Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s) underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ) fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0–48 hours), gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C) and during the phase transition to mold (22°C). This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.In response to temperature,  Blastomyces dermatitidis belongs to a group of human pathogenic fungi that convert between two forms, mold and yeast, in response to temperature. Growth as yeast (37°C) in tissue facilitates immune evasion, whereas growth as mold (22°C) promotes environmental survival, sexual reproduction, and generation of transmissible spores. Despite the importance of dimorphism, how fungi regulate temperature adaptation is poorly understood. We identified SREB, a transcription factor that regulates disparate processes including dimorphism. SREB null mutants, which lack SREB, fail to fully complete the conversion to mold at 22°C. The goal of our research was to characterize how SREB regulates transcription during the switch to mold. Gene expression microarray along with chromatin binding and biochemical analyses indicated that SREB affected several processes including iron homeostasis, lipid biosynthesis, and lipid droplet formation. In vivo, SREB directly bound and regulated genes involved with iron uptake, lipid biosynthesis, and transcription. Functional analysis suggested that lipid metabolism may influence filamentous growth at 22°C. In addition, SREB interacted with another transcription factor, HAPX. Blastomyces dermatitidis belongs to a group of medically important ascomycetes that adapt to shifts in temperature by undergoing a morphologic switch known as the phase transition [B. dermatitidis is able to evade host immune defenses to cause pneumonia and disseminate to other organs such as the bone or brain [Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Paracoccidioides brasiliensis, Penicillium marneffei, and Sporothrix schenckii [ansition . In soilansition ,2. In thor brain –5. This chenckii . The trachenckii .B. dermatitidis and H. capsulatum, DRK1 (dimorphism-regulating kinase-1) and RYP1-4 (required for yeast phase) promote the temperature-dependent conversion from mold to yeast, respectively [DRK1 renders B. dermatitidis and H. capsulatum avirulent during experimental murine pulmonary infection [Penicillium marneffei, HGRA  facilitates the conversion of yeast to hyphae at 25°C and TUPA promotes maintenance of mycelial phase morphology [B. dermatitidis, we discovered SREB (siderophore biosynthesis repressor in Blastomyces), which encodes a GATA transcription factor that affects the temperature-dependent morphologic switch and iron homeostasis [SREB null (SREB∆) and insertional mutants fail to fully complete the conversion from yeast to sporulating mold after a drop in temperature from 37°C to 22°C, and under iron-replete conditions cannot repress the biosynthesis of iron-gathering siderophores [SREB on the phase transition appears conserved in the dimorphic fungi; knockdown of an SREB homolog in H. capsulatum led to failure of yeast cells to fully convert to mold at ambient temperature [VMA1, which encodes a catalytic subunit in a vacuolar ATPase involved with iron homeostasis and virulence, also impairs the conversion of H. capsulatum yeast to mold [NGT1 and NGT2 transmembrane transporters [B. dermatitidis and H. capsulatum yeast to mold at room temperature [The phase transition is a complex process that involves alteration of transcription, metabolism, lipid content, and cell wall carbohydrate composition –13. In Bectively ,7,14,15.nfection . In Penirphology ,17. In Beostasis . SREB nurophores . The regperature . Deletio to mold . The kinsporters . Exogenoperature . AlthougASH1 in Saccharomyces cerevisiae inhibits mating type switching and under nitrogen poor conditions induces pseudohyphal growth [Cryptococcus neoformans CIR1, which is a homolog of SREB, regulates genes important for thermotolerance at 37°C, capsule formation, iron uptake, and mating [GATA transcription factors bind DNA via zinc finger motifs to induce or repress transcription in response to environmental stimuli. In fungi, they regulate adaptive responses to light, temperature, nitrogen, and iron –28. Morel growth ,23. Crypd mating ,25SREB regulon on a genome-wide scale using gene expression microarrays, identify a subset of genes directly bound and regulated by SREB in vivo, and functionally test the impact of SREB-regulated genes on the phase transition. Because the morphologic defect in SREB∆ occurred within 24–48 hours of a drop in temperature, we focused our attention to the early part of the phase transition where B. dermatitidis yeast adapt to 22°C and begin the conversion to mold. The data generated from our approach indicated that the GATA transcription factor encoded by SREB affects transcription at 37°C and 22°C, and binds to genes involved with diverse (iron and non-iron) processes. In addition to functioning as a major regulator of iron assimilation, SREB affected the biosynthesis of lipids at 37°C and 22°C, and lipid droplet formation at 22°C. Similar to filamentous fungi such as Aspergillus fumigatus, SREB was in a regulatory circuit with the bZIP transcription factor HAPX. Deletion of SREB or overexpression of HAPX resulted in a defect in the morphologic switch at 22°C.The goals of this study were to characterize the SREB∆, we compared it to an isogenic wild-type (WT) control at 37°C and at 6, 24, and 48-hrs following a drop in temperature to 22°C . At 37°C, WT and SREB∆ grew as budding yeast . After 6-hrs at 22°C, WT and SREB∆ continued to have a yeast morphology with 22% and 4% cells exhibiting early germ tube development, respectively . At 24 and 48-hrs at 22°C, the germ tubes of WT cells had elongated to become hyphae . In contrast, sharp morphologic differences between SREB∆ and WT became apparent at 24 and 48-hrs at 22°C with SREB∆ demonstrating a delay in germ tube formation and stunted, misshapen germ tubes and hyphae . Deletion of SREB did not affect cell viability (To investigate the kinetics of the phase transition defect in SREB∆ occurred early in the phase transition (conversion of wild-type B. dermatitidis to sporulating mold requires 10–14 days incubation at 22°C). To optimize detection of transcriptional events associated with the phase transition , we focused on the first 48-hrs at 22°C, which was when the morphologic defect in SREB∆ first became apparent. Microarray analyses of later time points  were not performed because the transcriptional differences between SREB∆ and WT would reflect differences in filamentous growth rather than the morphologic switch. We harvested RNA from 3 biological replicates for WT and SREB∆ at 37°C, and 6, 24, and 48-hrs following a drop in temperature to 22°C. The 37°C time point served a baseline, whereas the 22°C time points provided insight into transcription at the beginning of germ tube formation (6-hrs) and during the transition from germ tubes to hyphae (24-hrs and 48-hrs) . All cultures were grown under iron-replete conditions (10 μM FeSO4) to optimize transcription of SREB in the isogenic WT isolate. Iron induces transcription of SREB and is required for the binding of SREB homologs, such as H. capsulatum SRE1, to DNA [On the basis of the time-course data, we postulated that transcriptional alterations responsible for the morphologic defect in , to DNA ,29.SREB∆ versus WT isolates at each time point, [Table 1and S1 Table). All DE genes identified by EBarrays were also DE by LIMMA and, consequently, to be conservative, subsequent analyses herein include genes that were classified as DE by EBarrays (S1 Table). The majority of DE genes in SREB∆ exhibited a 1.5-fold or greater change in transcript abundance when controlling the false discovery rate at 5% (Table 1). In the yeast phase (37°C), the ratio of DE genes with increased to decreased transcription was 1.56:1. At 22°C, this ratio was nearly equal . At 22°C, the majority of DE genes  did not overlap with DE genes found at baseline (37°C). Thus, differential expression of these genes was unique to the 22°C time points. Collectively, these data indicate that deletion of SREB affected transcription at 37°C and 22°C.LIMMA and EBarrays statistical packages were used to identify differentially expressed genes in e point, ,31. Two B. dermatitidis genome using InterProScan. GO analysis revealed DE genes in SREB∆ were enriched for 17 GO terms and the number of enriched GO terms increased across the time course, which likely reflected the sharp morphologic differences between SREB∆ and WT at 22°C ( and S3 Table). Catalytic activity (GO:0003824), metabolic process (GO:0008152), oxidoreductase activity (GO:0016491), and oxidation-reduction process (GO:0055114) contained the largest number of DE genes that were enriched across all 22°C time points . To assess potential function for genes within GO categories, we integrated GO term, TBLASTN (PubMed and Saccharomyces cerevisiae databases), and PFAM analyses (S2 Table). The predicted functions these genes were diverse (S2 Table) and many DE genes were shared among the enriched gene ontologies (S2 Table and S4 Table). Integrative analysis suggested several biological themes including iron ion binding, lipid biosynthesis, and amino acid metabolism.To facilitate functional classification of DE genes, gene ontology (GO) terms were assigned to proteins encoded by the SREB∆ . Genes encoding proteins with ferric reductase domains had increased transcription in SREB∆  including an NADPH oxidase (BDBG_06745), a metalloreductase (BDBG_07462), and a FRE family reductase (BDBG_09322). Genes involved with stress response included those encoding peroxidases and catalases. Four peroxidases were DE including 3 with unknown function and 1 with a haem peroxidase domain. Similar to H. capsulatum, B. dermatitidis encodes 3 catalase isoforms . In SREB∆, these isoenzymes  are DE at select time points and not throughout the time course . Similarly, superoxide dismutase was DE only at 48-hrs 22°C .Differentially expressed genes predicted to encode iron-binding proteins included those with electron-carrier activity and stress response. The majority of DE genes encoding proteins with electron carrier functions contained cytochrome , acyl-CoA dehydrogenase/oxidase , and ferric reductase  domains. Cytochrome and acyl-CoA dehydrogenase/oxidase encoding genes exhibited reduced transcription  and known functions were limited to sterol biosynthesis  and beta-oxidation , respectively. Transcription of cytochromes involved with oxidative phosphorylation were unaffected by deletion of SREB decreased transcription of genes in the triacylglycerol (TAG) and ergosterol biosynthetic pathways, but had limited impact on genes involved with phospholipid biosynthesis.Deletion of SREB∆, 57.1% (8 / 14) of genes in the triacylglycerol (TAG) biosynthetic pathway and 66.7% (14 / 21) of genes in the ergosterol biosynthetic pathways were DE. The majority of these DE genes  had reduced transcript abundance in SREB∆ versus WT. In contrast, deletion of SREB had limited affect on phosopholipid biosynthesis in the Kennedy and cytidine diphosphate diacylglycerol (CDP-DAG) pathways . In the Kennedy pathway, two choline transporters and two biosynthetic genes for phosphatidylcholine (PC) were DE in SREB∆, but the impact on transcription was divergent . Transcription of genes for phosphadidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) biosynthesis were not altered. Moreover, homologs of S. cerevisiae OPI3 and PIS1, which are involved with conversion of PE to PC had similar transcript abundance in SREB∆ and WT . The only genes with altered transcription in the CPD-DAG pathway were involved in the biosynthesis of cardiolipin. These two genes, which were homologs of S. cerevisiae CRD1 (BDBG_07720) and PGS1 (BDBG_03699) had 0.77 and 0.95 log2 decrease in transcription in SREB∆ (versus WT) at 24 and 48-hrs 22°C, respectively . Homologs for S. cerevisiae INO2, INO4, and OPI1, which are transcription factors that regulate phospholipid biosynthesis [B. dermatitidis genome by tBLASTn analysis.In ynthesis , were noSREB∆ cells had reduced transcription for genes in 16 of 21 amino acid biosynthetic pathways at 24 and 48-hrs 22°C . For example, nearly all genes  involved with branched chain amino acid biosynthesis  had decreased transcript abundance . Biosynthetic genes for methionine, glutamate, and serine were not altered by deletion of SREB∆ at 24 or 48-hrs 22°C, whereas cysteine and asparagine biosynthetic genes had increased transcription . At 37°C, deletion of SREB had limited effect on biosynthetic genes; however, an amino acid acetyltransferase (BDBG_00643) involved with first step in ornithine biosynthesis had a 1.1 log2 increase ; ornithine serves as a substrate for siderophore biosynthesis.γ-aminobutyrate (BDBG_03404), and heterogenous for methionine/cysteine  as well as for transporters with unknown function . In addition, 2 putative mitochondrial transporters, BDBG_04548, BDBG_05901, had decreased transcription. Of the predicted amino acid transporters, BDBG_08000, which is a homolog of S. cerevisiae GAP1 , had the largest change in transcript abundance with log2 1.76–2.65 increase at 22°C in SREB∆ (versus WT).Transcript abundance for DE genes encoding amino acid transporters was also altered at 24-hrs 22°C (14 / 20 genes) and 48-hrs 22°C (13 / 20 genes). At these time points, transcript abundance for transporters was decreased for proline (BDBG_04825), increased for cationic amino acids  and SREB∆, DE genes involved with carbohydrate metabolism, including hydrolysis of O-glycosyl linkages, were enriched at 22°C (Table 2). Approximately 73% (33 / 45) of DE carbohydrate metabolic genes had hydrolase activity and nearly 65% (29 / 45) of these were involved with hydrolysis of O-glycosyl compounds with 72.4% (21 / 29) predicted to hydrolyze O-glycosyl linkages in chitin or glucan (Table 2and S4 Table). In SREB∆, the majority of DE chitinases  demonstrated increased expression, whereas most DE glucanases  were down-regulated (S1 Table and S2 Table). These genes were predicted to be involved with cell wall remodeling or maintenance. Deletion of SREB did not alter the transcription of genes involved with the biosynthesis of beta--glucan, beta--glucan, and alpha--glucan including FKS1 (BDBG_00352), RHO1 (BDBG_02597), KRE6 (BDBG_05692), AGS1 (BDBG_00595), and AMY1 (BDBG_07493). Similarly, deletion of SREB had limited impact on chitin biosynthesis, affecting 3 of 8 chitin synthases .In S2 Table). For example, the majority of short chain dehydrogenases/reductases (SDRs) had unknown function  (S2 Table). Similarly, functions for most medium chain dehydrogenases (MDRs), lyases, hydrolases, acyl carrier proteins, and acetyltransferases were unknown (S2 Table). Notable exceptions included an acyl carrier protein (BDBG_00048) and a GNAT acetyltransferase (BDBG_00051) involved with siderophore biosynthesis, and isocitrate lyase (BDBG_04918). B. dermatitidis encodes 2 isoforms of isocitrate lyase  and 1 malate synthase (BDBG_05338). At 24- and 48-hrs 22°C, non-mitrochondrial isocitrate lyase (BDBG_04918) and malate synthase (BDBG_05338) had > 1 log2 reduction in transcript compared to WT.Although GO analysis illuminated some common themes, detailed functional information for many DE genes was limited (Table 3). In 4 of these 11 modules, the majority of genes were differentially expressed . Moreover, 3 modules were enriched for GO terms previously identified including metabolic process (midnight blue module), fatty acid biosynthesis / catalytic activity (green-yellow module), and transmembrane transport (blue module) . Although these modules were enriched for specific GO terms, the predicted functions for DE genes within each module was diverse. Moreover, a substantial number of DE genes in each module had unknown function  and failed to properly increase across the 22°C time points when compared to WT . In contrast, DE genes in SREB∆ exhibited a more precipitous decrease in transcription across the time course than WT in the green-yellow module . For the blue module, the increase in transcription across the time points at 22°C was blunted for DE genes in SREB∆ versus WT . Transcription of DE genes in SREB∆ in the turquoise module increased during the time course compared to WT .The paucity of DE genes in modules enriched for nucleic acid binding, acid anhydride hydrolases, signal transduction, DNA replication, and nucleosome suggested that the deletion of B. dermatitidis is unclear and one study concluded that iron failed to enhance growth of yeast during iron starvation; however, a high concentration of ferric iron was used [B. dermatitidis, yeast were grown under iron-poor media or media containing 3, 5, and 10 μM FeCl3. B. dermatitidis yeast exhibited slow growth under iron-deplete conditions, whereas the addition of 3 μM FeCl3 improved growth (. Treatment of yeast with deferiprone (DFP), an iron chelator, impaired growth in a dose-dependent manner and the degree of growth inhibition was influenced by the concentration of exogenous iron . Collectively, these findings indicate that the acquisition of iron is important for the growth of B. dermatitidis.The acquisition of iron from the host or the environment is important for growth, and fungi have developed several mechanisms for iron uptake . The infwas used . To furtSREB functioned as a major regulator of iron assimilation. SREB∆ failed to repress genes involved with siderophore biosynthesis and transport under iron-replete conditions at 37°C and 22°C . Moreover, a siderophore biosynthetic gene cluster (SBGC) containing 10 genes across 31.295 kb was identified . With the exception of a gene predicted to encode a conserved hypothetical protein with unknown function (BDBG_00049), all genes in the predicted SBGC were differentially expressed in SREB∆ when compared to a WT isolate . DELTA-BLAST analysis against the NCBI  database predicted that genes in the SBGC were involved with the biosynthesis of siderophores (dimerum acid and coprogen) secreted into the extracellular environment. Quantitative real-time PCR confirmed derepression of DE genes in the SBGC under iron-replete conditions in SREB∆ . Two genes located outside the SBGC, BDBG_09503, BDBG_08208, were predicted to biosynthesize ferricrocin, an intracellular storage siderophore. Transcript abundance for BDBG_09503, which encodes a siderophore biosynthetic protein, was similar in SREB∆ and WT . In contrast, transcript for a non-ribosomal siderophore biosynthesis peptide synthase SIDC (BDBG_08208) was 1.4 and 2.5-fold higher in SREB∆ than WT by microarray and qRT-PCR analyses, respectively . Reverse-phase HPLC demonstrated elevated concentration of intracellular ferricrocin in SREB∆ compared to WT under iron-replete conditions .Gene expression microarray analysis indicated B. dermatitidis genome were predicted to be involved with siderophore uptake  and were derepressed in SREB∆ under iron-replete conditions when compared to WT . One of the siderophore transporters (BDBG_00050) was located in the SBGC .Four genes in the Saccharomyces cerevisiae, Cryptococcus neoformans, Candida albicans, and A. fumigatus [B. dermatitidis genome encodes 6 putative ferric reductases and a glutathione-dependent ferric reductase (GGT1) (. Ferric reductases are often involved with reductive iron assimilation (RIA) and function as part of a complex that includes a ferrous transporter/permease and a multicopper oxidase [SREB resulted in derepression of 2 of the 6 putative ferric reductases  under iron-replete conditions . To assess the effect of SREB on cell surface ferric reductase activity, SREB∆ and WT yeast were grown in the presence of triphenyltetrazolium chloride (TTC). No enhancement of red color was observed for SREB∆ compared to WT under iron-replete conditions . Translated BLAST analysis against the B. dermatitidis genome failed to yield homologs of S. cerevisiae FTR1 and A. fumigatus FTRA, which encode ferrous permeases involved with RIA. Similar analysis for Coccidioides immitis, Coccidioides posadasii, and Paracoccidioides brasiliensis also failed to identify FTR1/FTRA homologs. H. capsulatum strains G186AR, H88, and H143 contained FTR1/FTRA homologs, whereas NAM1 and G217B did not. Microarray analysis identified a putative B. dermatitidis Zn2+-Fe2+ transporter (BDBG_05224) that was DE in SREB∆  . The transcription of B. dermatitidis ferro-O2-oxidoreductase (BDBG_06775), which is a homolog of S. cerevisiae FET5 multicopper oxidase, was repressed in SREB∆ (versus WT) at 37°C and 22°C under iron-replete conditions . Collectively, these data indicate that SREB has a limited impact on the transcription of genes involved with ferric reduction.The reduction of ferric to ferrous iron represents an important mechanism of iron assimilation for several ascomycete fungi including umigatus –38. The  oxidase . DeletioH. capsulatum, the genome of B. dermatitidis encodes a γ-glutamyltransferase , a glutathione-dependent ferric reductase, whose mechanism for iron acquisition is independent of RIA [B. dermatitidis GGT1 transcript abundance in SREB∆ is similar to WT at 37°C, but is decreased at 22°C under iron-replete conditions . These data suggest that unlike siderophore biosynthesis and transport, SREB does not repress GGT1 transcription under iron-replete conditions.Similar to t of RIA . B. dermSREB affected lipid metabolism at 37°C and 22°C. To investigate for defects in lipid biosynthesis, glycerolipids (triacylglycerol (TAG) and diacylglycerol (DAG)), phospholipids, free fatty acids, and ergosterol were extracted from WT and SREB∆ at 37°C and 6, 24, and 48-hrs at 22°C. Gene expression microarray analysis indicated 8 of 14 genes in the glycerolipid pathway involved with TAG biosynthesis were DE in SREB∆ versus WT . Seven DE genes had decreased transcript abundance at 22°C, whereas a homolog of S. cerevisiae AYR1 (BDBG_09292) had increased transcription . Quantitative RT-PCR analysis validated a subset of these DE genes; log2 fold changes were similar for qRT-PCR and microarray analyses at 48-hrs 22°C . Gas chromatographic analysis of extracted lipids from SREB∆ suggested decreased TAG concentrations at 37°C and 22°C, and similar levels of DAG compared to WT .GO enrichment and WGCNA analyses suggested that deletion of SREB altered the transcription for 14 of 21 genes predicted to be involved with the biosynthesis of ergosterol , a medically important sterol in the plasma membrane targeted by polyene and azole antifungals. Nearly all DE genes (12 / 14) in the ergosterol biosynthetic pathway had decreased transcription. In SREB∆, ergosterol concentrations were decreased at 37°C and 22°C compared to the WT isolate . Moreover, ergosterol concentrations failed to increase in SREB∆ following a drop in temperature to 22°C . To assess the effect of decreased ergosterol concentrations in SREB∆ to antifungal drugs, disc diffusion testing using amphotericin B deoxycholate  and voriconazole  was performed. No difference in antifungal drug susceptibility was observed in SREB∆ versus WT at 37°C . Antifungal susceptibility testing at 22°C was precluded because SREB∆ exhibited a growth defect at this temperature; it failed to expand by radial growth or accumulate significant biomass [Deletion of  biomass .SREB∆ and WT were similar following a drop in temperature from 37°C to 22°C . Alterations in free fatty acid content were restricted to a single time point (48-hrs 22°C) in SREB∆ , which was due to an increase in stearic acid (18:0) without alteration in oleic acid (18:1n9).In contrast to TAG and ergosterol, the concentration of phospholipids in SREB∆ prompted investigation for alteration in lipid droplets at 37°C and 22°C. Lipid droplets are organelles that consist of a neutral lipid core of TAG and sterol esters surrounded by a phospholipid monolayer intercalated with proteins [SREB∆ was analyzed for defective lipid droplet formation using BODIPY, a fluorescent dye that is specific for lipid droplets (LDs) [SREB∆ compared to WT, LDs were quantified per 10 μm segments along the length of the filament. At 37°C and 6-hrs 22°C, there were no differences in LD abundance in yeast cells between SREB∆ and WT . At 24-hrs 22°C, the median LD abundance was sharply reduced in SREB∆ filaments versus WT (0 versus 7); however, the median number of LD per yeast cell body for SREB∆ was similar to WT (16.5 versus 18) . At 48-hrs 22°C, the median number of LDs per 10 μm segment (0 versus 6) and yeast cell body (10 versus 17) was decreased in SREB∆ compared to WT . At 24 and 48-hrs 22°C, more than 80% of 10 μm segments analyzed for SREB∆ had ≤ 4 LD, whereas for WT, less than 33% of 10 μm segments had 4 or fewer LD .The reduction in TAG and ergosterol in proteins . Sterol proteins . SREB∆ wts (LDs) . LDs werSREB∆ cells were treated with saturated  and unsaturated  fatty acids. Experiments with exogenous TAG were not performed because fungal cells are unable to uptake this glycerolipid. The poor solubility of saturated FAs in aqueous solutions necessitated dissolving 16:0 and 18:0 in DMSO prior to supplementation of iron-replete HMM. Treatment of SREB∆ with 0.5 mM palmitic (16:0) or 0.5 mM stearic (18:0) acid accelerated the morphologic switch at 22°C compared to control strains  without affecting the phase transition of WT cells  . At 24-hrs 22°C, exogenous 16:0 and 18:0 increased germ tube formation in SREB∆ versus SREB∆ untreated and SREB∆ DMSO . At 24 and 48-hrs 22°C, SREB∆ treated with 16:0 or 18:0 exhibited increased conversion to hyphae and decreased number of yeast cells compared to controls  . The morphology of yeast cells at 37°C was unaffected by 16:0 or 18:0 . Although DMSO did not affect the phase transition , higher concentrations of DMSO needed to solubilize ≥ 1 mM 16:0 or 18:0 hindered the morphologic switch to mold for WT and SREB∆. BODIPY staining demonstrated that exogenous 16:0 and 18:0 restored LDs in the growing filaments of SREB∆ at 24-hrs 22°C . Median LD per 10 μm increased from 1 for SREB∆ controls  to 5 for SREB∆ 16:0 and 2.5 for SREB∆ 18:0. Moreover, the percentage of 10 μm filament segments with 5 LDs increased 4-fold for SREB∆ 16:0 and 2.2-fold for SREB∆ 18:0 compared to controls  . The increase in LDs was transient and limited to the 24-hr 22°C time point . At 48-hrs 22°C, the median number of LDs per 10 μm for SREB∆ 16:0 and 18:0 declined to 2 and the percentage of 10 μm filament segments with ≤ 4 LDs increased . Exogenous 16:0 or 18:0 did not affect the number of lipid droplets for SREB∆ at 37°C and 6-hrs 22°C . WT cells treated with 16:0 or 18:0 had similar number of LDs as controls (median 6–7 LD per 10 μm filament segment at 24 and 48-hrs 22°C) . SREB∆ cells treated with 0.5 mM oleic acid (18:1n9) exhibited similar morphologic and LD defects as untreated SREB∆ cells at 48-hrs 22°C . Treatment of cells with 0.5 mM palmitoleic acid (16:1n7) was lethal for WT and SREB∆; reducing 16:1n7 concentrations to 0.250 mM or 0.125 mM did not improve cell viability .In eukaryotic cells, fatty acids (FAs) serve as an important substrate for TAG and sterol ester biosynthesis ,45. To iin vivo. For ChIP, SREB was engineered to contain an in-frame 3x-hemagglutinin (HA) epitope tag at the C-terminus. To minimize mis-expression or abnormal cellular localization, SREB-3xHA was placed under the control of its native promoter and contained the 3’-untranslated region, respectively. Retransformation of SREB∆ with SREB-3xHA complemented the defects in phase transition  and siderophore biosynthesis indicating the construct was functional. Western blot analysis using a ChIP-grade rabbit polyclonal HA antibody demonstrated specificity for 26199 strains transformed with SREB-3xHA and absence of antibody hybridization for wild type 26199 . To identify potential sites of DNA binding by SREB, gene expression microarray data were integrated with genome-wide in silico analysis of extended GATA binding motifs (ATC-w-gAta-a) in promoters of differentially expressed genes; approximately 12% of DE genes possessed an extended GATA motif. Use of the classic GATA motif (A/T-GATA-A/G) was limited because this motif was found upstream of nearly all genes (DE and non-DE) in the B. dermatitidis genome [ChIP-qPCR was used to identify genes bound and directly regulated by SREB s genome .4) using ChIP-qPCR . These included genes involved with extracellular  and intracellular (SIDC) siderophore biosynthesis, siderophore transport (MIRB), ferric reduction (FRE1 and MR), ferrous iron/zinc transporter (ZFT), a WD-repeat protein of unknown function (WD), and a bZIP transcription factor (HAPX) . For ChIP-qPCR, SREB-3xHA did not bind the GAPDH promoter, which lacked GATA motifs and served as a negative control. ChIP-seq analysis of yeast grown in iron-replete media (10 μM FeSO4) also demonstrated enrichment of SREB-3xHA binding for genes located in the siderophore biosynthetic gene cluster including BDBG_00046 , BDBG_00047 (ATRH), BDBG_00048 (NRPS), BDBG_00053 (SID1), BDBG_00054 (long chain fatty acid CoA ligase), and BDBG_00055 (ACE1) . In addition, SREB-3xHA bound to the promoters of SIDC (BDBG_08208) and siderophore transporters MIRB (BDBG_05798), MIRC (BDBG_08034), and SIT1 (BDBG_06965) . ChIP-seq also showed enrichment of SREB-3xHA upstream of genes unrelated to iron homeostasis including a glycolipid surface protein predicted to have 1,3-beta-glucanosyltransferase activity (BDBG_000734), GNAT acyltransferase (BDBG_06912), a thioesterase-domain containing protein (BDBG_07971), and a cation diffusion facilitator (BDBG_07469) . These genes were DE in the microarray analysis (S6 Table). In addition, SREB-3xHA was enriched upstream of an amino acid acetyltransferase, BDBG_00643, which is derepressed in SREB∆ and is predicted to be involved in the biosynthesis of ornithine. ChIP-qPCR confirmed enrichment of a subset of these genes including the glycolipid surface protein (BDBG_000734) and amino acid acetyltransferase (BDBG_00643) ( and S6 Table).Using this integrated approach, we identified a subset of genes bound and regulated by SREB-3xHA under iron-replete conditions  contained an extended GATA motif in the promoter region. This gene was DE at 37°C (0.80 log2 decrease) and ChIP-qPCR demonstrated 4.2-fold enrichment in yeast . Surprisingly, enrichment for this gene was not observed by ChIP-seq analysis at 37°C. This discrepancy may be related to differences in chromatin shearing. However, SREB-3xHA was enriched by ChIP-seq analysis upstream of a C-5 sterol desaturase  in the ergosterol biosynthetic pathway  and binding was confirmed by ChIP-qPCR . Differentially expressed genes involved with conversion of DAG to TAG in the glycerolipid biosynthetic pathway  were analyzed by ChIP-qPCR at 48-hrs 22°C. These genes possessed classic GATA motifs but lacked extended motifs. No enrichment was observed for BDBG_02489 and BDBG_07403 at 22°C. Analysis of BDBG_08114 was precluded due to difficulty in designing high-quality primers targeting the region containing the GATA motif.The reduction of ergosterol and TAG concentrations in SREB altered the phase transition independent of VMA1 and HGRA; these genes are not DE in SREB∆. To test the importance of a subset of SREB-bound genes on the phase transition, we overexpressed WD and HAPX in wild-type strain 26199 under the control of an H2B promoter. WD was selected for overexpression because WD-repeat proteins can be involved with transcriptional regulation and affect fungal development [HAPX is also involved with transcriptional regulation and in Aspergillus spp., it is in a negative regulatory circuit with SREA, a homolog of SREB [SREB∆. The H2B promoter was active at 37°C, 22°C, and during the phase transition when measured by qRT-PCR . WD overexpression strains grew as yeast and converted normally to mold at 22°C. HAPX overexpression strains  were morphologically similar to SREB∆ following a drop in temperature from 37°C to 22°C . Quantitative RT-PCR analysis demonstrated 15.5–53.1-fold increase in HAPX transcript abundance in OE strains compared to WT . SREB transcript abundance was decreased in OE 8 and OE 18, but was similar to WT and empty vector for OE 22 . In HAPX OE 8 and 18, the reduction in SREB transcript was unstable and normalized to WT levels over serial passage; however, the morphologic defect and HAPX overexpression persisted. Unlike deletion of SREB, overexpression of HAPX did not adversely affect LD formation at 24 and 48-hrs 22°C.Gene expression microarray data indicated that elopment ,47. HAPX of SREB ,49. ThesB. dermatitidis SREB and HAPX are in a negative regulatory circuit similar to Aspergillus nidulans and A. fumigatus [B. dermatitidis does not encode a homolog to C. albicans SEF1, which forms a regulatory circuit with SFU1 and HAP43 [SREB did not alter the transcription of HAPB, HAPC, or HAPE homologs, which form a DNA binding complex that recruits HAPX to CCAAT promoter sites of target genes [HAPX transcript abundance was substantially higher in the OE strains (15.5–53.1-fold) than SREB∆ (1.7–3.5-fold) compared to WT . To investigate if less extreme changes in transcript abundance for either HAPX or SREB affected the phase transition, SREB was targeted for gene silencing using a GFP sentinel RNAi system [SREB-GFP silenced strains had a 1.8–2.5-fold reduction in transcript abundance compared to controls  . SREB-GFP RNAi strains had increased SID1 transcript abundance under iron-replete (10 um FeSO4) conditions compared to controls . HAPX transcript was increased 1.7–3.2-fold in SREB RNAi versus controls . Following a drop in temperature, SREB-GFP silenced strains converted to mold at 22°C . Moreover, there was no delay in the phase transition. This data suggested that the 1.7–3.5-fold increase in HAPX transcript in SREB∆ was not responsible for the defect in phase transition at 22°C; larger changes in HAPX transcript abundance are required to impact morphologic development at 22°C.Integration of microarray, qRT-PCR, and ChIP data suggested that umigatus ,49. B. dnd HAP43 . Moreoveet genes . HAPX tri system . SREB-GFSREB regulon in B. dermatitidis following a drop in temperature from 37°C to 22°C. The microarray experiments captured the transcriptional response early in the phase transition (0 48-hrs) and under conditions (10 μM FeSO4) that promote binding of SREB to DNA. This facilitated analysis for how SREB affected disparate processes including the morphologic switch and iron assimilation; prior research demonstrated exogenous iron neither suppressed nor enhanced the defect in phase transition observed for SREB∆ [SREB regulon was accomplished using ChIP-qPCR integrated with microarray and MAST analyses  to identify a subset of genes directly bound and regulated by SREB in vivo. This included genes involved with the regulation of siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. The results herein represent the first genome-wide transcriptional analysis integrated with in vivo transcription factor binding data during the transition from yeast to mold in B. dermatitidis. Functional analysis of SREB∆ suggested that alterations in lipid metabolism contribute to the defect in the phase transition. In addition, chromatin immunoprecipitation data along with RNAi and overexpression analyses indicated that SREB was in a regulatory circuit with HAPX. Alteration of this circuit by either deletion of SREB or strong overexpression of HAPX impaired the conversion to mold at 22°C.We used whole genome gene expression microarrays and bioinformatics analyses to identify and characterize the or SREB∆ . FurtherSREB had pleiotropic effects on transcription during growth as yeast at 37°C and during the phase transition following a drop in temperature to 22°C. Deletion of SREB affected the transcription of 11.6–19.9% of genes  across the time course. Similarly, deletion of CIR1, a homolog of SREB important for thermotolerance at 37°C, impacted transcription for 21.0% of genes  in the C. neoformans genome under iron-replete conditions [SRE1 in H. capsulatum, which also affects the conversion from yeast to mycelia, altered transcription of 9.6% genes (364 / 3810) [SREA in A. fumigatus and SFU1 in Candida albicans altered transcription for 0.6% (49 / 8975) and 2.0% (139 / 7116) genes when iron was abundant, respectively [CIR1∆ or SRE1 knockdowns, SREA∆ mutants are viable at 37°C and SFU1∆ mutants convert to hyphae under inducing conditions [SREB, SRE1, and CIR1 affect the transcription of genes involved with diverse processes, whereas SREA and SFU1 regulate a limited set of genes.Analysis of the microarray data demonstrated nditions . RNA intectively ,53. Unlinditions ,53. A shnditions ,52,53. HSREB, the transcription factors encoded by SRE1, CIR1, SREA, and SFU1 can either induce or repress gene transcription [CIR1∆ and SREB∆ yeast, the ratio of genes with increased to decreased transcription was slightly greater than 1 [SREB∆ was close to 1. In contrast, the majority of DE genes in A. fumigatus SREA∆ were increased [C. albicans, conflicting results have been obtained from different SFU1∆ strains [SREB affected transcription during the phase transition, WGCNA was performed to identify groups of co-expressed genes. This demonstrated that deletion of SREB affected transcription differently among co-expressed gene sets. This included subsets of DE genes in SREB∆ that failed to properly increase or decrease following a drop in temperature as well as genes with reduced transcript abundance at 37°C and 22°C.Similar to cription ,24,52,53 strains ,53. To fSREB affected the transcription of genes with diverse functions. Analysis of DE genes identified 17 GO terms enriched in SREB∆ ranging from iron ion binding, catalytic activity, fatty acid biosynthetic process to carbohydrate metabolic process. Integrative analysis of genes within the enriched GO categories suggested several themes including iron ion binding, lipid biosynthesis, and amino acid metabolism. Genes that encoded iron ion binding proteins were involved with oxidative stress response and lipid metabolism. The absence of a coordinated or sustained transcriptional pattern for catalase isoforms and superoxide dismutase suggested that SREB∆ cells were not under substantial iron-related oxidative stress. Moreover, prior research demonstrated that exogenous iron concentrations did not affect the defect in the morphologic switch in SREB∆ [CIR1∆ and our analysis of SREB∆ indicated involvement of GATA transcription factors with lipid metabolism; CIR1∆ was enriched for fatty acid metabolism (GO:0006631) and SREB∆ for fatty acid biosynthetic process (GO:0006633) [SREB affected genes involved with the biosynthesis of 16 amino acids. Strikingly, the majority of DE genes had reduced transcript abundance at 24 and 48-hrs 22°C. This finding may reflect the distinct morphologic differences between SREB∆ and WT at these time points. B. dermatitidis and H. capsulatum amino acid concentrations are approximately 3-fold higher in mycelia than yeast [GO analysis offered additional support that deletion of in SREB∆ . GO enri0006633) . In addian yeast ,55.S. cerevisiae are involved with cell wall remodeling or maintenance. The impact of altered cell wall remodeling on SREB∆ at 22°C was likely limited because the addition of exogenous saturated fatty acids was able to accelerate filamentous growth at 22°C. Similarly, the effect of reduced transcription of BDBG_00734, a homolog of S. cerevisiae GAS5, which is bound and regulated by SREB at 37°C had limited impact; prior research demonstrated similar growth rate of SREB∆ and WT as yeast [Genes involved with carbohydrate metabolism, including hydrolysis of O-glycosyl linkages, were enriched at 24 and 48-hrs 22°C. Three patterns were observed: increased transcription of chitinases, reduced transcription of glucanases, and no impact on biosynthetic genes for alpha or beta-glucan. Homologs of these genes in as yeast .SREB was to regulate iron homeostasis by repressing the transcription of genes important for the biosynthesis and transport of siderophores when iron was abundant. Genes predicted to be involved with the biosynthesis of secreted siderophores were located in a gene cluster, whereas biosynthetic genes for ferricrocin, an intracellular storage siderophore, were not clustered. This arrangement is conserved in other fungi including H. capsulatum and A. fumigatus [in vivo that SREB bound upstream of clustered  and non-clustered  siderophore biosynthetic genes. For genes in the SBGC, SREB bound regions with extended GATA motifs (ATC-w-gAta-a) as predicted by gel electrophoretic-mobility shift [GFP reporter [in vivo that SREB binds the promoter SIDC, which has the classic GATA motif, A-GATA-A. Thus, SREB regulates extracellular and intracellular siderophore biosynthetic gene transcription by binding to regions with extended and classic GATA motifs.One of the major functions of umigatus ,56. Usinty shift  and GFP reporter  assays. C. neoformans, C. albicans, and S. cerevisiae, which do not produce siderophores [Schizosaccharomyces pombe can synthesize siderophores and use RIA to acquire iron [FRE1), ferrous permease (e.g. FTR1), and multicopper oxidase (e.g. FET3) [FTR1/FTRA are absent in the sequenced B. dermatitidis genomes . Similarly, the genomes of other closely related dimorphic fungi did not encode FTR1/FTRA homologs; however, some H. capsulatum genomes were predicted to contain FTR1/FTRA homologs. Collectively, this suggests that ferrous transport for RIA in B. dermatitidis occurs either through an unrecognized transmembrane transporter such as ZFT (BDBG_05224) or that RIA is dispensable. Moreover, B. dermatitidis has an RIA-independent mechanism for acquiring iron by secreting a glutathione-dependent ferric reductase (GGT1) [H. capsulatum, GGT1-mediated ferric reduction is important for virulence [GGT1 transcription was not under the control of SREB at 37°C. This indicated SREB regulates some, but not all mechanisms for iron assimilation in B. dermatitidis.Reductive iron assimilation (RIA) serves as a primary mechanism for iron uptake in rophores ,38. Someire iron . The trig. FET3) . Despitee (GGT1) . In H. cB. dermatitidis, H. capsulatum, and Paracoccidioides brasiliensis is associated with changes in lipid concentration. Lipid content is often ≥ 2-fold higher in mycelia than yeast [SREB reduced TAG and ergosterol concentrations, but had limited effect on phospholipids and free fatty acids (except for an increase in stearic acid at 48-hrs 22°C). As the time course progressed, the quantitative differences for ergosterol and TAG between SREB∆ and WT became more pronounced, which likely reflected the increased number of genes in these pathways that had reduced transcript abundance at 22°C versus 37°C. ChIP-qPCR and ChIP-seq analyses suggested that SREB had direct and indirect effects on genes involved with the biosynthesis of neutral lipids .The morphologic switch between yeast and mold in an yeast ,60. MicrSREB∆; hydrolases involved with LD breakdown were not upregulated. Lipid droplet formation in eukaryotes including fungi is linked to TAG content as well as fatty acids, which serve as a substrate for TAG and sterol ester biosynthesis [S. cerevisiae, the biosynthesis of TAG contributes to lipid droplet formation to a greater degree than the biosynthesis of sterol esters [SREB∆ transiently restored LD formation and accelerated the phase transition at 22°C. Importantly, exogenous 16:0 and 18:0 did not increase LD content or accelerate the conversion to hyphae in WT cells. The increase in LD content at 24-hrs followed by a decrease at 48-hrs 22°C in SREB∆ suggested that LDs are biosynthesized and degraded during the phase transition. The presence of LDs during filamentous growth in SREB∆ cells treated with 16:0 or 18:0 indicated that the loss of LDs in untreated cells was due to a reduction in biosynthesis and not from impaired intracellular LD transport. Thus, deletion of SREB disrupted LD homeostasis at 22°C in which reduced biosynthesis could not meet catabolic demands during filamentous growth. TAG and sterol esters in LDs can serve as a source of carbon for energy and lipids for membrane biosynthesis [SREB∆ may represent a compensatory response to altered LD biosynthesis; however, it was not enough to overcome reduced concentrations of neutral lipids. Collectively, these data suggest that lipid metabolism including LD homeostasis may contribute to the morphologic switch from yeast to mold following a drop in temperature to 22°C.The decrease of neutral lipids was likely responsible for the reduction in LDs at 22°C in ynthesis ,45,61–63ynthesis . The incH. capsulatum during the transition to yeast following an increase in temperature to 37°C [HSP82 transcription in a temperature sensitive strain, whereas unsaturated fatty acids blunted the transcription of HSP82 in response to elevated temperature in a thermotolerant strain [SREB∆, the addition of saturated fatty acids enhanced the morphologic shift following a drop in temperature to 22°C, whereas unsaturated fatty acids did not impact the phase transition. Taken together, these data suggest that fatty acids and lipid metabolism influence the response to temperature at 37°C and 22°C.Prior research on saturated and unsaturated fatty acids in the dimorphic fungi has focused on the heat shock response and thermotolerance of  to 37°C ,66. The B. dermatitidis SREB and HAPX likely form a negative regulatory circuit similar to A. fumigatus and A. nidulans. Functional analysis demonstrated a novel role for this circuit in B. dermatitidis. Deletion of SREB or overexpression of HAPX impaired the temperature-dependent morphologic switch. In A. fumigatus and A. nidulans, the SREA-HAPX circuit governs the cellular response to exogenous iron with SREA repressing HAPX transcription when iron is abundant, and HAPX inhibiting SREA transcription under iron poor conditions [C. albicans, the regulatory circuit involving HAP43, SFU1, and SEF1 mediates the ability of this pathogen to adapt to niches within the mammalian host that have radically different iron concentrations [,A. fumigatus, A. nidulans, and C. albicans, transcript abundance of B. dermatitidis HAPX and SREB is influenced by the presence or absence of exogenous iron [SREA-HAPX and SFU1-SEF1-HAP43 regulatory circuits mediate adaptation to changing environmental conditions. In addition to responding to exogenous iron, B. dermatitidis SREB and HAPX influence morphogenesis at 22°C. The defect in the phase transition in SREB∆ is unlikely to be due to HAPX derepression because SREB knockdown strains, which had similar levels of HAPX transcript as SREB∆, converted to mold. Moreover, the morphologic defect in HAPX OE strains was unlikely to be mediated through SREB because its repression was transient. These findings suggest that the amount of SREB and HAPX transcript influenced the morphologic response to ambient temperature.nditions ,49. In Ctrations ,67,68.ous iron . In the SREB regulon in B. dermatitidis during growth as yeast at 37°C and during the early part of the phase transition at 22°C. Deletion of SREB affected the transcription of genes involved with iron homeostasis, iron ion binding, amino acid biosynthesis, carbohydrate metabolism, lipid biosynthesis, and the transcriptional regulator HAPX. Moreover, deletion of SREB altered transcript differently among subsets of DE genes including those that failed to properly increase or decrease following a shift in temperature to 22°C. Integration of gene expression microarray and chromatin immunoprecipitation studies demonstrated that SREB binds and regulates genes involved with siderophore biosynthesis and iron uptake as well as genes unrelated to iron assimilation. SREB∆ cells had reduced TAG and ergosterol concentration along with decreased numbers of lipid droplets at 22°C. Moreover, experimental analysis suggested that LDs are biosynthesized and degraded during the phase transition and that lipid metabolism may contribute to filamentous growth at 22°C. Exogenous palmitic (16:0) and stearic (18:0) accelerated the morphologic shift and transiently restored LD formation in SREB∆ at 22°C. Deletion of SREB resulted in derepression of HAPX and functional analysis suggested that SREB and HAPX are in a negative regulatory circuit. Both SREB and HAPX affected the morphogenesis of B. dermatitidis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required.In summary, we characterized the B. dermatitidis strains used included American Type Culture Collection (ATCC) strain 26199, SREB∆, SREB-3xHA, and HAPX-OE. B. dermatitidis 26199 was originally isolated from a patient from South Carolina and is virulent in a murine model of pulmonary infection (ATCC 26199 has been used as a standard laboratory strain since the 1970’s and was a gift from the laboratory of Bruce S. Klein. This strain was not isolated from a patient for this specific study) [SREB∆ was generated by targeted deletion of SREB in strain 26199 [HAPX in strain 26199. B. dermatitidis cultures were maintained as yeast on Histoplasma macrophage medium (HMM) at 37°C containing 3 μM or 10 μM FeSO4 with or without hygromycin 25 μg ml-1 [4. F-12 Ham’s nutrient mixture lacking FeSO4 was used for preparation of HMM. For iron chelation experiments, deferiprone -pyridone; Sigma-Aldrich, Saint Louis, MO) 0.5 mM or 1.0 mM was added to liquid HMM supplemented with 1, 5, or 10 μM FeSO4. Agrobacterium tumefaciens strain LBA1100 harboring the Ti helper plasmid pAL1100  was maintained on Luria-Bertani (LB) medium supplemented with 0.1% glucose, spectinomycin 100 ug/ml, and kanamycin 100 ug/ml after transformation with a binary vector [c study) –71. SREBin 26199 . SREB-3x μg ml-1 ,72. Culty vector ,73.To quantify the percentage of cells with yeast morphology, germ tube development, and hyphal growth, cells were stained with calcofluor white (Sigma Aldrich) . CalcoflSREB∆ and an isogenic WT isolate, 385K whole genome gene expression microarrays  were custom designed for B. dermatitidis using the annotated sequence of strain SLH14081 . A total of 9,583 genes  were represented on each array in quadruplicate with 9,571 genes having a probe-set comprised of 10 unique oligonucleotide probes; 12 genes had less than 10 unique oligonucleotide probes. Oligonucleotide probes with sufficient specificity were unable to be designed for a mitochondrial laglidadg endonuclease (BDBG_17014), mitochondrial ATP synthase F0 subunit 8 (BDBG_17017), mitochondrial NADH dehydrogenase subunit 5 (BDBG_17032), and a predicted protein (BDBG_03106). Empty features on the array were filled with randomly generated probes with similar length and G+C content as the NimbleGen internal controls.To compare transcription between SREB∆ and the isogenic wild-type control at 37°C and 6 hours, 24 hours, and 48 hours following a drop in temperature to 22°C . All cultures were grown in liquid HMM under iron replete conditions (10 μM FeSO4). All glassware was pre-treated with 2N HCl and extensively washed with ddH2O. Prior investigation of SREB demonstrated that transcription was induced in the presence of exogenous iron [B. dermatitidis cells were washed with PBS (pre-warmed to 37°C or 22°C), frozen in liquid nitrogen, lysed by grinding in a mortar and pestle, and treated with TRI Reagent followed by 1-bromo-3-chloropropane . Total RNA was precipitated using a 1:1 concentration of isopropanol and high salt solution , and washed with 75% ethanol. Following resuspension in water pre-treated with diethyl pyrocarbonate , total RNA was treated with turboDNAase , and purified using an RNeasy kit . To optimally remove guanidine salts, an extra wash of RPE buffer was performed during RNAeasy purification. RNA quality was assessed using an Agilent bioanalyzer . Double-stranded cDNA was synthesized and purified at the Gene Expression Center at the University of Wisconsin–Madison. Roche NimbleGen performed Cy3 labeling, hybridization, scanning, and RMA  normalization for all 24 microarrays at their facility in Reykjavík, Iceland.Total RNA was isolated from 3 biological replicates of ous iron . In addious iron ,75. The ous iron . In brieSREB∆ versus WT at each time point, two different statistical programs were used, LIMMA (Linear Models for Microarray data) and EBarrays  [,SREB∆ and WT using a moderated t-statistic [µ1 = µ2) or different expression (µ1µ2) in SREB∆ versus WT [SREB∆ and WT at each time point. An individual gene was considered differentially expressed if statistical significance was met for both LIMMA  and EBarrays (posterior probability > 0.95) analyses. Heat maps were generated using CIMminer from the Genomics and Bioinformatics Group  (http://discover.nci.nih.gov). Gene expression microarray data have been deposited to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) (accession number: GSE67724).To identify differentially expressed genes in oarrays) ,31. Botatistic . We usedtatistic . EBarrayB. dermatitidis genome using InterProScan [GO terms were assigned to the proteome encoded by the rProScan . The annrProScan . The enrallez to determine GO and KEGG enrichment in each module, and calculated correlation between module eigengenes and time points.We used WGCNA  to find SREB∆ and WT yeast were grown in liquid HMM supplemented with 10 μM FeSO4 at 37°C. Once cells reached stationary growth when measured by spectrophotometric absorbance at 600 nm, they were washed 3 times in 1x phosphate buffered saline (PBS) to remove extracellular siderophores from the medium, flash frozen in liquid nitrogen, ground in a mortar and pestle, resuspended in PBS, and centrifuged to pellet the cellular debris. The supernatant was collected, treated with 2% ferric chloride, and applied to a column packed with Amberlite XAD-2 resin . Resin and column preparation along with siderophore isolation was performed as previously described [escribed . FollowiSREB∆ were incubated (5 x 105 cells) on iron poor HMM (IPM) and iron-replete (10 μM FeCl3) HMM media supplemented with 50 μM CuSO4 at 37°C for 4 days prior to exposure to 0.1% triphenyltetrazolium chloride  [Wild type and Aldrich) ,82. TTC SREB∆ and WT were grown at 37°C and 22°C in liquid HMM supplemented with 10 μM FeSO4. Cells were harvested, washed with H2O twice, resuspended in 0.97% KCl, and beaten with beads prewashed with methanol-chloroform (1:1). Lipids were extracted using chloroform-methanol (2:1) containing 5mg/L butylated hydroxytoluene (Sigma-Aldrich) in organic solvent-washed glassware (no plastics were used). Samples were mixed by vortexing for 3 minutes and incubated at 4°C for 2 hours. The samples were then treated with 0.97% KCl, vortexed for 30 seconds, and incubated at 4°C for 1 hour. Following centrifugation (300 x g for 5 minutes), the organic layer was removed and washed x 3 with 0.97% KCl. The collected lipid extracts were dried under nitrogen.2O and centrifuged (850 x g for 5 minutes) at room temperature. The top hexane layer, which contained methyl ester lipid derivatives, was transferred to a new glass vial, dried under nitrogen, resuspended in 100 μl hexane, and transferred to gas chromatography vials.After drying, samples were reconstituted in 2:1 chloroform:methanol and lipid classes were separated by thin layer chromatography  using heptane:isopropyl ether:acetic acid (60:40:3). After spraying TLC plates with 0.2% 2’,7’-dichlorofluorescein in ethanol, lipid classes  were visualized by UV light and scraped off the TLC plate. Lipids were reextracted overnight at room temperature with chloroform: methanol (4: 1) containing 100 mg/L butylated hydroxytoluene (Sigma-Aldrich), and centrifuged 850 x g to remove silica gel particles. The internal standard, pentadecanoic acid (100 μl of 0.05 mg/ml in chloroform) was added to each sample and the organic solvents were evaporated under nitrogen. Isolated lipids were then methylated using 0.5 ml 14% boron trifluoride-methanol solution (Sigma-Aldrich), boiled for 30 minutes at 100°C, cooled, mixed with 1 ml hexane, vortexed for 30 seconds, mixed with 0.5 ml HFatty acid methyl esters were identified by gas chromatography using a Hewlett-Packard 6890  equipped with a capillary column coated with DB-225  . Column temperature was kept at 70°C for 1 min, increased to 180°C at a rate of 20°C/min and then to 220°C at a rate of 3°C/min. The temperature was kept at 220°C for 15 min. The front inlet was set at 250ºC and the detector temperature was set at 300ºC. Peaks were identified by a comparison of retention times with a set of authentic FA standards . The abundance of FAs was calculated from the relative peak areas . The fat2O, the unsaponifiable fraction was extracted three times with 3 volumes of C6H6. The sterols were identified by FAST GC-MS and quantified by the peak area ratio of sterol/internal standard (cholesterol) in the GC-FID chromatogram. The characterization of sterol was performed on a Shimadzu 2010 Plus system equipped with a Shimadzu QP 2010 Ultra mass spectrometer detector. The separation was performed on a Zebron ZB-5MS capillary column . The system was operated in the constant linear velocity (70 cm/s) using helium as a carrier gas and the sample was injected in the split mode (Split ratio 20:1). The GC oven temperature was started at 80 °C, increased to 220 °C at a rate of 60 °C min−1, then to 300 °C at a rate of 20 °C min−1 and finally held to 300 °C for 1.5 min. The injection port, transfer line and ion source temperature were maintained at 285, 300 and 250 °C, respectively. The MS was run in the mode of electron impact (EI) mode with an electron energy of 70 eV and the spectra were scanned in the range of m/z 50–500. The sterols were identified by comparing their mass spectra with the standard mass spectra in the NIST MS library. Additional FID detector was allowed the quantitative analysis of sterol. The FID temperature was 320 °C and the oven temperature program was the same as to the GC–MS operation.Sterols were extracted and analyzed according to the protocol described by Zarnowski et al. . BrieflyB. dermatitidis SREB∆ and WT yeast (1.2 x 106) were uniformly spread on 6-well plates  containing HMM supplemented with 10 μM FeSO4. Antibiotic assay paper discs  were impregnated with 0–10 μg Amphotericin B deoxycholate dissolved in dimethyl sulfoxide  or 0–1 μg voriconazole dissolved in water , placed in the center of each well, overlaid with 0.5% agarose top agar, and incubated at 37°C. Antifungal susceptibility was assessed by the diameter of the clear zone surrounding the discs impregnated with amphotericin B deoxycholate or voriconazole.SREB∆ and WT cells were stained with BODIPY 493/503  at 37°C and 22°C [4, washed in 1x PBS, and fixed in 2% paraformaldehyde to optimize intracellular entry of BODIPY 493/503. Following fixation, cells were washed and resuspended in 1x PBS. Yeast cells grown at 37°C (and 6-hrs 22°C) were stained with 1:50 dilution of 0.5 mM BODIPY 493/503 and cells grown at 22°C (24-hrs and 48-hrs) were stained with a 1:25 diluation of 0.5 mM BODIPY 493/503. Optimal image capture of LDs by the microscopy camera  required a higher concentration of BODIPY 493/503 for WT and SREB∆ cells harvested at 24 and 48-hrs 22°C versus 37°C. Intracellular lipid droplets were visualized using fluorescent microscopy  using an excitation filter with a range of 460–490 nm. ImageJ was used to facilitate quantitative LD analysis.Lipid droplets in and 22°C . CultureSREB∆ and WT cells grown in iron-replete (10 μM FeSO4) liquid HMM at 37°C were supplemented with 0.5–1.0 mM palmitic acid (16:0), palmitoleic acid (16:1n7), stearic acid (18:0), and oleic acid (18:1n9)  prior to incubation at 22°C. Palmitic and stearic acid were nearly insoluble in aqueous solutions and required solubilization in DMSO (Sigma-Aldrich) prior to addition to iron-replete liquid HMM . Palmitoleic and oleic acid were soluble in liquid HMM. SREB∆ and WT cells treated with fatty acids along with controls  were harvested at 37°C and 6, 24, and 48-hrs at 22°C. Harvested cells were washed twice with 1x PBS, fixed in 2% paraformaldehyde, and stained with either BODIPY 493/503 or calcofluor for analysis of LDs and morphology, respectively.SREB coding region using primers SREB_F1 and 3xHA_RC (S7 Table), the PCR product was purified using QIAquick gel extraction kit , sequenced by the DNA Sequencing Laboratory at the University of Wisconsin Biotechnology Center, and ligated into a vector that contained the native SREB promoter and 3’ UTR to create pBTS47-SREB-3xHA. Agrobacterium tumefaciens-mediated transformation was used to transform B. dermatitidis strain 26199 [S8 Fig). In brief, wild-type and SREB-3xHA transformed cells were beaten with beads, suspended in 1x SDS-PAGE loading buffer, boiled for 10 minutes, and loaded onto an SDS-PAGE gel  for separation by electrophoresis. Separated proteins were transferred to Immobilon-P PVDF membrane (Millipore), probed with primary  and secondary (1:2000 dilution of peroxidase affinipure goat anti-rabbit IgG (H+L); Jackson ImmunoResearch Laboratories, West Grove, PA), developed using ECL western blotting substrate , and imaged using VersaDoc imaging systems (Bio-Rad). To confirm the SREB-3xHA construct was functional, SREB∆ was transformed with pBTS47-SREB-3xHA using A. tumefaciens-mediated DNA transfer.To perform ChIP, we engineered SREB to contain a 3x-HA tag located in-frame at the C-terminus. Following PCR amplification of the in 26199 . TransfoCaenorhabditis elegans protocol published by Mukhopadhyay et al. [B. dermatitidis SREB-3xHA and WT cells grown in liquid HMM supplemented with 10 μM FeSO4 at 37°C and 22°C were treated with 1% formaldehyde (Sigma-Aldrich) for 30 minutes to crosslink HA-tagged SREB to its DNA binding site. Formaldehyde crosslinking was quenched with 0.125 M glycine for 5 minutes. Cells were washed twice in ice-cold 1x PBS, washed once in ice-cold 1x PBS containing protease inhibitor cocktail , flash frozen in liquid nitrogen, ground to a fine powder using a mortar and pestle, and resuspended in FA lysis buffer containing PIC plus phenylmethylsulfonyl fluoride (PMSF). The cell lysate was centrifuged 13,600 x g for 15 minutes at 4°C, supernatant discarded, pellet resuspended in FA lysis buffer with PIC plus PMSF, and stored at -80°C. Chromatin was sheared using probe sonication . Cells grown at 37°C were sonicated for a total of 25 minutes of “on time”; whereas cells at 22°C were sonicated for a total of 15 minutes of “on time”. Each round of sonication consisted of 1 minute of “on time”, which was divided into 15 seconds of sonication (amplitude 30) x 4 with intervening rest periods of 30 seconds. Cells were kept ice cold during sonication and between each round of sonication, the cells were chilled for 10 seconds in a dry ice-ethanol bath to prevent overheating. From each sample, 50 μl was saved as an “input” sample and 50 μl was analyzed for efficacy of chromatin shearing by 0.8% agarose gel electrophoresis.For ChIP-qPCR, we modified the y et al. . B. dermS7 Table). Potential GATA motifs were identified by MAST analysis [For ChIP, protein A agarose/salmon sperm DNA beads were prepared according to the manufacturer’s instructions  and resuspended in FA lysis buffer containing PIC plus PMSF. Sonicated chromatin (300 μg measured by nanodrop) was pre-cleared for 1 hour at 4°C prior to adding 10 μl anti-HA along with freshly prepared beads. Following an overnight incubation at 4°C with orbital rotation, the samples were washed twice with buffer #1 (FA lysis buffer), once with buffer #2 (FA lysis buffer containing 0.5 M NaCl), once with buffer #3 , and thrice with Tris-EDTA pH 8. All washes were 3 minutes at 4°C with orbital rotation. To reverse cross-linking, ChIP’d samples were treated with proteinase K for 2 hours at 45°C, and then 65°C overnight. Input samples were treated with proteinase K at 55°C for 4 hours then 65°C overnight. DNA was isolated and purified according to the protocol in Mukhopadhyay et al., and resuspended in Tris-HCl pH 8 . For qPCanalysis . Fold enanalysis .4) was prepared, sheared and immunoprecipitated similar to the protocol described for ChIP-qPCR; however, protein A agarose beads without salmon sperm DNA were used . Immunoprecipitated DNA  from WT and SREB-3xHA strains were submitted to the University of Wisconsin Biotechnology Center for sequencing using Illumina HiSeq 2000 platform. Two biological replicates were analyzed for WT and SREB-3xHA. We aligned the ChIP and input reads using Bowtie (version 0.12.7 with parameters “-p 4-q-v 2-a—best—strata-m 2”) to the genome sequence of Blastomyces dermatitidis . ChIP-seq data have been deposited to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) (accession number: GSE68680).For ChIP-seq, chromatin from yeast cells grown under iron-replete conditions , digested with ClaI and BamHI-HF , and ligated into pBTS47 using T4 DNA ligase  resulting in pBTS47-Woods. pBTS47 contains a nousethricin resistance cassette and was a gift from Dr. Bruce Klein. Open reading frames of candidate genes were amplified using forward and reverse primers containing AscI and SbfI restriction site sequences, respectively (S7 Table). The amplicons were separated by gel electrophoresis, purified by QiaQuick PCR purification kit, digested with AscI and SbfI, and ligated into pBTS47-Woods using T4 DNA ligase. A. tumefaciens-mediated DNA transfer was used to transform B. dermatitidis strain 26199 with pBTS47-Woods containing the candidate gene of interest (S7 Table). Transformants were selected on HMM supplemented with 25 μg/ml nourseothricin and 200 mM cefotaxime.Plasmid pLBZ1, which was a gift from Dr. Jon Woods, was used to generate pBTS47-Woods to overexpress candidate genes in B. dermatitidis cells grown in liquid HMM supplemented with 10 μM FeSO4 at 37°C and 22°C as described in the microarray section. One microgram of DNase-treated total RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR was performed with SsOFast EvaGreen Supermix (Bio-Rad) using a MyiQ real-time PCR detection system (Bio-Rad). Reactions were performed in triplicate using the following conditions: 1 cycle 95°C x 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 10 sec. Primers used for qRT-PCR are listed in HAPX were normalized relative to the transcript abundance of GAPDH, whereas genes involved with glycerolipid biosynthesis analyzed at 37°C and 48-hrs 22°C were normalized to L34. For analyzing gene transcription at two different temperatures, 37°C and 22°C, L34 transcript abundance demonstrated the most stability . Relative expression (RE) was calculated as RE = 2-∆Ct, ∆Ct = Cttarget gene−CtGAPDH or L34 [Total RNA was extracted from H or L34 .S1 Table(XLS)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S1 FigDE genes were enriched for 4 GO terms across all 22°C time points : catalytic activity, metabolic process, oxidation-reduction process, and oxidoreductase activity. Putative function for DE genes within these broad ontologies was assigned by integration of GO term, tBLASTn, and pFAM analyses. Number of DE genes in each ontology is listed in parentheses. NOC is not otherwise categorized.(EPS)Click here for additional data file.S2 Fig(A) Kennedy and cytidine diphosphate diacylglycerol (CDP-DAG) pathways are involved with the biosynthesis of phospholipids including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and cardiolipin. (B) Heat map of fluorescent intensity (log2) values of putative genes in the Kennedy and CDP-DAG pathways. DE genes are highlighted by green text. Genes numbered in (A) correspond to genes numbered in (B). Homologs of genes in S. cerevisiae are in parentheses. PIT is phosphatidyltransferase. Adjacent to the heat map, log2 fold change values for DE genes are shown; genes that are not DE are labeled as “—”.(EPS)Click here for additional data file.S3 Fig2 fold change values for genes involved with amino acid biosynthesis (SREB∆ versus wild-type). Log2 fold change values for DE genes are shown. Genes that are not DE are labeled as “—”.Heat map of log(EPS)Click here for additional data file.S4 FigSREB∆ yeast (5 x 105 cells) grown on iron poor HMM (IPM) and iron-replete (10 μM FeCl3) media were exposed to 0.1% triphenyltetrazolium chloride (TTC) for 15, 30, 60, and 90 minutes. Reduction of TTC results in the production of red pigmentation (formazan).Wild type and (TIF)Click here for additional data file.S5 Fig2 fold change values for genes in the ergosterol biosynthetic pathway (SREB∆ versus wild-type). Differentially expressed genes are highlighted in green text. Adjacent to the heat map, log2 fold change values for DE genes are shown; genes that are not DE are labeled as “—”.Heat map of log(EPS)Click here for additional data file.S6 FigDisc diffusion testing for amphotericin B deoxycholate (0–10 μg) and voriconazole (0–1 μg).(TIF)Click here for additional data file.S7 FigA) Morphology and lipid droplet straining (BODIPY 493/503) of wild type and SREB∆ cells treated with 0.5 mM palmitic (16:0) and stearic (18:0) acid at 37°C. Controls included untreated cells and cells grown in DMSO. Corresponding bright field microscopic images (light) are below the fluorescent images. Scale bar equals 10 μm. (B) Morphology and lipid droplet straining (BODIPY 493/503) of wild type and SREB∆ cells treated with 0.5 mM palmitic (16:0) and stearic (18:0) acid at 6-hrs 22°C. Controls included untreated cells and cells grown in DMSO. Corresponding bright field microscopic images (light) are below the fluorescent images. Black arrows indicate germ tubes. Scale bar equals 10 μm.((TIF)Click here for additional data file.S8 FigA) Morphology and lipid droplet staining of SREB∆ untreated and SREB∆ 18:1n9 treated cells at 48-hrs 22°C. (B) WT and SREB∆ cells grown in iron-replete liquid HMM with or without supplementation of 0.125 mM palmitoleic acid (16:1n7) at 37°C. Growth was measured by absorbance at 600 nm. Results are averaged from 2 biological replicates from a representative experiment.((TIF)Click here for additional data file.S9 FigA) The morphologic defect of SREB∆ at 22°C was successfully complemented by the SREB-3xHA construct. SREB∆, empty vector, complemented strains, and wild type strains were imaged at 17 days 22°C. Scale bar is equals 10 μM. (B) Western blot and coomassie gel analyses of wild type 26199 and 26199 transformed with SREB-3xHA. Predicted molecular weight of SREB-3xHA was 71.2 kDa.((TIF)Click here for additional data file.S10 FigATRH, NRPS, SID1, Ligase, ACE1) as well as genes involved with ferricrocin biosynthesis (SIDC), siderophore uptake , FRE1, and HAPX. Below each tracing is the motif within the bound region along with its location with respect to the upstream start codon. Solid lines represent immunoprecipitated chromatin for the control (26199 WT) and SREB-3xHA. Dashed lines represent the input control. Tracings are from two biological replicates, A and B.ChIP-seq enrichment of SREB-3xHA binding upstream of genes located in the siderophore biosynthetic gene cluster Click here for additional data file.S11 FigChIP-seq enrichment of SREB-3xHA binding upstream of genes encoding a WD repeat protein, cation diffusion facilitator, GNAT family acetyltransferase, thioesterase, glycolipid surface protein, C-5 sterol desaturase, and an amino acid acetyltransferase. Below each tracing is the motif within the bound region along with its location with respect to the upstream start codon. Solid lines represent immunoprecipitated chromatin for the control (26199 WT) and SREB-3xHA. Dashed lines represent the input control. Tracings are from two biological replicates, A and B.(EPS)Click here for additional data file.


The aim of this study was to investigate and evaluate the role of magnetic resonance (MR) diffusion kurtosis imaging (DKI) in characterizing breast lesions.2 and dynamic contrast-enhanced (DCE) MR imaging. Breast lesions were histologically characterized and DKI related parameters—mean diffusivity (MD) and mean kurtosis (MK)—were measured. The MD and MK in normal fibroglandular breast tissue, benign and malignant lesions were compared by One-way analysis of variance (ANOVA) with Tukey's multiple comparison test. Receiver operating characteristic (ROC) analysis was performed to assess the sensitivity and specificity of MD and MK in the diagnosis of breast lesions.One hundred and twenty-four lesions in 103 patients (mean age: 57±14 years) were evaluated by MR DKI performed with 7 b-values of 0, 250, 500, 750, 1,000, 1,500, 2,000 s/mmP<0.001), and, in contrast, MD for benign lesions (1.97±0.35 (10−3 mm2/s)) was higher than that for malignant lesions (1.20±0.31 (10−3 mm2/s)) (P<0.001). At a cutoff MD/MK 1.58 (10−3 mm2/s)/0.69, sensitivity and specificity of MD/MK for the diagnosis of malignant were 79.3%/84.2% and 92.9%/92.9%, respectively. The area under the curve (AUC) is 0.86/0.92 for MD/MK.The benign lesions (n = 42) and malignant lesions (n = 82) had mean diameters of 11.4±3.4 mm and 35.8±20.1 mm, respectively. The MK for malignant lesions (0.88±0.17) was significantly higher than that for benign lesions (0.47±0.14) (DKI could provide valuable information on the diffusion properties related to tumor microenvironment and increase diagnostic confidence of breast tumors. Breast MRI has been increasingly used in diagnosing patients with suspicious breast lesions owing to its non-invasive nature and excellent soft-tissue contrast as compared with other diagnostic imaging modalities Tumor invasiveness (grade and aggressiveness) is highly related to the pathophysiologic features of tumor tissues such as cytological patterns or organization, cellular structure and density Recently, DKI parameters have been proved to be able to indicate microstructural changes within cerebral glioma tissue that affect the way that molecule diffuse and to allow better differentiation among cerebral glioma grades than those of conventional DWI All research procedures were approved by our institutional review boards . Written informed consent for each study was obtained independently for all patients.N = 124) were included in this study from September 2012 to June 2014. Following breast MRI, needle or excision biopsies were performed on patients to obtain lesion samples. All biopsy specimens were fixed in 10% formalin and embedded in paraffin. The specimens were then cut and stained with hematoxylin and eosin (H&E), followed by histological examination. The lesion classification based on pathological analysis of biopsy specimens was considered as standard reference. Each breast lesion was assigned as either benign or malignant. There were 42 benign lesions in 30 patients, including 17 fibroadenomas, 9 fibrocystic changes, 16 cysts; and 82 malignant lesions in 73 patients, including 27 infiltrating ductal carcinomas, 21 ductal carcinomas in situ, 23 infiltrating lobular carcinomas, 11 lobular carcinoma in situ.In total, 103 female patients  with suspicious breast lesions  or Sensitivity Encoding , number of averages (NA)  = 2, and acquisition time  = 2 minutes. For DKI, interleaved multi-slice DWI was performed using a single-shot spin-echo echo planar imaging (EPI) sequence with 7 b-values of 0, 250, 500, 750, 1000, 1500 and 2000 s/mm2 in x, y and z directions with TR  = 4000 ms, TE  = 100 ms, receiver bandwidth  = 1184 Hz/Pix, FOV  = 166×380 mm2, acquisition matrix  = 84×192, slice thickness  = 7.0 mm, number of slices  = 12, parallel imaging acceleration factor  = 2 using GRAPPA or SENSE, fat suppression achieved by spectral adiabatic inversion recovery (SPAIR), “distortion correction”  =  Yes, NA  = 1, and acquisition time  = 2.7 minutes. Dynamic contrast-enhanced T1-weighted MRI was then performed using fat-saturated three-dimensional (3D) fast low-angle shot (FLASH) sequence with TR  = 4.73 ms, TE  = 1.71 ms, flip angle (FA)  = 10°, FOV  = 320×320 mm2, acquisition matrix  = 384×384, slice thickness  = 1.2 mm, number of slices  = 128, parallel imaging acceleration factor  = 2 using GRAPPA or SENSE, NA  = 1, and acquisition time  = 50 sec for each time point, before (pre-contrast) and five times after injection (post-contrast) of gadolinium chelate  at a dose of 0.1 mmol/kg at a rate of 2 mL/s followed by a flush of saline solution.Bilateral breast MR imaging was performed on two 3.0 Tesla MR systems  with a maximum gradient strength of 45(40) mT/m and a maximum slew rate of 200 T/m/s. 46 patients  were scanned in a prone position on Siemens scanner with a dedicated phased-array 4-channel bilateral breast coil; while 57 patients  were scanned in prone position on Philips scanner with a dedicated phased-array 7-channel bilateral breast coil. T2-weighted images were first acquired with interleaved multi-slice turbo spin-echo (TSE) sequence with repetition time (TR)  = 3000 ms, echo time (TE)  = 61 ms, Inversion Time (TI)  = 230 ms, turbo factor  = 16, field of view (FOV)  = 320×320 mm0 is the signal without diffusion weighting. Note that we excluded the b0  images from the numerical fitting to minimize blood perfusion effects on the DW signal decay. Background voxels with signal intensities below a threshold value in the b0 images were excluded from the fitting to increase efficiency. MD and MK maps were then calculated as the average of the parametric ADC and AKC maps along all applied diffusion gradient directions, respectively 2) images with reference to the contrast-enhanced T1-weighted images. Care was taken to avoid region's cyst and necrosis that were hyperintense on both T2W images and MD maps, as well as regions of adipose tissue that were hypointense on b0 image. ROI with similar size was also defined in normal fibroglandular breast tissue in the contralateral breast. Lesion and normal fibroglandular breast tissue ROIs were then transferred and used for MD and MK measurements on the MD and MK maps. Lesion sizes were defined as the longest dimension on DCE-MRI. Results were expressed as mean ± standard deviation (SD). Prior to the analysis of DKI parameters for lesions, the study performed on two different scanners necessitates a pilot analysis to evaluate the vendor-specific differences of DKI. An independent samples t-test was used to compare DKI parameters for the ROIs of normal fibroglandular breast tissue, which were divided into two groups according to the scanners. Then, One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was performed to compare the MD and MK values among normal fibroglandular breast tissue, benign and malignant tissues. A P-value of less than 0.05 was considered statistically significant. In addition, receiver operating characteristic (ROC) analysis was carried out to determine suitable MD and MK cutoff values for discrimination between benign and malignant lesions and to assess the diagnostic performance of DKI.MR images were analyzed in MATLAB . Apparent diffusion coefficient (ADC) and apparent kurtosis coefficient (AKC) along each diffusion gradient direction were derived per voxel simultaneously by least-square fitting the DW signals non-linearly to: −3 mm2/s) and 0.55±0.15 for MD and MK respectively, while 67 ROIs of normal fibroglandular breast tissue  obtained on Philips scanner had mean values of 2.14±0.36 (10−3 mm2/s) and 0.53±0.14 for MD and MK respectively. No significant differences were found for MD (P = 0.23) and MK (P = 0.54) from these two groups.57 ROIs of normal fibroglandular breast tissue  obtained on Siemens scanner had mean values of 2.22±0.41 (10−3 mm2/s), 1.97±0.35 (10−3 mm2/s) and 1.20±0.31 (10−3 mm2/s), while MK values are 0.54±0.18, 0.47±0.14, 0.88±0.17 for contralateral fibroglandular tissues, benign and malignant lesions, respectively. The results reveals that both MD and MK values in fibroglandular tissue, benign and malignant lesions are significantly different (P<0.001).−3 mm2/s) as MD cutoff value between benign and malignant lesions, the sensitivity was 79.3% and specificity was 92.9%; 20.7% were misclassified as benign, while 7.1% were misclassified as malignant. For MK, the AUC was 0.92 and the 95% confidence interval was 0.85–0.96. Using 0.69 as MK cutoff value between benign and malignant lesions, the sensitivity was 84.2% and specificity was 92.9%; 15.8%were misclassified as benign, while 7.1% were misclassified as malignant.Accurate diagnosis and classification of breast lesions have always been challenging using conventional mammography and ultrasound, especially in dense fibroglandular breasts Our study results demonstrate the potential utility of DKI for the characterization of breast lesions. The results of ROC analysis suggest that the use of MK lead to a higher sensitivity and specificity and lower percentage for misclassification than those of MD in determining benign and malignant breast lesions, indicating a better diagnostic performance, The behavior of DW signal with higher b-values, whether in benign or malignant lesions, is better characterized by DKI model rather than mono-exponential model, Although DCE-MRI using gadolinium chelates offers high sensitivity in detecting breast lesions and hence has been widely used for locating multiple small breast tumors, it suffers from low specificity in characterizing tumor tissues 2) to minimize the effects of pseudorandom vascular perfusion It is worthwhile to note that pronounced microvascular perfusion associated with tumor angiogenesis may lead to bi-exponential DW signal decay in breast tumor tissue Breasts consist of an exceptionally high content of fatty tissue. It has been shown that adipose tissue in breasts yields significantly lower MD value than normal fibroglandular tissue In our study, DKI was performed based on a single-shot spin-echo EPI sequence with 7 b-values in three orthogonal directions, and the MK was computed by averaging AKC over all directions. Indeed, the use of average AKC for the approximation of MK took an assumption of isotropic diffusion for simplicity, which might introduce the rotation variations and hence deteriorate the validity of MK in the cases of anisotropic diffusion. For improvement, a tensor could be used to describe the dependency of the measured diffusional kurtosis on the direction of the diffusion sensitizing gradients Several potential limitations exist in the current study. First, we did not evaluate the influences of menopausal status and menstrual cycle on DKI parameters in breast tumor tissues. Slight variation in normal breast MD owing to hormonal fluctuations and hence water content throughout menstrual cycle has been reported previously In conclusion, the results of this study suggest that DKI could provide valuable information on the diffusion properties related to tumor microenvironment by the simultaneous quantification of both MD and MK. DKI could improve tissue characterization of breast lesions and increase diagnostic confidence of breast tumors. Further studies with larger sample size are warranted to explore the full potential of DKI for non-invasive imaging of human breast lesions in clinical setting. Once fully validated, breast DKI may also serve as a non-contrast breast screening imaging technique, avoiding unnecessary biopsies.