Transcriptomics Reveals the Effect of Thymol on the Growth and Toxin Production of Fusarium graminearum.

Fusarium graminearum is a harmful pathogen causing head blight in cereals such as wheat and barley, and thymol has been proven to inhibit the growth of many pathogens. This study aims to explore the fungistatic effect of thymol on F. graminearum and its mechanism. Different concentrations of thymol were used to treat F. graminearum. The results showed that the EC50 concentration of thymol against F. graminearum was 40 µg/mL. Compared with the control group, 40 µg/mL of thymol reduced the production of Deoxynivalenol (DON) and 3-Ac-DON by 70.1% and 78.2%, respectively. Our results indicate that thymol can effectively inhibit the growth and toxin production of F. graminearum and cause an extensive transcriptome response. Transcriptome identified 16,727 non-redundant unigenes and 1653 unigenes that COG did not annotate. The correlation coefficients between samples were all >0.941. When FC was 2.0 times, a total of 3230 differential unigenes were identified, of which 1223 were up-regulated, and 2007 were down-regulated. Through the transcriptome, we confirmed that the expression of many genes involved in F. graminearum growth and synthesis of DON and other secondary metabolites were also changed. The gluconeogenesis/glycolysis pathway may be a potential and important way for thymol to affect the growth of F. graminearum hyphae and the production of DON simultaneously.

Pydiflumetofen Co-Formulated with Prothioconazole: A Novel Fungicide for Fusarium Head Blight and Deoxynivalenol Control.

Fusarium head blight (FHB) is an important disease of small grain cereals worldwide, resulting in reduced yield and quality as well as the contamination of harvested grains with mycotoxins. The key mycotoxin of concern is deoxynivalenol (DON), which has legislative and advisory limits in numerous countries. Cereal growers have a number of control options for FHB including rotation, cultivation, and varietal resistance; however, growers are still reliant on fungicides applied at flowering as part of an IPM program. Fungicides currently available to control FHB are largely restricted to triazole chemistry. This study conducted three field experiments to compare a new co-formulation of pydiflumetofen (a succinate dehydrogenase inhibitor (SDHI) with the tradename ADEPIDYN™) and prothioconazole (a triazole) against current standard fungicides at various timings (flag leaf fully emerged, mid-head emergence, early flowering, and late flowering) for the control of FHB and DON. Overall, the co-formulation showed greater efficacy compared to either pydiflumetofen alone or current fungicide chemistry. This greater activity was demonstrated over a wide range of spray timings (flag leaf fully emerged to late flowering). The availability of an SDHI with good activity against FHB and the resulting DON contamination of harvested grain will give growers an additional tool within an IPM program that will provide a greater flexibility of spray application windows and reduce fungicide resistance selection pressure.

Fatty Acids and Oxylipins as Antifungal and Anti-Mycotoxin Agents in Food: A Review.

Fungal contamination of food, especially by mycotoxigenic fungi, not only reduces the quality of the food, but can also cause serious diseases, thus posing a major food safety challenge to humans. Apart from sound food control systems, there is also a continual need to explore antifungal agents that can inhibit fungal growth and mycotoxin production in food. Many types of fatty acids (FAs) and their oxidized derivatives, oxylipins, have been found to exhibit such effects. In this review, we provide an update on the most recent literature on the occurrence and formation of FAs and oxylipins in food, their effects on fungal growth and mycotoxin synthesis, as well as the genetic and molecular mechanisms of actions. Research gaps in the field and needs for further studies in order to realizing the potential of FAs and oxylipins as natural antifungal preservatives in food are also discussed.

Antifungal Peptides and Proteins to Control Toxigenic Fungi and Mycotoxin Biosynthesis.

The global challenge to prevent fungal spoilage and mycotoxin contamination on food and feed requires the development of new antifungal strategies. Antimicrobial peptides and proteins (AMPs) with antifungal activity are gaining much interest as natural antifungal compounds due to their properties such as structure diversity and function, antifungal spectrum, mechanism of action, high stability and the availability of biotechnological production methods. Given their multistep mode of action, the development of fungal resistance to AMPs is presumed to be slow or delayed compared to conventional fungicides. Interestingly, AMPs also accomplish important biological functions other than antifungal activity, including anti-mycotoxin biosynthesis activity, which opens novel aspects for their future use in agriculture and food industry to fight mycotoxin contamination. AMPs can reach intracellular targets and exert their activity by mechanisms other than membrane permeabilization. The mechanisms through which AMPs affect mycotoxin production are varied and complex, ranging from oxidative stress to specific inhibition of enzymatic components of mycotoxin biosynthetic pathways. This review presents natural and synthetic antifungal AMPs from different origins which are effective against mycotoxin-producing fungi, and aims at summarizing current knowledge concerning their additional effects on mycotoxin biosynthesis. Antifungal AMPs properties and mechanisms of action are also discussed.

Mycosubtilin Produced by Bacillus subtilis ATCC6633 Inhibits Growth and Mycotoxin Biosynthesis of Fusarium graminearum and Fusarium verticillioides.

Fusarium graminearum and Fusarium verticillioides are fungal pathogens that cause diseases in cereal crops, such as Fusarium head blight (FHB), seedling blight, and stalk rot. They also produce a variety of mycotoxins that reduce crop yields and threaten human and animal health. Several strategies for controlling these diseases have been developed. However, due to a lack of resistant cultivars and the hazards of chemical fungicides, efforts are now focused on the biocontrol of plant diseases, which is a more sustainable and environmentally friendly approach. In the present study, the lipopeptide mycosubtilin purified from Bacillus subtilis ATCC6633 significantly suppressed the growth of F. graminearum PH-1 and F. verticillioides 7600 in vitro. Mycosubtilin caused the destruction and deformation of plasma membranes and cell walls in F. graminearum hyphae. Additionally, mycosubtilin inhibited conidial spore formation and germination of both fungi in a dose-dependent manner. In planta experiments demonstrated the ability of mycosubtilin to control the adverse effects caused by F. graminearum and F. verticillioides on wheat heads and maize kernels, respectively. Mycosubtilin significantly decreased the production of deoxynivalenol (DON) and B-series fumonisins (FB1, FB2 and FB3) in infected grains, with inhibition rates of 48.92, 48.48, 52.42, and 59.44%, respectively. The qRT-PCR analysis showed that mycosubtilin significantly downregulated genes involved in mycotoxin biosynthesis. In conclusion, mycosubtilin produced by B. subtilis ATCC6633 was shown to have potential as a biological agent to control plant diseases and Fusarium toxin contamination caused by F. graminearum and F. verticillioides.

Ammoides pusilla Essential Oil: A Potent Inhibitor of the Growth of Fusarium avenaceum and Its Enniatin Production.

Enniatins are mycotoxins produced by Fusarium species contaminating cereals and various agricultural commodities. The co-occurrence of these mycotoxins in large quantities with other mycotoxins such as trichothecenes and the possible synergies in toxicity could lead to serious food safety problems. Using the agar dilution method, Ammoides pusilla was selected among eight Tunisian plants for the antifungal potential of its essential oil (EO) on Fusarium avenaceum mycelial growth and its production of enniatins. Two EO batches were produced and analyzed by GC/MS-MS. Their activities were measured using both contact assays and fumigant tests (estimated IC50 were 0.1 µL·mL-1 and 7.6 µL·L-1, respectively). The A. pusilla EOs and their volatiles inhibited the germination of spores and the mycelial growth, showing a fungistatic but not fungicidal activity. The accumulation of enniatins was also significantly reduced (estimated IC50 were 0.05 µL·mL-1 for the contact assays and 4.2 µL·L-1 for the fumigation assays). The most active batch of EO was richer in thymol, the main volatile compound found. Thymol used as fumigant showed a potent fungistatic activity but not a significant antimycotoxigenic activity. Overall, our data demonstrated the bioactivity of A. pusilla EO and its high potential to control F. avenaceum and its enniatins production in agricultural commodities.

Biosynthetic Studies of Phomopsins Unveil Posttranslational Installation of Dehydroamino Acids by UstYa Family Proteins.

UstYa family proteins (DUF3328) are widely and specifically distributed in fungi. They are known to be involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and nonribosomal peptides, and possibly catalyze various reactions, including oxidative cyclization and chlorination. In this study, we focused on phomopsin A, a fungal RiPP consisting of unique nonproteinogenic amino acids. Gene knockout experiments demonstrated that three UstYa homologues, phomYc, phomYd, and phomYe, are essential for the desaturation of amino acid moieties, showing unprecedented function among UstYa family proteins. Sequence similarity network analysis indicated that their amino acid sequences are highly diverged and that most remain uncharacterized, paving the way for genome mining of fungal metabolites with unique modifications.

Phytopathogenic Fungi and Toxicity.

Phytopathogen fungi are responsible for serious plant diseases which might negatively affect crop productivity [...].

Biocontrol Agents Reduce Progression and Mycotoxin Production of Fusarium graminearum in Spikelets and Straws of Wheat.

The aim of this study was to evaluate the interactions between wheat plant (spikelets and straws), a strain of mycotoxigenic pathogen Fusarium graminearum and commercial biocontrol agents (BCAs). The ability of BCAs to colonize plant tissue and inhibit the pathogen or its toxin production was observed throughout two phases of the life cycle of pathogens in natural conditions (colonization and survival). All evaluated BCAs showed effective reduction capacities of pathogenic traits. During establishment and the expansion stage, BCAs provoked an external growth reduction of F. graminearum (77-93% over the whole kinetic studied) and mycotoxin production (98-100% over the whole kinetic studied). Internal growth of pathogen was assessed with digital droplet polymerase chain reaction (ddPCR) and showed a very strong reduction in the colonization of the internal tissues of the spikelet due to the presence of BCAs (98% on average). During the survival stage, BCAs prevented the formation of conservation perithecia of the pathogen on wheat straw (between 88 and 98% of perithecia number reduction) and showed contrasting actions on the ascospores they contain, or perithecia production (-95% on average) during survival form. The mechanisms involved in these different interactions between F. graminearum and BCAs on plant matrices at different stages of the pathogen's life cycle were based on a reduction of toxins, nutritional and/or spatial competition, or production of anti-microbial compounds.

Production of Alternaria Toxins in Yellow Peach (Amygdalus persica) upon Artificial Inoculation with Alternaria alternate.

The yellow peach (Amygdalus persica), an important fruit in China, is highly susceptible to infection by Alternaria sp., leading to potential health risks and economic losses. In the current study, firstly, yellow peaches were artificially inoculated with Alternariaalternate. Then, the fruits were stored at 4 °C and 28 °C to simulate the current storage conditions that consumers use, and the Alternaria toxins (ATs) contents from different parts of the fruits were analyzed via ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The results showed that the growth of A. alternate and the ATs production were dramatically affected by the storage temperature. At 28 °C, the fungi grew rapidly and the lesion diameter reached about 4.0 cm within 15 days of inoculation, while, at 4 °C, the fungal growth was noticeably inhibited, with no significant change in the lesion diameter. To our surprise, high contents of ATs were produced under both storage conditions even though the fungal growth was suppressed. With an increase in the incubation time, the amounts of ATs showed a steady tendency to increase in most cases. Remarkably, alternariol monomethyl ether (AME), alternariol (AOH), and tenuazonic acid (TeA) were detected in the rotten tissue and also in the surrounding tissue, while a large amount of TeA could also be found in the healthy tissue. To the best of our knowledge, this is the first report regarding the production of ATs by the infection of Alternaria sp. in yellow peach fruits via artificial inoculation under regulated conditions, and, based on the evidence herein, it is recommended that ATs be included in monitoring and control programs of yellow peach management and food safety administration.

Current Approaches for Advancement in Understanding the Molecular Mechanisms of Mycotoxin Biosynthesis.

Filamentous fungi are able to synthesise a remarkable range of secondary metabolites, which play various key roles in the interaction between fungi and the rest of the biosphere, determining their ecological fitness. Many of them can have a beneficial activity to be exploited, as well as negative impact on human and animal health, as in the case of mycotoxins contaminating large quantities of food, feed, and agricultural products worldwide and posing serious health and economic risks. The elucidation of the molecular aspects of mycotoxin biosynthesis has been greatly sped up over the past decade due to the advent of next-generation sequencing technologies, which greatly reduced the cost of genome sequencing and related omic analyses. Here, we briefly highlight the recent progress in the use and integration of omic approaches for the study of mycotoxins biosynthesis. Particular attention has been paid to genomics and transcriptomic approaches for the identification and characterisation of biosynthetic gene clusters of mycotoxins and the understanding of the regulatory pathways activated in response to physiological and environmental factors leading to their production. The latest innovations in genome-editing technology have also provided a more powerful tool for the complete explanation of regulatory and biosynthesis pathways. Finally, we address the crucial issue of the interpretation of the combined omics data on the biology of the mycotoxigenic fungi. They are rapidly expanding and require the development of resources for more efficient integration, as well as the completeness and the availability of intertwined data for the research community.

Cross-talk between Fusarium verticillioides and Aspergillus flavus in vitro and in planta.

Driven by increasing temperatures and the higher incidences of heat waves during summer, an increased incidence of Aspergillus flavus next to Fusarium verticillioides in European maize can be expected. In the current study, we investigated the interaction between both species. Colonies of A. flavus/F. verticillioides were grown in a single culture, in a dual culture, and in a mixed culture. The growth rate of A. flavus and F. verticillioides grown in a dual or mixed culture with the other species was clearly slower compared to the growth rate in a single culture. Mycotoxin production was in most cases negatively affected by dual or mixed inoculation. In planta, a dual inoculation resulted in reduced lesions of A. flavus, whereas the lesion size and toxin production of F. verticillioides were unaffected in the presence of A. flavus. The lesions as a result of a mixed inoculation were 112% bigger than a single A. flavus inoculation and 9% smaller than a single F. verticillioides inoculation. The fumonisin levels were 17% higher compared to a single inoculation. In case A. flavus was present two days before F. verticillioides, the lesion size of F. verticillioides was 55% smaller compared to a single F. verticillioides inoculation, and fumonisin production was almost completely inhibited. The interaction between A. flavus and F. verticillioides is highly dynamic and depends on the experimental conditions, on the variables measured and on the way they colonize the host, in two inoculation points, simultaneously in one inoculation point, or sequentially one species colonizing an existing lesion made by the other.

Tick defensin γ-core reduces Fusarium graminearum growth and abrogates mycotoxins production with high efficiency.

Fusarium graminearum is a major fungal pathogen affecting crops of worldwide importance. F. graminearum produces type B trichothecene mycotoxins (TCTB), which are not fully eliminated during food and feed processing. Therefore, the best way to minimize TCTB contamination is to develop prevention strategies. Herein we show that treatment with the reduced form of the γ-core of the tick defensin DefMT3, referred to as TickCore3 (TC3), decreases F. graminearum growth and abrogates TCTB production. The oxidized form of TC3 loses antifungal activity, but retains anti-mycotoxin activity. Molecular dynamics show that TC3 is recruited by specific membrane phospholipids in F. graminearum and that membrane binding of the oxidized form of TC3 is unstable. Capping each of the three cysteine residues of TC3 with methyl groups reduces its inhibitory efficacy. Substitutions of the positively-charged residues lysine (Lys) 6 or arginine 7 by threonine had the highest and the lesser impact, respectively, on the anti-mycotoxin activity of TC3. We conclude that the binding of linear TC3 to F. graminearum membrane phospholipids is required for the antifungal activity of the reduced peptide. Besides, Lys6 appears essential for the anti-mycotoxin activity of the reduced peptide. Our results provide foundation for developing novel and environment-friendly strategies for controlling F. graminearum.

The transcription regulator ACTR controls ACT-toxin biosynthesis and pathogenicity in the tangerine pathotype of Alternaria alternata.

The host-selective ACT toxin is essential for the pathogenesis of the citrus fungal pathogen Alternaria alternata. However, the mechanism of ACT-toxin gene clusters ACT-toxin biosynthesis regulated by is still poorly understood. The biosynthesis of ACT toxin is mainly regulated by multiple ACT toxin genes located in the secondary metabolite gene cluster. In this study, we reported a transcription regulator ACTR contributes ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 in Alternaria alternata. We generated ACTR-disrupted and -silenced mutants in the tangerine pathotype of A. alternata. Phenotype analysis showed that the ACTR mutants displayed a significant loss of ACT toxin production and a decreased virulence on citrus leaves whereas the vegetative growth and sporulation were not affected, indicating an essential role of ACTR in both ACT toxin biosynthesis and pathogenicity. To elucidate the transcription network of ACTR, we performed RNA-Seq experiments on wild-type and ACTR null mutant and identified genes that were differentially expressed between two genotypes. Transcriptome profiling and RT-qPCR analysis demonstrated that the ACT toxin biosynthetic gene ACTS4 is down-regulated in ACTR mutant. We generated ACTS4 knock-down mutant and found that the pathogenicity of ACTS4 mutant was severely impaired. Interestingly, both ACTR and ACTS4 are not involved in the response to different abiotic stresses including oxidative stress, salt stress, cell-wall disrupting regents, and metal ion stress, indicating the function of these two genes is highly specific. In conclusion, our results highlight the important regulatory role of ACTR in ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 and underline the essential role of in the tangerine pathotype of A. alternata.

Effect of Debaryomyces hansenii and the antifungal PgAFP protein on Alternaria spp. growth, toxin production, and RHO1 gene expression in a tomato-based medium.

Tomato fruit is susceptible to Alternaria spp. spoilage, which poses a health risk due to their mycotoxin production. Biopreservation relies on the use of whole microorganisms or their metabolites to manage spoilage microorganisms including filamentous fungi. However, the use of treatments at fungistatic level might activate intracellular pathways, which can cause an increment in mycotoxin accumulation. The objective of this work was to evaluate the effect of two strains of Debaryomyces hansenii and the antifungal protein PgAFP at 10 and 40 µg/mL. Both growth and production of two of the most common mycotoxins (tenuazonic acid and alternariol monomethyl ether) by Alternaria tenuissima sp.-grp. and Alternaria arborescens sp.-grp. on a tomato-based matrix, were analysed at 12 °C. Additionally, the impact of these biocontrol agents on the stress-related RHO1 gene expression was assessed. All treatments reduced mycotoxin accumulation (from 27 to 92% of inhibition). Their mode of action against Alternaria spp. in tomato seems unrelated to damages to fungal cell wall integrity at the genomic level. Therefore, the two D. hansenii strains (CECT 10352 and CECT 10353) and the antifungal protein PgAFP at 10 µg/mL are suggested as biocontrol strategies in tomato fruit at postharvest stage.

Synthesis of (12R,13S)-pyriculariol and (12R,13S)-dihydropyriculariol revealed that the rice blast fungus, Pyricularia oryzae, produces these phytotoxins as racemates.

Synthesis of assumed natural (12R,13S)-enantiomers of pyriculariol (1) and dihydropyriculariol (2), phytotoxins isolated from rice blast disease fungus, Pyricularia oryzae, was achieved using Wittig reaction or microwave-assisted Stille coupling reaction as the key step. The synthesis revealed that the natural 1 and 2 are racemates. Foliar application test on a rice leaf indicated that both the salicylaldehyde core and side chain were necessary for phytotoxic activity. The fungus is found to produce optically active phytotoxins when incubated with rotary shaker, but racemic ones when cultured using an aerated jar fermenter.

Controlling the production of phytotoxin pyriculol in Pyricularia oryzae by aldehyde reductase.

Pyricularia oryzae is one of the most devastating plant pathogens in the world. This fungus produces several secondary metabolites including the phytotoxin pyriculols, which are classified into 2 types: aldehyde form (pyriculol and pyriculariol) and alcohol form (dihydropyriculol and dihydropyriculariol). Although interconversion between the aldehyde form and alcohol form has been predicted, and the PYC10 gene for the oxidation of alcohol form to aldehyde is known, the gene responsible for the reduction of aldehyde to alcohol form is unknown. Furthermore, previous studies have predicted that alcohol analogs are biosynthesized via aldehyde analogs. Herein, we demonstrated that an aldo/keto reductase PYC7 is responsible for the reduction of aldehyde to alcohol congeners. The results indicate that aldehyde analogs are biosynthesized via alcohol analogs, contradicting the previous prediction. The results suggest that P. oryzae controls the amount of pyriculol analogs using two oxidoreductases, PYC7 and PYC10, thereby controlling the bioactivity of the phytotoxin.

Screening of Wood/Forest and Vine By-Products as Sources of New Drugs for Sustainable Strategies to Control Fusarium graminearum and the Production of Mycotoxins.

Fusarium graminearum is a fungal pathogen that can colonize small-grain cereals and maize and secrete type B trichothecene (TCTB) mycotoxins. The development of environmental-friendly strategies guaranteeing the safety of food and feed is a key challenge facing agriculture today. One of these strategies lies on the promising capacity of products issued from natural sources to counteract crop pests. In this work, the in vitro efficiency of sixteen extracts obtained from eight natural sources using subcritical water extraction at two temperatures was assessed against fungal growth and TCTB production by F. graminearum. Maritime pine sawdust extract was shown to be extremely efficient, leading to a significant inhibition of up to 89% of the fungal growth and up to 65% reduction of the mycotoxin production by F. graminearum. Liquid chromatography/mass spectrometry analysis of this active extract revealed the presence of three families of phenolics with a predominance of methylated compounds and suggested that the abundance of methylated structures, and therefore of hydrophobic compounds, could be a primary factor underpinning the activity of the maritime pine sawdust extract. Altogether, our data support that wood/forest by-products could be promising sources of bioactive compounds for controlling F. graminearum and its production of mycotoxins.

Sarocladium and Lecanicillium Associated with Maize Seeds and Their Potential to Form Selected Secondary Metabolites.

The occurrence and diversity of Lecanicillium and Sarocladium in maize seeds and their role in this cereal are poorly understood. Therefore, the present study aimed to investigate Sarocladium and Lecanicillium communities found in endosphere of maize seeds collected from fields in Poland and their potential to form selected bioactive substances. The sequencing of the internally transcribed spacer regions 1 (ITS 1) and 2 (ITS2) and the large-subunit (LSU, 28S) of the rRNA gene cluster resulted in the identification of 17 Sarocladium zeae strains, three Sarocladium strictum and five Lecanicillium lecanii isolates. The assay on solid substrate showed that S. zeae and S. strictum can synthesize bassianolide, vertilecanin A, vertilecanin A methyl ester, 2-decenedioic acid and 10-hydroxy-8-decenoic acid. This is also the first study revealing the ability of these two species to produce beauvericin and enniatin B1, respectively. Moreover, for the first time in the present investigation, pyrrocidine A and/or B have been annotated as metabolites of S. strictum and L. lecanii. The production of toxic, insecticidal and antibacterial compounds in cultures of S. strictum, S. zeae and L. lecanii suggests the requirement to revise the approach to study the biological role of fungi inhabiting maize seeds.

Specialized Flavoprotein Promotes Sulfur Migration and Spiroaminal Formation in Aspirochlorine Biosynthesis.

Epidithiodiketopiperazines (ETPs) are a class of ecologically and medicinally important cyclodipeptides bearing a reactive transannular disulfide bridge. Aspirochlorine, an antifungal and toxic ETP isolated from Aspergillus oryzae used in sake brewing, deviates from the common ETP scaffold owing to its unusual ring-enlarged disulfide bridge linked to a spiroaminal ring system. Although this disulfide ring system is implicated in the biological activity of ETPs the biochemical basis for this derailment has remained a mystery. Here we report the discovery of a novel oxidoreductase (AclR) that represents the first-in-class enzyme catalyzing both a carbon-sulfur bond migration and spiro-ring formation, and that the acl pathway involves a cryptic acetylation as a prerequisite for the rearrangement. Genetic screening in A. oryzae identified aclR as the candidate for the complex biotransformation, and the aclR-deficient mutant provided the biosynthetic intermediate, unexpectedly harboring an acetyl group. In vitro assays showed that AclR alone promotes 1,2-sulfamyl migration, elimination of the acetoxy group, and spiroaminal formation. AclR features a thioredoxin oxidoreductase fold with a noncanonical CXXH motif that is distinct from the CXXC in the disulfide forming oxidase for the ETP biosynthesis. Crystallographic and mutational analyses of AclR revealed that the CXXH motif is crucial for catalysis, whereas the flavin-adenine dinucleotide is required as a support of the protein fold, and not as a redox cofactor. AclR proved to be a suitable bioinformatics handle to discover a number of related fungal gene clusters that potentially code for the biosynthesis of derailed ETP compounds. Our results highlight a specialized role of the thioredoxin oxidoreductase family enzyme in the ETP pathway and expand the chemical diversity of small molecules bearing an aberrant disulfide pharmacophore.