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Honey flavonoids inhibit Candida albicans morphogenesis by affecting DNA behavior and mitochondrial function Barbara Canonico1, Manila Candiracci2, Barbara Citterio2, Rosa Curci3,4,5, Stefano Squarzoni3,5, Annalisa Mazzoni6, Stefano Papa2 & Elena Piatti*,2
ABSTRACT: Aim: Candida albicans is a pathogenic yeast, which forms a range of polarized and expanded cell shapes. We aimed to determine the correlation between honey extract (HFE) activity and changes in C. albicans cell cycle, morphology and subcellular organelles. Materials & methods: HFE anticandidal properties were investigated using flow cytometry and scanning electron microscopy. Results: Flow cytometry and scanning electron microscopy analyses indicated that HFE may inhibit the growth of the three phenotypes displayed by C. albicans and reduce infection by affecting membrane integrity. HFE affects hyphal transition by reducing the G0/G1 phase and increasing the G2/M phase. Conversely, yeast and pseudohyphae do not appear to be affected. Modifications of vacuolization and mitochondrial activity, during yeast–hypha transition establish the involvement of vacuole and mitochondria. Conclusion: HFE improved mitochondrial functionality and reduced the vacuolization, modifying the branching process associated with virulence. It is hypothesized that HFE induces changes in cell cycle progress, membrane integrity, mitochondrial function and biogenesis. Candida albicans is a pleiomorphic fungus that can exist as either a commensal or an opportunistic pathogen, capable of causing superficial to life-threatening infections. A special feature of C. albicans is the morphological transition from unicellular yeast to an elongated, multinucleate hyphal form through the critical stage of germ-tube formation. In between these two extreme growth forms, the fungus can exhibit a variety of other forms that are collectively referred to as pseudohyphae. Pseudohyphae rarely form true hyphae and hyphae rarely produce pseudohyphal buds [1] . These morphogenetic transitions between the yeast, pseudohyphae and true hyphae are coordinated with the cell cycle. Conditions that arrest cell cycle progression result in a polarized growth phenotype: G1-arrested cells tend to be more hyphal-like, whereas S, G2 and M arrests tend to be pseudohyphal-like [2] . In addition, changes in subcellular structures, such as the vacuole and mitochondria, occur as a consequence of phenotypic switching [3] . In fact, previous studies have established that when C. albicans generates the germ-tube in subapical region, the vacuolar volume significantly increases [4] . The germ-tube extension is followed by protoplasm migration towards the hyphal tip, gradually losing the ‘empty’ look. This event suggests that hyphal cell vacuolization may be an important part of a ‘host invasion strategy’ because C. albicans mutants deficient in vacuolar biogenesis are defective in polarized hyphal growth and virulence [5] . Mitochondria play very important roles in the life
KEYWORDS
• Candida albicans • flavonoids • mitochondrial activity • morphogenesis • vacuole
Department of Earth, Life & Environmental Sciences, Urbino, Italy Department of Biomolecular Sciences, University “Carlo Bo” Urbino, Italy 3 CNR-National Research Council of Italy, Institute of Molecular Genetics, Bologna, Italy 4 Laboratory of Cell Biology-Ramses Laboratory, c/o Rizzoli Orthopaedic Institute, Bologna, Italy 5 Laboratory of Musculoskeletal Biology, c/o Rizzoli Orthopaedic Institute, Bologna, Italy 6 Department of Biomedicine, Unit of Dental Sciences & Biomaterials, University of Trieste, Italy *Author for correspondence. Tel.: +39 0722 305242; Fax: +39 0722 305324; [email protected] 1 2
10.2217/FMB.14.17 © 2014 Future Medicine Ltd
Future Microbiol. (2014) 9(4), 445–456
part of
ISSN 1746-0913
445
Research Article Canonico, Candiracci, Citterio et al. cycle of cells, which are involved in a range of processes. Moreover, mitochondria are also the main intracellular source of reactive oxygen species (ROS) during normal metabolism. We found that the mitochondria in C. albicans are capable of generating ROS depending on morphogenesis and their highest levels occur in hyphal forms, relevant to Candida pathogenesis [6] . Treatment of C. albicans infections has been greatly facilitated since the introduction of antifungal agents, in particular azole derivatives; however, their intensive clinical use for both therapy and prophylaxis has favored the emergence of resistant strains [7] . The phenomenon of drug resistance has prompted an increasing interest in traditional and nonconventional medical treatments. One treatment that has received much interest is honey. Since antiquity, raw honey has been shown to be active against a diverse range of microorganisms [8] . The main antibacterial factors of the honey have been reported to be hydrogen peroxide [9] and, recently, defensin-1 [10] ; however, other antimicrobial factors are present and they include nonperoxide components, such as flavonoids and phenolic acids [11] . The amount of these components may be small or diluted in the honey; however, when extracted with organic solvents, they become concentrated and therefore exhibit more activity [12,13] . We have demonstrated that the flavonoid extract of Italian honey (HFE) possess antifungal activity against C. albicans [6,14] . Both yeast-form and mycelial growth are inhibited by HFE and this effect has been shown to depend on antioxidant action of the flavonoids that support intracellular glutathione levels, whose alteration plays a significant role in the yeast to hyphal conversion. In this study, we treated C. albicans with HFE and investigated the correlation between the activity of HFE and changes in cell cycle, cell morphology and subcellular organelles, in particular, the vacuole and mitochondria. Vacuolization and mitochondrial activity, such as mitochondrial membrane potential (ΔΨm), mass/number of mitochondria and ROS production were also investigated. Materials & methods ●●Extraction of honey flavonoids
Unprocessed multifloral honey was locally obtained from Associazione Marchigiana Apicoltori (Marche, Italy), harvested in 2011 and stored in the darkn at room temperature (RT) to minimize any alterations. Phenolic compounds
446
Future Microbiol. (2014) 9(4)
were extracted as reported by Fiorani et al. [12] . Extract was stored at -80°C and, just before use, aliquots were diluted with dimethyl sulfoxide (DMSO). The volume of DMSO ranges in function of the HFE volume; however, its final concentration is always lower than 2%. Our previous results report that DMSO negative control at used concentrations as been utilized and then the DMSO did not interfere with Candida growth [14] . ●●Fungal strain & growth conditions
C. albicans ATCC 10123 strain was grown in YNB medium (Sigma-Aldrich Chemie Steinheim, Germany), supplemented with 5% (weight/volume) glucose, up to early exponential phase at 37°C. For growth in the yeast form, C. albicans cells (1 × 105 cells/ml) were inoculated in YNB and incubated at 37°C. For hyphal and pseudohyphal proliferation, Candida cells (1 × 105 cells/ml) were inoculated in RPMI-1640 and incubated at 37 or 28°C, respectively. All incubations were performed for 6 h in the absence and presence of 48 μg/ml HFE, corresponding to MIC50 value [14] . Hyphae and pseudohyphae formation was detected by an Axioplane optic microscope (Zeiss, Oberkochen, Germany). ●●Flow cytometric analysis
Scatter characteristics, DNA content, ΔΨm, mitochondria mass/number and acidic compartment were evaluated by flow cytometry (FC) using a FACS calibur flow cytometer equipped with a 15 mW, 488 nm, air-cooled argon-ion laser and a second, red diode laser at 635 nm, using CellQuest™ Software (Becton Dickinson, CA, USA). A gate that excluded debris on clusters was adjusted on a dot plot showing forward (light) scatter (FSC) versus side (light) scatter (SSC). A total of 10,000 yeast cells were analyzed. ●●Evaluation of scatter characteristics
Candida suspensions were harvested by centrifugation, washed with phosphate-buffered saline and resuspended in the specific media to be analyzed. Dual-parameter contour plots were used to distinguish and define the morphologic forms. For each sample analyzed we recorded FSC and SSC associated with morphological aspects. Light scattered in the forward direction is collected by a lens known as the forward scatter channel; FSC serves as an indirect measure of cell size diameter and volume. By contrast, light measured approximately at a 90° angle to
future science group
Honey flavonoids inhibit Candida albicans morphogenesis the excitation beam is called side scatter and it represents a direct measure of cell surface texture and internal structure. These contour plots provided a series of isometric lines to show the number of cells at selected levels. In particular, FSC and SSC analysis allows the detection of cell shrinkage [15,16] and/or an increase in cellular size over time [17] during morphogenic transition. ●●Evaluation of DNA content
Candida cells were fixed in ethanol (70%) and stored at -20°C for up to 1 month. Samples were resuspended in 50 mM Tris-HCl and treated with 1 mg/ml RNAse-A at 37°C for 30 min. After incubation, samples were pelleted and resuspended in 0.05 M HCl containing 5 mg/ml of pepsin for 1 h at 25°C. Fixed cells were washed in NaCl 180 mM with MgCl2 70 mM, Tris-HCl 180 mM, resuspended in 400 μl of citrate buffer and stained with 20 μg/ml propidium iodide (Sigma-Aldrich Chemie Steinheim). Samples were incubated at 37°C for 30 min, and then analyzed. DNA content was evidenced as fluorescence intensity (FI) and possible events in the subdiploid peak identified the percentage of apoptosis [4] . ●●Scanning electron microscopy analysis
Candida suspensions were prefixed with 2.5% glutaraldehyde in 0.1 M cacodylate pH 7.3 (buffer A) for 1 h at 4°C, seeded on precoated poly-l-lysine coverslips, washed with buffer A and re-fixed on coverslips with 2.5% glutaraldehyde in buffer A for 1 h at 4°C. After a second wash with buffer A, the samples were postfixed with 1% osmium tetroxide in buffer A for 1 h at RT. After rinsing with buffer A, the samples were dehydrated in an ethanol series and critical point dried. The samples were then mounted on aluminium stubs with silver adhesive paint, sputter-coated with 4-nm gold in argon atmosphere using Edwards S150B (CA, USA) apparatus and observed at a 0° tilt angle with a Cambridge Stereoscan 200 (Cambridge, UK) scanning electron microscope operated at 20 kV. ●●Evaluation of mitochondrial activity
Determination of mitochondria mass/number & ΔΨm
Candida cells were stained with 100 nM MitoTracker-Green (MTG; Sigma-Aldrich Chemie Steinheim) for 30 min at RT. MTG passively diffuses across the plasma membrane and accumulates in active mitochondria, defining
future science group
Research Article
their mass/number. ΔΨm was analyzed using 80 nM tetra-methyl-rhodamine-methyl-ester (Sigma-Aldrich Chemie Steinheim) for 15 min at RT. The results are reported as arbitrary units of fluorescence. Measurement of cardiolipin content
To monitor changes in mitochondrial cardiolipin (CL), Candida cells were resuspended in the specific media containing 100 nM nonyl-acridineorange (Sigma-Aldrich Chemie Steinheim) and incubated for 30 min at 37°C. FI was quantified by FC. Determination of acidic vacuoles
The dynamics of vacuole development and inheritance in C. albicans were investigated by acridine-orange (AO; Sigma-Aldrich Chemie). When AO is bound to acid compartments, such as lysosomes and acidic vacuoles, red fluorescence is emitted (FL3–650 nm) with proportional intensity to the acidity degree. Candida suspensions were incubated with AO to the final concentration of 1 μg/ml for 15–20 min a RT. After incubation, cells were washed, resuspended in phosphate-buffered saline and analyzed by FC. The results are reported as arbitrary units of fluorescence. ●●Determination of ROS levels
The intracellular ROS content was determined by 0.5 μM of 2´,7´-dichlorofluorescin-diacetate (DCFH-DA; Molecular Probes, Inc., OR, USA; final concentration) [6] . FI was measured using a spectrafluor instrument (Perkin-Elmer, CT, USA; LS-5) with λex 485 nm and λem520 nm. ROS production was calculated by subtracting the FI value of cells not treated with DCFH-DA by the FI value of cells treated with DCFH-DA and expressed as FI/μg of proteins [6] . ●●Protein determination
After incubation, Candida cells were extensively washed and lysed by several freeze–thaw cycles and again by a sonicator (three cycles of 30 s at 100 W) [6] . After centrifugation (13,000 rpm for 10 min at 4°C), pellets were resuspended in 0.5 N NaOH for the determination of proteins using Coomassie brilliant blue reagent from BioRad Laboratories (CA, USA) [18] . ●●Statistics
All samples were prepared in triplicate and the experiments were repeated at least twice. Data
www.futuremedicine.com
447
Research Article Canonico, Candiracci, Citterio et al. are presented as mean FI ratios of the HFEtreated samples and the controls from three separate experiments ± one standard error of the mean [19,20] . Comparisons between HFE-treated and untreated samples were evaluated using the Student t-test. A value of p
Honey flavonoids inhibit Candida albicans morphogenesis by affecting DNA behavior and mitochondrial function Barbara Canonico1, Manila Candiracci2, Barbara Citterio2, Rosa Curci3,4,5, Stefano Squarzoni3,5, Annalisa Mazzoni6, Stefano Papa2 & Elena Piatti*,2
ABSTRACT: Aim: Candida albicans is a pathogenic yeast, which forms a range of polarized and expanded cell shapes. We aimed to determine the correlation between honey extract (HFE) activity and changes in C. albicans cell cycle, morphology and subcellular organelles. Materials & methods: HFE anticandidal properties were investigated using flow cytometry and scanning electron microscopy. Results: Flow cytometry and scanning electron microscopy analyses indicated that HFE may inhibit the growth of the three phenotypes displayed by C. albicans and reduce infection by affecting membrane integrity. HFE affects hyphal transition by reducing the G0/G1 phase and increasing the G2/M phase. Conversely, yeast and pseudohyphae do not appear to be affected. Modifications of vacuolization and mitochondrial activity, during yeast–hypha transition establish the involvement of vacuole and mitochondria. Conclusion: HFE improved mitochondrial functionality and reduced the vacuolization, modifying the branching process associated with virulence. It is hypothesized that HFE induces changes in cell cycle progress, membrane integrity, mitochondrial function and biogenesis. Candida albicans is a pleiomorphic fungus that can exist as either a commensal or an opportunistic pathogen, capable of causing superficial to life-threatening infections. A special feature of C. albicans is the morphological transition from unicellular yeast to an elongated, multinucleate hyphal form through the critical stage of germ-tube formation. In between these two extreme growth forms, the fungus can exhibit a variety of other forms that are collectively referred to as pseudohyphae. Pseudohyphae rarely form true hyphae and hyphae rarely produce pseudohyphal buds [1] . These morphogenetic transitions between the yeast, pseudohyphae and true hyphae are coordinated with the cell cycle. Conditions that arrest cell cycle progression result in a polarized growth phenotype: G1-arrested cells tend to be more hyphal-like, whereas S, G2 and M arrests tend to be pseudohyphal-like [2] . In addition, changes in subcellular structures, such as the vacuole and mitochondria, occur as a consequence of phenotypic switching [3] . In fact, previous studies have established that when C. albicans generates the germ-tube in subapical region, the vacuolar volume significantly increases [4] . The germ-tube extension is followed by protoplasm migration towards the hyphal tip, gradually losing the ‘empty’ look. This event suggests that hyphal cell vacuolization may be an important part of a ‘host invasion strategy’ because C. albicans mutants deficient in vacuolar biogenesis are defective in polarized hyphal growth and virulence [5] . Mitochondria play very important roles in the life
KEYWORDS
• Candida albicans • flavonoids • mitochondrial activity • morphogenesis • vacuole
Department of Earth, Life & Environmental Sciences, Urbino, Italy Department of Biomolecular Sciences, University “Carlo Bo” Urbino, Italy 3 CNR-National Research Council of Italy, Institute of Molecular Genetics, Bologna, Italy 4 Laboratory of Cell Biology-Ramses Laboratory, c/o Rizzoli Orthopaedic Institute, Bologna, Italy 5 Laboratory of Musculoskeletal Biology, c/o Rizzoli Orthopaedic Institute, Bologna, Italy 6 Department of Biomedicine, Unit of Dental Sciences & Biomaterials, University of Trieste, Italy *Author for correspondence. Tel.: +39 0722 305242; Fax: +39 0722 305324; [email protected] 1 2
10.2217/FMB.14.17 © 2014 Future Medicine Ltd
Future Microbiol. (2014) 9(4), 445–456
part of
ISSN 1746-0913
445
Research Article Canonico, Candiracci, Citterio et al. cycle of cells, which are involved in a range of processes. Moreover, mitochondria are also the main intracellular source of reactive oxygen species (ROS) during normal metabolism. We found that the mitochondria in C. albicans are capable of generating ROS depending on morphogenesis and their highest levels occur in hyphal forms, relevant to Candida pathogenesis [6] . Treatment of C. albicans infections has been greatly facilitated since the introduction of antifungal agents, in particular azole derivatives; however, their intensive clinical use for both therapy and prophylaxis has favored the emergence of resistant strains [7] . The phenomenon of drug resistance has prompted an increasing interest in traditional and nonconventional medical treatments. One treatment that has received much interest is honey. Since antiquity, raw honey has been shown to be active against a diverse range of microorganisms [8] . The main antibacterial factors of the honey have been reported to be hydrogen peroxide [9] and, recently, defensin-1 [10] ; however, other antimicrobial factors are present and they include nonperoxide components, such as flavonoids and phenolic acids [11] . The amount of these components may be small or diluted in the honey; however, when extracted with organic solvents, they become concentrated and therefore exhibit more activity [12,13] . We have demonstrated that the flavonoid extract of Italian honey (HFE) possess antifungal activity against C. albicans [6,14] . Both yeast-form and mycelial growth are inhibited by HFE and this effect has been shown to depend on antioxidant action of the flavonoids that support intracellular glutathione levels, whose alteration plays a significant role in the yeast to hyphal conversion. In this study, we treated C. albicans with HFE and investigated the correlation between the activity of HFE and changes in cell cycle, cell morphology and subcellular organelles, in particular, the vacuole and mitochondria. Vacuolization and mitochondrial activity, such as mitochondrial membrane potential (ΔΨm), mass/number of mitochondria and ROS production were also investigated. Materials & methods ●●Extraction of honey flavonoids
Unprocessed multifloral honey was locally obtained from Associazione Marchigiana Apicoltori (Marche, Italy), harvested in 2011 and stored in the darkn at room temperature (RT) to minimize any alterations. Phenolic compounds
446
Future Microbiol. (2014) 9(4)
were extracted as reported by Fiorani et al. [12] . Extract was stored at -80°C and, just before use, aliquots were diluted with dimethyl sulfoxide (DMSO). The volume of DMSO ranges in function of the HFE volume; however, its final concentration is always lower than 2%. Our previous results report that DMSO negative control at used concentrations as been utilized and then the DMSO did not interfere with Candida growth [14] . ●●Fungal strain & growth conditions
C. albicans ATCC 10123 strain was grown in YNB medium (Sigma-Aldrich Chemie Steinheim, Germany), supplemented with 5% (weight/volume) glucose, up to early exponential phase at 37°C. For growth in the yeast form, C. albicans cells (1 × 105 cells/ml) were inoculated in YNB and incubated at 37°C. For hyphal and pseudohyphal proliferation, Candida cells (1 × 105 cells/ml) were inoculated in RPMI-1640 and incubated at 37 or 28°C, respectively. All incubations were performed for 6 h in the absence and presence of 48 μg/ml HFE, corresponding to MIC50 value [14] . Hyphae and pseudohyphae formation was detected by an Axioplane optic microscope (Zeiss, Oberkochen, Germany). ●●Flow cytometric analysis
Scatter characteristics, DNA content, ΔΨm, mitochondria mass/number and acidic compartment were evaluated by flow cytometry (FC) using a FACS calibur flow cytometer equipped with a 15 mW, 488 nm, air-cooled argon-ion laser and a second, red diode laser at 635 nm, using CellQuest™ Software (Becton Dickinson, CA, USA). A gate that excluded debris on clusters was adjusted on a dot plot showing forward (light) scatter (FSC) versus side (light) scatter (SSC). A total of 10,000 yeast cells were analyzed. ●●Evaluation of scatter characteristics
Candida suspensions were harvested by centrifugation, washed with phosphate-buffered saline and resuspended in the specific media to be analyzed. Dual-parameter contour plots were used to distinguish and define the morphologic forms. For each sample analyzed we recorded FSC and SSC associated with morphological aspects. Light scattered in the forward direction is collected by a lens known as the forward scatter channel; FSC serves as an indirect measure of cell size diameter and volume. By contrast, light measured approximately at a 90° angle to
future science group
Honey flavonoids inhibit Candida albicans morphogenesis the excitation beam is called side scatter and it represents a direct measure of cell surface texture and internal structure. These contour plots provided a series of isometric lines to show the number of cells at selected levels. In particular, FSC and SSC analysis allows the detection of cell shrinkage [15,16] and/or an increase in cellular size over time [17] during morphogenic transition. ●●Evaluation of DNA content
Candida cells were fixed in ethanol (70%) and stored at -20°C for up to 1 month. Samples were resuspended in 50 mM Tris-HCl and treated with 1 mg/ml RNAse-A at 37°C for 30 min. After incubation, samples were pelleted and resuspended in 0.05 M HCl containing 5 mg/ml of pepsin for 1 h at 25°C. Fixed cells were washed in NaCl 180 mM with MgCl2 70 mM, Tris-HCl 180 mM, resuspended in 400 μl of citrate buffer and stained with 20 μg/ml propidium iodide (Sigma-Aldrich Chemie Steinheim). Samples were incubated at 37°C for 30 min, and then analyzed. DNA content was evidenced as fluorescence intensity (FI) and possible events in the subdiploid peak identified the percentage of apoptosis [4] . ●●Scanning electron microscopy analysis
Candida suspensions were prefixed with 2.5% glutaraldehyde in 0.1 M cacodylate pH 7.3 (buffer A) for 1 h at 4°C, seeded on precoated poly-l-lysine coverslips, washed with buffer A and re-fixed on coverslips with 2.5% glutaraldehyde in buffer A for 1 h at 4°C. After a second wash with buffer A, the samples were postfixed with 1% osmium tetroxide in buffer A for 1 h at RT. After rinsing with buffer A, the samples were dehydrated in an ethanol series and critical point dried. The samples were then mounted on aluminium stubs with silver adhesive paint, sputter-coated with 4-nm gold in argon atmosphere using Edwards S150B (CA, USA) apparatus and observed at a 0° tilt angle with a Cambridge Stereoscan 200 (Cambridge, UK) scanning electron microscope operated at 20 kV. ●●Evaluation of mitochondrial activity
Determination of mitochondria mass/number & ΔΨm
Candida cells were stained with 100 nM MitoTracker-Green (MTG; Sigma-Aldrich Chemie Steinheim) for 30 min at RT. MTG passively diffuses across the plasma membrane and accumulates in active mitochondria, defining
future science group
Research Article
their mass/number. ΔΨm was analyzed using 80 nM tetra-methyl-rhodamine-methyl-ester (Sigma-Aldrich Chemie Steinheim) for 15 min at RT. The results are reported as arbitrary units of fluorescence. Measurement of cardiolipin content
To monitor changes in mitochondrial cardiolipin (CL), Candida cells were resuspended in the specific media containing 100 nM nonyl-acridineorange (Sigma-Aldrich Chemie Steinheim) and incubated for 30 min at 37°C. FI was quantified by FC. Determination of acidic vacuoles
The dynamics of vacuole development and inheritance in C. albicans were investigated by acridine-orange (AO; Sigma-Aldrich Chemie). When AO is bound to acid compartments, such as lysosomes and acidic vacuoles, red fluorescence is emitted (FL3–650 nm) with proportional intensity to the acidity degree. Candida suspensions were incubated with AO to the final concentration of 1 μg/ml for 15–20 min a RT. After incubation, cells were washed, resuspended in phosphate-buffered saline and analyzed by FC. The results are reported as arbitrary units of fluorescence. ●●Determination of ROS levels
The intracellular ROS content was determined by 0.5 μM of 2´,7´-dichlorofluorescin-diacetate (DCFH-DA; Molecular Probes, Inc., OR, USA; final concentration) [6] . FI was measured using a spectrafluor instrument (Perkin-Elmer, CT, USA; LS-5) with λex 485 nm and λem520 nm. ROS production was calculated by subtracting the FI value of cells not treated with DCFH-DA by the FI value of cells treated with DCFH-DA and expressed as FI/μg of proteins [6] . ●●Protein determination
After incubation, Candida cells were extensively washed and lysed by several freeze–thaw cycles and again by a sonicator (three cycles of 30 s at 100 W) [6] . After centrifugation (13,000 rpm for 10 min at 4°C), pellets were resuspended in 0.5 N NaOH for the determination of proteins using Coomassie brilliant blue reagent from BioRad Laboratories (CA, USA) [18] . ●●Statistics
All samples were prepared in triplicate and the experiments were repeated at least twice. Data
www.futuremedicine.com
447
Research Article Canonico, Candiracci, Citterio et al. are presented as mean FI ratios of the HFEtreated samples and the controls from three separate experiments ± one standard error of the mean [19,20] . Comparisons between HFE-treated and untreated samples were evaluated using the Student t-test. A value of p
