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Jun 15, 2017 - Here we address this knowledge gap using the plant pathogen, Aspergillus parasiticus, our laboratory's model for studying fungal second...
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Citrate-coated silver nanoparticles growth- independently inhibit aflatoxin synthesis in Aspergillus parasiticus Chandrani Mitra, Phani M Gummadidala, Kamelia Afshinnia, Ruth Corrin Merrifield, Mohammed A Baalousha, Jamie R Lead, and Anindya Chanda Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

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Citrate-coated silver nanoparticles growth-

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independently inhibit aflatoxin synthesis in

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Aspergillus parasiticus

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Chandrani Mitra1, Phani M. Gummadidala1, Kamelia Afshinnia2 , Ruth C. Merrifield2,

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Mohammed Baalousha2, Jamie R. Lead2 and Anindya Chanda1*

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1

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South Carolina, Columbia, USA

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Department of Environmental Health Sciences, Arnold School of Public Health, University of

Center for Environmental Nanoscience and Risk, Arnold School of Public Health, University of

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South Carolina, Columbia, USA

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Corresponding author, [email protected]

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Phone: 803-777-8263

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Fax: 803-777-3391

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Abstract

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Manufactured silver nanoparticles (Ag NPs) have long been used as antimicrobials. However,

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little is known about how these NPs affect fungal cell functions. While multiple previous studies

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reveal that Ag NPs inhibit secondary metabolite syntheses in several mycotoxin producing

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filamentous fungi, these effects are associated with growth repression and hence need sub-lethal

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to lethal NP doses, which besides stopping fungal growth, can potentially accumulate in the

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environment. Here we demonstrate that citrate coated Ag NPs of size 20 nm, when applied at a

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selected nonlethal dose, can result in a > 2 fold inhibition of biosynthesis of the carcinogenic

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mycotoxin and secondary metabolite, aflatoxin B1 in the filamentous fungus and an important

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plant pathogen, Aspergillus parasiticus, without inhibiting fungal growth. We also show that the

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observed inhibition was not due to Ag ions, but was specifically associated with the mycelial

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uptake of Ag NPs. The NP exposure resulted in a significant decrease in transcript levels of five

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aflatoxin genes and at least two key global regulators of secondary metabolism, laeA and veA,

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with a concomitant reduction of total reactive oxygen species (ROS). Finally, the depletion of Ag

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NPs in the growth medium allowed the fungus to regain completely, its ability of aflatoxin

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biosynthesis. Our results therefore demonstrate the feasibility of Ag NPs to inhibit fungal

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secondary metabolism at nonlethal concentrations, hence providing a novel starting point for

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discovery of custom designed engineered nanoparticles that can efficiently prevent mycotoxins

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with minimal risk to health and environment.

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Keywords: Aflatoxin biosynthesis, Secondary metabolism, Citrate coated nanoparticles, reactive

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oxygen species, oxidative stress

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Conflict of interest statement: The authors declare no conflict of interest

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Environmental Science & Technology

Introduction

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Silver nanoparticles (Ag NPs) are one of the most studied manufactured nanoparticles 1-5

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due to their antimicrobial properties

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resistant microbes

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cytotoxicity 9, 10, along with their ease of synthesis and use. However, as popularity of Ag NPs

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continues to increase in medicine, pharmaceutical, cosmetics, electronics and diverse other

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consumer product industries

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essential to judge comprehensively, their beneficial effects on, for instance, food preservation as

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well as their potential harmful impact on environmental health.

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6-8

and their efficacy in controlling diverse and multi-drug

. They are generally preferred to silver ions due to their lower mammalian

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, additional studies on their effects on microbial metabolism are

To this end, a significant number of studies have shown that Ag NPs can alter the 12

13

14, 15

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metabolite profiles in bacteria

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effects

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antifungal effects

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well. However, while these studies have all focused on growth-dependent repression of

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secondary metabolism using high NP concentrations, the growth-independent effects of Ag NPs,

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typically at low NP concentrations, on fungal secondary metabolism remain unclear. We

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emphasize here that environmentally relevant concentrations of Ag NPs are much lower than

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those typically used in toxicological and ecotoxicolgoical studies, are largely variable depending

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on the environmental compartment of concern, the local conditions and weather events such as

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tidal flooding

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4 µg kg−1 to 14 mg kg−1 in soils

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concentrations below 50 µg L-1 have not been reported to the best of our knowledge. While NP

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exposure pathways for fungi are still unclear at this point, it is possible that multiple exposure

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and algae

either through growth inhibitory

or hormetic

. Multiple studies conducted on fungal strains suggest that Ag NPs also demonstrate

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9, 17-20

and as a result, can repress their secondary metabolite synthesis

21-24

as

, and can range from 10 pg L-1 to 1 µg L-1 in water columns and from 26, 27

. However, effects of Ag NPs on fungal cells at

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pathways exist in fungi for high and low concentrations leading to differential biological

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outcomes. Hence, studying biological behaviors of fungi at sub-lethal doses are critical. In

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particular it is essential to know whether fungi, and specifically, the mycotoxin producers,

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which are very resistant to harsh conditions in the environment 28, alter their metabolic profiles in

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response to Ag NPs. This information is critical in the realistic evaluation of the impact of NP

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accumulation on ecological physiology and human health. Here we address this knowledge gap

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using the plant pathogen, Aspergillus parasiticus, our laboratory’s model for studying fungal

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secondary metabolism

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mycotoxin that contaminates billions of dollars’ worth of crops worldwide including corn,

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peanuts, treenuts and cotton

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investigating molecular mechanisms underlying fungal secondary metabolite syntheses that are

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connected with significant economic and health implications.

29-31

. This filamentous fungus synthesizes aflatoxin B1, a carcinogenic

32, 33

. Hence, it is a well characterized and established model for

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The primary goal of this study was to investigate whether and how aflatoxin production

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in A. parasiticus is impacted by 20 nm sized citrate-coated silver nanoparticles (cit-Ag NPs).

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The cit- Ag NP s has been routinely used as model manufactured nanoparticles in several

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previous studies34-38 that have examined their uptake and toxicity in microbes, aquatic animals

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and other eukaryotic cells. The specific aims of this study were: (a) to investigate the effect of a

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working cit-Ag NP concentration (50 nM) on mycelial growth and aflatoxin biosynthesis at the

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levels of transcription activation of the aflatoxin gene cluster and (b) to investigate the possible

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mechanism for inhibition of aflatoxin biosynthesis.

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MATERIALS AND METHODS:

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Nanoparticle synthesis

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The cit-Ag NPs were synthesized by a standard chemical reduction method described previously

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39, 40

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sodium citrate (0.31 mM) and 10 mL of 0.25mM of sodium borohydride was prepared and stored

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at 4oC in absence of light for 30 min. The silver nitrate and sodium citrate solutions were mixed

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together in a conical flask and stirred vigorously. Subsequently, 6 mL of the sodium borohydride

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was added to this reaction mix as the reducing agent. After 10 min of stirring, the reaction mix

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was heated slowly to boiling. The solution was then heated for an extra 90 min, left overnight

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and cooled (4 °C, in the dark). The Ag NPs thus formed (as indicated by the change of solution

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color from colorless to yellow) were then cleaned, to remove the excess reagents, by

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ultrafiltration (Amicon, 1 kDa regenerated cellulose membrane, Millipore) in a diafiltration

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mode to prevent NP aggregation and drying. Ag NPs were re-dispersed in 0.31 mM sodium

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citrate solution to avoid further growth and the washing process was repeated at least three times

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and was performed each time before use. The concentration of Ag NPs was finally measured by

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inductively coupled plasma-optical emission spectroscopy.

. Briefly, 100 mL aqueous solution of silver nitrate (0.25 mM), 100 mL aqueous solution of

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Nanoparticle characterization

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Presence of Ag NP suspension was determined in the NP suspension using surface plasmon UV-

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Vis spectrophotometry (wavelength 200nm to 800nm). The z-average hydrodynamic diameter

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and polydispersity index (PDI) were determined using dynamic light scattering (DLS) using a

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Malvern Zetasizer (Nanoseries, Malvern Instruments) as described previously 41. The z-average

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diameter and PDI were measured five times to calculate the standard error of mean. Particle size

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distribution, and shape were analyzed using transmission electron microscopy (TEM) as

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described previously

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. Briefly, NPs were drop deposited onto Formvar/carbon coated 50

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mesh Cu grids (S162-3, AGAR Scientific) and allowed to adsorb to the carbon coating for

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10 min (without drying). The grids were then gently immersed in ultrapure water to remove un-

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adsorbed particles, and air-dried under protective cover. Electron micrographs (10 micrographs

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from various locations of the grids) were acquired using TEM (Hitachi H-8000) at 200 keV. The

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cit-Ag NP aggregation in the test media was qualitatively monitored by UV-vis

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concentration of the synthesized Ag NPs was measured by by inductively coupled plasma-atomic

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emission spectroscopy (ICP-OES).

43

. Finally the

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Determination of total and dissolved silver concentrations

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Mycelia after NP exposure (50 mg) and fungal growth media (1 mL) samples were digested at

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room temperature (with 10 mL of 70 % nitric acid (AR grade; Fisher scientific) for 24 h. This

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reaction mixture was diluted with 15 mL double distilled water and heated at 80oC for 15min

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prior to cooling to room temperature and filtration (Whatman filter paper 2). The samples were

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finally diluted in 100 mL volumetric flask and then analyzed using an inductively coupled

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plasma-optical emission spectrometer (ICP-OES; Varian 710-ES). The detection limit was 5 µg

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L-1. Dissolved silver in the growth media was determined by centrifugation (4000xg, 45min) and

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ultrafiltration (Amicon; 1Kda cutoff) of the media and subsequent ICP-OES analyses of the

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filtrate. Nanoparticle concentration was determined by difference.

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Fungal strain, medium and growth conditions:

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The wild-type aflatoxin producer, Aspergillus parasiticus, SU-1 (NRRL 5862) was used for this

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study. For NP exposure experiments, 106 spores of A. parasiticus were inoculated in 100 mL

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yeast extract sucrose (YES) liquid growth medium containing cit- Ag NP (50 ng mL-1 ), and

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grown under our standard growth conditions (in an incubator shaker at 29oC in absence of light,

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with rpm 150). The choice of 50 ng mL-1 as the ideal working concentration for this study was

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based on an initial dose-response experiment (see results), which was used to determine the cit-

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Ag NP concentration that only inhibited aflatoxin biosynthesis without affecting fungal growth.

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Untreated control groups were grown under same growth conditions but in absence of cit- Ag

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NPs.

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Growth and aflatoxin measurements:

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Quantitative comparisons of growth was conducted using dry weight measurements of harvested

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mycelia as described previously

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quantified using Enzyme-Linked Immunosorbant Assay (ELISA) as described previously

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Since previous studies

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study have shown that 40 h cultures demonstrate aflatoxin biosynthesis and export into the

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growth medium at peak levels, a comparison of 40h aflatoxin levels in the presence and absence

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of cit- Ag NP s was conducted. Aflatoxin levels were measured at 48h time-point as well, to

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examine whether aflatoxin biosynthesis could revert to control levels after an almost complete

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removal (by uptake) of most or all cit- Ag NP s in the growth medium by the mycelium.

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44

. Aflatoxin B1 accumulation in the growth medium was 45

.

that have employed the fungal growth conditions used in the current

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NP-aflatoxin interaction assay:

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Interaction of cit-Ag NPs with aflatoxin was determined by incubating total aflatoxin extracted

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from a 40h A. parasiticus culture with the NPs at 50 ng mL-1 concentrations for 4 hours. This

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aflatoxin-cit-Ag NP mixture was ultra-filtered using amicon ultra-centrifugal filter unit (3Kda).

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The concentration aflatoxin in the filtrate was compared with an unfiltered mix and an aflatoxin-

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only control using ELISA. Significance of difference in aflatoxin measurements between the

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filtrate and the unfiltered mix was used to evaluate NP-aflatoxin interaction and the significance

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of difference in aflatoxin measurements between the unfiltered mix and the aflatoxin-only

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control was used to evaluate interference of the NPs in the aflatoxin measurements.

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Quantification of ROS:

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Total ROS generated by mycelia was quantified using a 2’,7’-dichlorofluorescein diacetate

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(DCFH-DA) protocol described previously

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mycelia in the presence of absence of cit- Ag NPs, equal weights of mycelia from each sample

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were placed into 1 mL freshly made 1 µM DCFH-DA in PBS. The yield of the fluorescent

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product dichlorofluorescin (DCF) upon oxidation of DCFH-DA due to generation of ROS was

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measured after a 5hour reaction at 37 °C using an excitation/emission wavelength of 490/525

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nm.

46

. For comparison of total ROS generated by

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Gene expression studies:

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a) RNA extraction, purification and cDNA synthesis. Total RNA was extracted from cells

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harvested in triplicates using a TRIzol (TRIzol Reagent; Invitrogen, Carlsbad, CA) based method

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previously described

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Qiagen® RNEasy Cleanup Kit (Qiagen, Valencia, CA), and then stored at -80°C. The cDNA

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was synthesized from RNA using a reverse transcription kit (New England BioLabs Inc.,

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Ipswich, MA) according to manufacturer’s instructions.

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. Within 24 hours of extraction, RNA cleanup was conducted using a

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b) Real-time PCR assays: The expressions of aflatoxin and superoxide dismutase genes were

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measured using quantitative real-time PCR assays using SsoAdvanced universal SYBR Green

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supermix (BioRad Laboratories, Hercules, CA) and gene specific forward and reverse primers

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(Table 1). The gene specific primers were designed using Primer3 online software

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(http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The aflatoxin gene primers were identical to

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those described previously

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Laboratories, Hercules, CA). Similar to the previous gene expression studies in A. parasiticus 47,

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β-tubulin gene was used as the housekeeping gene. Expression value of each gene was obtained

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from the threshold cycle values that were normalized against 18SrRNA expression in each

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sample. All RT-PCRs were performed in triplicates for each gene per sample. Data analysis of

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gene expression studies were performed using CFX Manager software (Bio-Rad Laboratories).

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. Reactions were performed in a CFX96 thermal cycler (Bio-Rad

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Statistical Analysis:

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Statistical analyses for this study were conducted using the GrpahPad Prism software (Graphpad,

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CA, USA). Statistical significance of two-tailed p-values were determined using an unpaired t-

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test, with n=3 and p < 0.05.

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Results and Discussion:

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Properties of pristine Ag NPs.

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The absorption spectrum of cit-Ag NPs ( figure 1a) showed a single peak centered on λmax 395

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nm (implicative of successful synthesis of the 20 nm Cit- Ag NP s 1, 48) and showed no signs of

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Ag NP aggregates in the synthesized suspension. TEM micrographs confirmed that cit- Ag NP s

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were present predominantly as single particles (representative TEM image shown in figure 1b).

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The average core size of the NPs as revealed by TEM was ca. 13.8 ± 2.3 nm (mean ± standard

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deviation, n= 500). The average hydrodynamic diameter and size PDI as revealed by DLS were

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ca. 20.1 ± 1.0 nm (figure 1c) and 0.11 ± 0.03 respectively, implicating a narrow size distribution.

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The total concentration of the Ag NP stock solution was 9.6 mg L-1.

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Choice of cit-Ag NP dose for studying the Ag NP effect on aflatoxin biosynthesis.

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Dose-response experiment demonstrated that cit- Ag NP did not inhibit A. parasiticus growth

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within 10-50 ng mL-1 concentration range (figure 2a). However, higher concentrations up to 500

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ng mL-1 (highest concentration measured), resulted in significant growth inhibition (~2 fold

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reduction of dry-weight). Also concentrations < 50 ng mL-1 did not result in significant reduction

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in aflatoxin levels in the growth medium. At 50 ng mL-1, cit- Ag NP resulted in significant

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reduction (~2 fold) of aflatoxin accumulation in the growth medium (figure 2b). Aflatoxin

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measurement and growth analyses in experiment was conducted with 40h cultures based on

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previous studies, which show that under our growth conditions aflatoxin is synthesized at peak

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levels at 40h. To confirm that reduction in of aflatoxin accumulation in the growth medium was

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not due to growth effects at time points earlier than 40 h time-point, the growth rates of the

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fungus (cit- Ag NP treated versus untreated) were compared during the 40h period. No

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significant difference in growth rate, expressed as dry weight accumulation per unit time (mg h-

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1

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suggesting that at the applied concentration, cit- Ag NP associated inhibition of aflatoxin

), was observed between the cit- Ag NP treated mycelia and untreated mycelia (figure 2c),

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accumulation in the growth medium was not a fungal growth inhibition effect. Also, NP-

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aflatoxin interaction assays indicated no significant differences in aflatoxin measurements

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between aflatoxin-NP mixtures, ultrafiltered aflatoxin-NP mixtures and aflatoxin-only controls

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indicating that the reduced aflatoxin measurements were not an artifact from aflatoxin-NP

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interactions (SI Fig 1). Hence the cit- Ag NP concentration of 50 ng mL-1 was chosen in this

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study as the ideal dose to examine the possibility of specific inhibitory effects of the NPs on

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aflatoxin synthesis.

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Interaction of cit- Ag NP with the growth medium.

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The possibility of whether the observed cit- Ag NP - mediated reduction in aflatoxin

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accumulation in the growth medium was caused by dissolution of the NPs into the growth

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medium was also explored. Residual silver in the growth medium obtained upon

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ultracentrifugation and ultrafiltration of the cit- Ag NP -containing medium was below the ICP-

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OES detection limit (5 ng mL-1, i.e. 2-fold reduction in transcripts of both laeA and

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veA, thereby demonstrating the ability of cit- Ag NP to inhibit secondary metabolism in A.

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parasiticus. Our results therefore suggest that inhibition of aflatoxin biosynthesis by cit- Ag NP

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was mediated at least in part, by the transcription inhibition of laeA and veA. These genes encode

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the protein LaeA and VeA, both of which are a part of the regulatory complex of secondary

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metabolism regulatory Velvet complex43, and which in addition to aflatoxin biosynthesis,

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regulate other secondary metabolite pathways of filamentous fungi as well 43. Hence our results

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demonstrate the feasibility of cit- Ag NP s to inhibit secondary metabolism in filamentous fungi

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without inhibiting fungal growth.

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Effect of cit- Ag NP on total mycelial ROS

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Quantitative comparison of total ROS generated by cit- Ag NP treated mycelia and the untreated

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control cells demonstrated that cit- Ag NP exposure resulted in a significant reduction (by

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~30%) of total ROS at 24h (figure 4b). In addition, we also explored whether the cit- Ag NP

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mediated drop in total ROS was associated with a concomitant reduction of transcriptional

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activation of superoxide dismutase genes (SOD genes), which are enzymes that remove

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superoxides (one class of ROS) through dismutation reactions

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significant reduction was observed in the transcriptional activation of the cytosolic Cu/Zn-SOD

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gene. No effect was observed on the transcriptional activations of the other SOD genes. Multiple

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studies

61-69

60

. As shown in SI figure 4,

suggest that activation of secondary metabolism is a response to an increase in

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intracellular oxidative stress. Hence it is possible that cit- Ag NP

mediated reduction in

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intracellular oxidative stress (as implicated by the drop in total ROS) and cytosolic Cu/Zn-SOD

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gene could result in the reduction in transcriptional activation of aflatoxin genes (observed in

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figure 4a). We reason that since only the cytosolic Cu/Zn-SOD gene demonstrated

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transcriptional downregulation, the cit- Ag NP possibly resulted in the reduction of ROS build-

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up in the cytosol. Interestingly these findings suggest an intracellular behavior (antioxidant

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effects) of Ag NP, which are contradictory to the effects of Ag NPs when applied at higher doses

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to induce cell toxicity 9, 17-20, and may partly explain hormetic responses found in other biological

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systems 16. It is possible that prior to uptake, the NPs- growth medium interaction resulted in a

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protein coating that resulted in the subsequent antioxidant effects. The molecular mechanisms

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that enable the NPs to reduce cellular ROS will be investigated in greater detail in our future

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studies.

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Aflatoxin biosynthesis upon cit- Ag NP removal from the growth medium.

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Finally, to confirm that the fungus is able to recover its ability of aflatoxin biosynthesis upon

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complete removal of cit- Ag NPs in the growth medium, a quantification of aflatoxin

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accumulation at 48h was conducted. As shown in figure 5a, both growth (figure 5a-i) and

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aflatoxin accumulation (figure 5a-ii) demonstrated no significant difference between the treated

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and control samples, thereby demonstrating that upon depletion of cit- Ag NP s in the growth

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medium (by ~24-30h), mycelia were able to regain their full ability of aflatoxin biosynthesis.

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Analysis of the total ROS at both 30h and 40h also confirmed the build of ROS in mycelia upon

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depletion of cit- Ag NP in the growth medium (figure 5b). Transcript levels of aflatoxin genes

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(SI figure 5) also increased to those observed in the control.

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Environmental Implications

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Filamentous fungi are found ubiquitously throughout all ecosystems on the planet and

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interact with a wide-range of different organisms that include other microbes, plants, animals and

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humans; a excellent recent review from Braga et al

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interactions in establishment and maintenance of an environment. Fungal secondary metabolites

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play significant regulatory roles in shaping the community profiles, chemical interactions and

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ecological physiology of their ecosystems28. Hence, to fully understand the effects of effluent

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Ag NPs on the environment and human health, it is essential to understand their impacts on

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fungal secondary metabolism, especially when the NPs are present at concentrations that do not

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inhibit growth of the fungal cells. Our results clearly demonstrate the potential of NPs to

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interfere with the production of secondary metabolites that are enormously significant to

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environmental and public health.

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sheds light on the importance of such

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Our results also provide the first demonstration of the feasibility of Ag NPs to interfere in

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the biosynthesis of natural products in filamentous fungi without affecting their growth effect.

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We showed that mycelial uptake of cit- Ag NP, resulted in reduction in intracellular ROS that

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associated with a down-regulation of the transcriptional activation of secondary metabolism and

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aflatoxin biosynthesis. This establishes a novel research direction that can generate custom

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designed engineered nanoparticles, which can efficiently control contamination of crops with

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aflatoxins and other mycotoxins with low NP doses that pose minimal human hazard and risk to

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the environment. Our future studies will explore the molecular mechanisms through which

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nanoparticles modulate fungal cellular stress and syntheses of mycotoxins that are enormously

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relevant to the safety of environmental and public health.

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Figure 1. Characterization of synthesized cit- Ag NP . a) UV-Vis spectral profile of the cit- Ag

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NP stock solution. b) Transmission electron microscopy image of a representative cit- Ag NP

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sample (scale bar, 50 nm) c) Dynamic Light Scattering of a representative cit- Ag NP sample.

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Figure 2. Effect of cit- Ag NP on A. parasiticus growth and aflatoxin biosynthesis. a) Analysis

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of different doses of cit- Ag NP exposures on A. parasiticus growth. Effect of growth upon

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exposure to each dose was compared with the untreated set of samples. Statistical significance of

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two-tailed p-values were determined using an unpaired t-test, with n=3 and p < 0.05 as

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significance level. b) Effect on aflatoxin biosynthesis. A dose of 50 ng mL-1 cit- Ag NP was

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spiked into the growth medium during the start of growth. Aflatoxin B1 accumulation in the

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growth medium upon cit- Ag NP exposure was compared with untreated samples at 40h.

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Statistical significance of two-tailed p-values were determined using an unpaired t-test, with n=3

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and p < 0.05 as significance level. ‘*’ denote significance. c) Effect on growth rates. A dose of

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50 ng mL-1 cit- Ag NP was spiked into the growth medium during the start of growth. Dry

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weights were monitored during a time span of 40h to derive the growth rates (ng h-1) by

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quantifying the slope of the growth curves during the exponential growth phase. The growth

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rates were compared with the untreated samples. Statistical significance of two-tailed p-values

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were determined using an unpaired t-test, with n=3 and p < 0.05 as significance level. ‘*’ denote

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significance.

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Figure 3. NP behavior and uptake during fungal growth. a. Time-course UV-Vis spectral

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profiles of the growth medium. Growth medium spiked with 50ng mL-1 cit- Ag NP (N+M)

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spectral profiles and the growth medium spiked with cit- Ag NP containing the growing mycelia

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(N+M+F) were compared with the 50ng mL-1 cit- Ag NP stock solution (N) to conduct a

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qualitative analysis of the cit- Ag NP absorbance peaks under each condition. b. Reduction of

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total [Ag] in the growth medium. A dose of 50 ng mL-1 cit- Ag NP was spiked into the growth

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medium during the start of growth. A time-course inductively coupled plasma optical emission

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spectroscopy (ICP-OES) was performed until minimum detection limit (3 ng mL-1 denoted by

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the dotted line) was reached at 30h. These readings were compared with total silver levels at the

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corresponding time-points in the ‘growth medium only’ control flasks. Statistical significance of

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two-tailed p-values were determined using an unpaired t-test, with n=3 and p < 0.05 as

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significance level. ‘*’ denote significance. c. Increase in total silver in mycelia. Inductively

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coupled plasma optical emission spectroscopy (ICP-OES) was performed with spores (0h) and

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mycelia (at 24h and 30h). No detectable silver was found in untreated mycelia (not shown)

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Figure 4. Effect of cit- Ag NP on aflatoxin biosynthesis regulatory gene transcripts and total

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ROS generation. a. mRNA levels at 24h for aflatoxin pathway genes (aflR, nor-1, vbs, ver-1 and

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omtA) and the global secondary metabolism regulators (laeA and veA) upon cit- Ag NP uptake

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were compared with the untreated controls. Statistical significance of two-tailed p-values were

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determined using an unpaired t-test, with n=3 and p < 0.05 as significance level. ‘+’, mycelia

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treated with cit- Ag NP , ‘-’ , untreated samples, ‘*’ denote significance. b. Quantitative

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comparison of total ROS at 24h. Total ROS was quantified using a DCFH-DA based protocol as

ACS Paragon Plus Environment

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Environmental Science & Technology

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described in A. parasiticus 46. Statistical significance of two-tailed p-values were determined

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using an unpaired t-test, with n=3 and p < 0.05 as significance level. ‘+’, mycelia treated with

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cit- Ag NP , ‘-’ , untreated samples, ‘*’ denote significance.

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Figure 5. Aflatoxin synthesis and total ROS generation post cit- Ag NP uptake. a. i) Aflatoxin

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B1 accumulation in the growth medium at 48h in the treated flasks was compared with untreated

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samples and ii) comparison of the dry weights of the treated and untreated mycelia. Statistical

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significance of two-tailed p-values were determined using an unpaired t-test, with n=3 and p