome localization at hyphal tips of germinated macroconidia in Neurospora crassa, and treatment with a high concentration of the microtubule-inhibitor benomyl results in the production of filamentous eisosome patterns. The defect in the cytoskeleton caused by the disassembly of microtubules or F-actin leads to an increased formation of eisosomes.

Two herbicides, two fungicides and spore-associated bacteria affect Funneliformis mosseae extraradical mycelium structural traits and viability.

The extraradical mycelium (ERM) produced by arbuscular mycorrhizal fungi is fundamental for the maintenance of biological fertility in agricultural soils, representing an important inoculum source, together with spores and mycorrhizal root fragments. Its viability and structural traits, such as density, extent and interconnectedness, which are positively correlated with the growth and nutrition of host plants, may be affected by different agronomic practices, including the use of pesticides and by different mycorrhizospheric communities. This work, carried out using a whole-plant experimental model system, showed that structural traits of ERM, such as length and density, were strongly decreased by the herbicides dicamba and glufosinolate and the fungicides benomyl and fenhexamid, while anastomosis frequency and hyphal branching were differentially modulated by singly inoculated mycorrhizospheric bacteria, depending on their identity.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

The genetic architecture in Saccharomyces cerevisiae that contributes to variation in drug response to the antifungals benomyl and ketoconazole.

The genetic basis of variation in drug response was investigated in individual Saccharomyces cerevisiae strains that exhibited different susceptibility to two antifungal agents: benomyl and ketoconazole. Following dose-response screening of 25 strains, 4 were selected on the basis of resistance or sensitivity relative to the standard laboratory strain BY. UWOPS87-2421 and L-1374 were respectively resistant and sensitive to benomyl; DBVPG6044 and Y12 were respectively resistant and sensitive to ketoconazole. We used advanced intercross lines and next generation sequencing-bulk segregant analysis to characterise the quantitative trait loci (QTL) underpinning drug responses after drug selection. Drug response was controlled by multiple QTL, ranging from a minimum of 5 to a maximum of 60 loci, almost all of which were not the primary drug target. For each drug, the resistant and the sensitive strain exhibited a number of shared loci, but also had strain-specific QTL. In our analysis, it was possible to estimate genetic effect of QTL, and a number of those shared between resistant and sensitive strains exhibited variable effect on the response phenotype. Thus, drug responses arise as a result of different genetic architectures, depending on the genetic background of the individual strain in question.

Mechanism of Action of the Benzimidazole Fungicide on Fusarium graminearum: Interfering with Polymerization of Monomeric Tubulin But Not Polymerized Microtubule.

Tubulins are the proposed target of clinically relevant anticancer drugs, anthelmintic, and fungicide. ß2-tubulin of the plant pathogen Fusarium graminearum was considered as the target of benzimidazole compounds by homology modeling in our previous work. In this study, α1-, α2-, and ß2-tubulin of F. graminearum were produced in Escherichia coli. Three benzimidazole compounds (carbendazim, benomyl, and thiabendazole) interacted with the recombinant ß2-tubulin and reduced the maximum fluorescence intensity of 2 µM ß2-tubulin 47, 50, and 25%, respectively, at saturation of compound-tubulin complexes. Furthermore, carbendazim significantly inhibited the polymerization of α1-/ß2-tubulins and α2-/ß2-tubulins 90.9 ± 0.4 and 93.5 ± 0.05%, respectively, in vitro. A similar result appeared with benomyl on the polymerization of α1-/ß2-tubulins and α2-/ß2-tubulins at 89.9 ± 0.1% and 92.6 ± 1.2% inhibition ratios, respectively. In addition, thiabendazole inhibited 81.6 ± 1% polymerization of α1-/ß2-tubulins, whereas it had less effect on α2-/ß2-tubulin polymerization, with 20.1 ± 1.9% inhibition ratio. However, the three compounds cannot destabilize the polymerized microtubule. To illuminate the issue, mapping the carbendazim binding sites and ß/α subunit interface on ß/α-tubulin complexes by homology modeling showed that the two domains were closed to each other. Understanding the nature of the interaction between benzimidazole compounds and F. graminearum tubulin is fundamental for the development of tubulin-specific anti-F. graminearum compounds.

Elucidation of novel budding yeast separase mutants.

The mitotic separase cleaves Scc1 in cohesin to allow sister chromatids to separate from each other upon anaphase onset. Separase is also required for DNA damage repair. Here, we isolated and characterized 10 temperature-sensitive (ts) mutants of separase ESP1 in the budding yeast Saccharomyces cerevisiae. All mutants were defective in sister chromatid separation at the restricted temperature. Some esp1-ts mutants were hypersensitive to the microtubule poison benomyl and/or the DNA-damaging agent bleomycin. Overexpression of securin alleviated the growth defect in some esp1-ts mutants, whereas it rather exacerbated it in others. The Drosophila Pumilio homolog MPT5 was isolated as a high-dosage suppressor of esp1-ts cells. We discuss various features of separase based on these findings.

Global analysis of core histones reveals nucleosomal surfaces required for chromosome bi-orientation.

The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere-specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae, 24 histone residues that conferred sensitivity to the microtubule-depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty-three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS-I, -II, and -III (TBZ/benomyl-sensitive regions I-III). Elevation of mono-polar attachment induced by prior nocodazole treatment was observed in H2A-I112A (TBS-I), H2A-E57A (TBS-II), and H4-L97A (TBS-III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi-orientation, occurred in H2A-I112A and H2A-E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4-L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS-I and -II, are necessary for Sgo1-mediated chromosome bi-orientation and that TBS-III is required for Htz1 function.

Isolation of a spontaneous cerulenin-resistant sake yeast with both high ethyl caproate-producing ability and normal checkpoint integrity.

In the brewing of high-quality sake such as Daiginjo-shu, the cerulenin-resistant sake yeast strains with high producing ability to the flavor component ethyl caproate have been used widely. Genetic stability of sake yeast would be important for the maintenance of both fermentation properties of yeast and quality of sake. In eukaryotes, checkpoint mechanisms ensure genetic stability. However, the integrity of these mechanisms in sake yeast has not been examined yet. Here, we investigated the checkpoint integrity of sake yeasts, and the results suggested that a currently used cerulenin-resistant sake yeast had a defect in spindle assembly checkpoint (SAC). We also isolated a spontaneous cerulenin-resistant sake yeast FAS2-G1250S mutant, G9CR, which showed both high ethyl caproate-producing ability and integrity/intactness of the checkpoint mechanisms. Further, morphological phenotypic robustness analysis by use of CalMorph supported the genetic stability of G9CR. Finally, we confirmed the high quality of sake from G9CR in an industrial sake brewing setting.

Sclerotinia sclerotiorum populations infecting canola from China and the United States are genetically and phenotypically distinct.

Genetic and phenotypic diversity and population differentiation of Sclerotinia sclerotiorum isolates infecting canola from China and the United States were investigated. Genetic diversity was assessed with eight microsatellite markers and mycelial compatibility groups (MCGs). Phenotypic diversity was assessed with sensitivity to three fungicides, production of oxalate and sclerotia, growth rate, and virulence on two canola cultivars. No shared MCGs or multilocus haplotypes were detected between the two populations, and populations differed significantly (P < 0.001). Recombination was detected in both populations but was greater in the Chinese population. A polymerase chain reaction detection assay showed that ~60% of the isolates were inversion-plus at the mating type locus. The two populations differed significantly (P < 0.05) for all of the phenotypic traits except for sensitivity to fungicide fluazinam and virulence. Isolates in the Chinese population were unique in several aspects. Despite the phenotypic differentiation, heritabilities of the phenotypic traits were similar for both populations. Significant correlations were found among five phenotypic traits. Cross resistance to benomyl and iprodione was detected. Virulence was not significantly correlated with any other phenotypic trait and had the least heritability. However, both populations were equally virulent on either a susceptible or a moderately resistant canola cultivars.

Genomic profiling of drug sensitivities via induced haploinsufficiency.

Lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any drug that acts on the product of this gene. This haploinsufficient phenotype thereby identifies the gene product of the heterozygous locus as the likely drug target. We exploited this finding in a genomic approach to drug-target identification. Genome sequence information was used to generate molecularly tagged heterozygous yeast strains that were pooled, grown competitively in drug and analysed for drug sensitivity using high-density oligonucleotide arrays. Individual heterozygous strain analysis verified six known drug targets. Parallel analysis identified the known target and two hypersensitive loci in a mixed culture of 233 strains in the presence of the drug tunicamycin. Our discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may have important consequences in pharmacogenomics and variable drug toxicity observed in human populations.

Efficiency of selected mutagens in generating Xanthophyllomyces dendrorhous strains hyperproducing astaxanthin.

Astaxanthin is a xanthophill pigment with commercial application in the aquaculture, pharmaceutical, food and cosmetic industries. The red yeast Xanthophyllomyces dendrorhous is one of the most promising microorganisms for its industrial production. However, astaxanthin content in wild yeast strains is low. Pigment production by X. dendrorhous can be improved by mutagenesis. The aim of the study was to assess the efficiency of four mutagens: UV radiation, benomyl, ethyl methanesulfonate and ethidium bromide in generating asthaxanthin-hyperproducing strains of the yeast Xanthophyllomyces dendrorhous DSM 5626. Mutations with benomyl, ethidium bromide and UV radiation generated a group of hyperpigmented mutants exhibiting increases up to 100% in astaxanthin content. Ethyl methanesulfonate turned out to be useless in this respect.

Benomyl-induced development and cardiac toxicity in zebrafish embryos.

Benomyl is a highly effective broad-spectrum fungicide widely used worldwide to control vegetable, fruit, and oil crop diseases. However, the mechanism of its toxicity to aquatic organisms and humans remains unknown. In this study, zebrafish were used to determine the toxicity of benomyl. It was found to be highly toxic, with a 72-h post-fertilization (hpf) lethal concentration 50 (LC50) of 1.454 mg/L. Benomyl induced severe developmental toxicity, including shorter body length, slower heart rate, and a reduced yolk absorption rate. Benomyl also increased oxidative stress in zebrafish, especially in the heart and head, as well as increasing malondialdehyde (MDA) content and decreasing catalase (CAT) and superoxide dismutase (SOD) activities. This indicates that benomyl induced reactive oxygen species (ROS) production and cell membrane peroxidation in vivo. Acridine orange (AO) staining and apoptosis factor detection further indicated that benomyl induced apoptosis in zebrafish. Overall, these findings demonstrate that benomyl disrupts cellular homeostasis by activating oxidative stress in zebrafish, resulting in an imbalance of cardiac development-related gene expression and apoptosis, which causes severe developmental toxicity and cardiac dysfunction. This study evaluated the in vivo toxicity of benomyl, which is a potential threat to aquatic organisms and humans. Possible toxicity mechanisms are explored, providing a valuable reference for the safe use of benomyl.

Disrupting microtubule network immobilizes amoeboid chemotactic receptor in the plasma membrane.

Signaling cascades are initiated in the plasma membrane via activation of one molecule by another. The interaction depends on the mutual availability of the molecules to each other and this is determined by their localization and lateral diffusion in the cell membrane. The cytoskeleton plays a very important role in this process by enhancing or restricting the possibility of the signaling partners to meet in the plasma membrane. In this study we explored the mode of diffusion of the cAMP receptor, cAR1, in the plasma membrane of Dictyostelium discoideum cells and how this is regulated by the cytoskeleton. Single-particle tracking of fluorescently labeled cAR1 using Total Internal Reflection Microscopy showed that 70% of the cAR1 molecules were mobile. These receptors showed directed motion and we demonstrate that this is not because of tracking along the actin cytoskeleton. Instead, destabilization of the microtubules abolished cAR1 mobility in the plasma membrane and this was confirmed by Fluorescence Recovery after Photobleaching. As a result of microtubule stabilization, one of the first downstream signaling events, the jump of the PH domain of CRAC, was decreased. These results suggest a role for microtubules in cAR1 dynamics and in the ability of cAR1 molecules to interact with their signaling partners.

A further study on chromosome minimization by protoplast fusion in Aspergillus oryzae.

Our goal in this work was to develop a method to minimize the chromosomes of Aspergillus oryzae, to arrive at a deeper understanding of essential gene functions that will help create more efficient industrially useful strains. In a previous study, we successfully constructed a highly reduced chromosome 7 using multiple large-scale chromosomal deletions (Jin et al. in Mol Genet Genomics 283:1-12, 2010). Here, we have created a further reduced chromosome A. oryzae mutant harboring a reduced chromosome 7 and a reduced chromosome 8 both of which contain a large number of non-syntenic blocks. These are the smallest A. oryzae chromosomes that have been reported. Protoplast fusion between the two distinct chromosome-reduced mutants produced a vigorous and stable fusant which was isolated. PCR and flow cytometry confirmed that two kinds of nuclei, derived from the parent strains, existed in this fusant and that the chromosome DNA per nucleus was doubled, suggesting that the fusant was a heterozygous diploid strain. By treating the cell with 1 µg/ml benomyl, cell nuclei haploidization was induced in the stable diploid strain. Array comparative genomic hybridization and pulsed-field gel electrophoresis confirmed that the reduced chromosomes 7 and 8 co-existed in the haploid fusant and that no other chromosomal modifications had occurred. This method provides a useful tool for chromosome engineering in A. oryzae to construct an industry-useful strain.

Global analysis for functional residues of histone variant Htz1 using the comprehensive point mutant library.

Histone variants perform unique functions and are deposited onto DNA by mechanisms distinct from those of canonical histones. The H2A variant, H2A.Z, also known as Htz1 in Saccharomyces cerevisiae, is not uniformly distributed across the genome but facilitates transcriptional activation at target gene promoters and anti-silencing at heterochromatin loci. Htz1 is also involved in DNA replication, DNA repair, chromosome segregation and cell cycle control. Its sequence identity to canonical H2A is only ∼60%, and it is likely that the nonconserved residues are responsible for Htz1-specific functions. However, precise roles of these variant-specific residues are not well understood. To gain insights into the molecular basis underlying the functional differences between canonical and variant histones, 117 alanine-scanning point mutants of Htz1 were constructed for this study, and chemical genetic screens were carried out. Consequently, seven Htz1 residues that conferred one or more abnormal phenotypes when mutated were identified. Based on primary sequence and functional conservation between H2A and Htz1, two of these residues (F32 and I109) appear to have an Htz1-specific role, whereas the rest seem to have functions shared between H2A and Htz1. This study provides a useful resource for future investigations into functional convergence and divergence between canonical and variant histones.

The sesquiterpenoid valerenic acid protects neuronal cells from the detrimental effects of the fungicide benomyl on apoptosis and DNA oxidation.

BACKGROUND: Valerenic acid (VA), a sesquiterpenoid of the plant Valeriana officinalis, has attracted attention of the research community due to its potential positive role against neurodegenerative diseases induced by chemicals. However, the relevant evidence in the literature is scarce. Therefore, this study aimed to examine the putative protective role of VA on the toxic effects of the fungicide benomyl on SH-SY5Y neural cells. METHODS: Cell viability was determined via the MTT and NRU assays, DNA damage was assessed via comet assay and apoptosis was evaluated through the expression of relevant genes. RESULTS: According to the results, exposure of the cells to benomyl enhanced viability inhibition and promoted DNA damage and apoptosis since the expression levels of the genes coding for MAPK8, NF-kB, Bax, Caspase-9 and Caspase-3 were increased. Treatment of the cells with VA ameliorated these effects in a concentration dependent manner. CONCLUSION: It is concluded that the molecular mechanism through which benomyl exerts its toxic action appears to depend on DNA oxidation and apoptosis induction. Furthermore, VA, a plant-derived compound is a protective antioxidant against pesticide-induced toxicity. Therefore, herbs, extracts and compounds of plant origin could be used as nutritional supplements that back up the beneficial role of medicine in neurodegenerative diseases.

Transcriptional Response of Candida auris to the Mrr1 Inducers Methylglyoxal and Benomyl.

Candida auris is an urgent threat to human health due to its rapid spread in health care settings and its repeated development of multidrug resistance. Diseases that increase risk for C. auris infection, such as diabetes, kidney failure, or immunocompromising conditions, are associated with elevated levels of methylglyoxal (MG), a reactive dicarbonyl compound derived from several metabolic processes. In other Candida species, expression of MG reductase enzymes that catabolize and detoxify MG are controlled by Mrr1, a multidrug resistance-associated transcription factor, and MG induces Mrr1 activity. Here, we used transcriptomics and genetic assays to determine that C. auris MRR1a contributes to MG resistance, and that the main Mrr1a targets are an MG reductase and MDR1, which encodes a drug efflux protein. The C. auris Mrr1a regulon is smaller than Mrr1 regulons described in other species. In addition to MG, benomyl (BEN), a known Mrr1 stimulus, induces C. auris Mrr1 activity, and characterization of the MRR1a-dependent and -independent transcriptional responses revealed substantial overlap in genes that were differentially expressed in response to each compound. Additionally, we found that an MRR1 allele specific to one C. auris phylogenetic clade, clade III, encodes a hyperactive Mrr1 variant, and this activity correlated with higher MG resistance. C. auris MRR1a alleles were functional in Candida lusitaniae and were inducible by BEN, but not by MG, suggesting that the two Mrr1 inducers act via different mechanisms. Together, the data presented in this work contribute to the understanding of Mrr1 activity and MG resistance in C. auris. IMPORTANCE Candida auris is a fungal pathogen that has spread since its identification in 2009 and is of concern due to its high incidence of resistance against multiple classes of antifungal drugs. In other Candida species, the transcription factor Mrr1 plays a major role in resistance against azole antifungals and other toxins. More recently, Mrr1 has been recognized to contribute to resistance to methylglyoxal (MG), a toxic metabolic product that is often elevated in different disease states. MG can activate Mrr1 and its induction of Mdr1 which can protect against diverse challenges. The significance of this work lies in showing that MG is also an inducer of Mrr1 in C. auris, and that one of the major pathogenic C. auris lineages has an activating Mrr1 mutation that confers protection against MG.

A role for the karyopherin Kap123p in microtubule stability.

Several components of the nuclear transport machinery play a role in mitotic spindle assembly in higher eukaryotes. To further investigate the role of this family of proteins in microtubule function, we screened for mutations in Saccharomyces cerevisiae that confer sensitivity to microtubule-destabilizing drugs. One mutant exhibiting this phenotype lacked the gene encoding the karyopherin Kap123p. Analysis of kap123Delta cells revealed that the drug sensitivity was caused by a defect in microtubule stability and/or assembly. In support of this idea, we demonstrated genetic interactions between the kap123Delta mutation and mutated alleles of genes encoding alpha-tubulins and factors controlling microtubule dynamics. Moreover, kap123Delta cells exhibit defects in spindle structure and dynamics as well as nuclear positioning defects during mitosis. Cultures of kap123Delta strains are enriched for mononucleated large-budded cells often containing short spindles and nuclei positioned away from the budneck, phenotypes indicative of defects in both cytoplasmic and nuclear microtubules. Finally, we identified a gene, CAJ1, which when deleted in combination with KAP123 exacerbated the microtubule-related defects of the kap123Delta mutants. We propose that Kap123p and Caj1p, a member of the Hsp40 family of proteins, together play an essential role in normal microtubule function.