Self-Assembly of Artificial Sweetener Aspartame ... - ACS Publications

Subscriber access provided by BUFFALO STATE

Article

Self-Assembly of Artificial Sweetener Aspartame Yields Amyloid-Like Cytotoxic Nanostructures Bibin Gnanadhason Anand, Kailash Prasad Prajapati, Kriti Dubey, Naseem Ahamad, Dolat Singh Shekhawat, Pramod Chandra Rath, George Kodimattam Joseph, and Karunakar Kar ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b02284 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Self-Assembly of Artificial Sweetener Aspartame Yields Amyloid-Like Cytotoxic Nanostructures Short title: Self-assembly of Aspartame into Nanostructures Bibin Gnanadhason Anand¶, Kailash Prasad Prajapati†, Kriti Dubey†, Naseem Ahamad†, Dolat Singh Shekhawat¶, Pramod Chandra Rath†, George Kodimattam Joseph¶, Karunakar Kar†*

Author affiliations:

†School

of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India

¶Department

of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur,

Jodhpur-342037, India *Corresponding

author: Karunakar Kar, Biophysical and Biomaterials Research Laboratory,

School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India phone: +91-1126704517; email: [email protected], [email protected]

1 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 45

ABSTRACT Recent reports have revealed the intrinsic propensity of single aromatic metabolites to undergo self-assembly and form nanostructures of amyloid nature. Hence, identifying whether aspartame, a universally consumed artificial sweetener, is inherently aggregation prone becomes an important area of investigation. Though the reports on aspartame-linked side effects describe a multitude of metabolic disorders, the mechanistic understanding of such destructive effects is largely mysterious. Since aromaticity, an aggregation promoting factor, is intrinsic to aspartame’s chemistry, it is important to know whether aspartame can undergo self-association and if such a property can predispose any cytotoxicity to biological systems. Our study finds that aspartame molecules, under mimicked physiological conditions, undergo a spontaneous selfassembly process yielding regular -sheet-rich cytotoxic nanofibrils of amyloid nature. The resultant aspartame fibrils were found triggering amyloid cross-seeding and becoming a toxic aggregation trap for globular proteins, A peptides and aromatic metabolites that converts native structures to β-sheet-rich fibrils. Aspartame fibrils were also found inducing hemolysis and causing DNA damage resulting in both apoptosis and necrosis mediated cell death. Specific spatial arrangement between aspartame molecules is predicted to form regular amyloid-like architecture with sticky exterior which is capable of promoting viable H-bonds, electrostatic interactions, and hydrophobic contacts with biomolecules leading to onset of protein aggregation and cell death. Results reveal that aspartame molecule is inherently amyloidogenic and the selfassembly of aspartame becomes a toxic trap for proteins and cells, exposing the bitter side of such a ubiquitously used artificial sweetener. Keywords: aspartame, artificial sweetener, amyloid-like nanostructures, cross-seeding, amyloid aggregates, cytotoxicity 2 ACS Paragon Plus Environment

Page 3 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Current evidences substantiate the propensity of single metabolites to undergo a self-assembly process that can yield toxic nanostructures of amyloid nature.1-4 Since the aggregation promoting factor aromaticity5 is inherent to aspartame (Figure 1a), identifying whether this universally consumed artificial sweetener is inherently aggregation-prone becomes an important area of inquiry. Aspartame is chemically recognized as a dipeptide consisting of aspartic acid and the methyl ester form of phenylalanine, and several reports based on in vivo studies have revealed diverse side effects of aspartame consumption (Table S1). The self-assembly of single aromatic metabolites such as phenylalanine and tyrosine has already been reported, and furthermore, it is observed that aromatic molecules, under physiological buffer conditions, can form amyloid structures that can effectively initiate amyloid cross-seeding resulting in cytotoxic fibrils made up of proteins and metabolites.1-4 Many studies have proposed direct links between neurophysiological symptoms and aspartame usage suggesting that aspartame may be responsible for adverse neurobehavioral health issues,6, 7 most of which are also seen in amyloid linked pathologies. Though a number of aspartame-linked adverse side effects7-11 have been reported (Table S1), the fundamental mechanism that mediates these complications is poorly understood. Furthermore, it is also unknown whether such problems arise from amyloid-linked consequences. It remains important to know whether aspartame is inherently amyloidogenic because recent reports have revealed the phenylalanine’s surprising ability to form hemolytic and cytotoxic amyloid fibrils which can effectively trigger amyloid formation in several globular proteins and metabolites.1, 2 Recently, our laboratory has revealed similar amyloid cross-seeding potential of tyrosine generated nanostructures under mimicked physiological conditions of buffer and temperature.4 Such research revelations on the ability of aromatic metabolites such as


ACS Nano

mM) at different pH values

30 30 0.2

15

15

0

-1

-1

-1

-1

45

0 30 0

0

Asp + Asp fibrils

100

60

CD Signal

Thioflavin T signal

2020

90

30

-1

-1

-1

60 60 90 90 120 120 150 150 30 Time (h) (h) Time (h) Time

CD spectra of aspartame solution (13.5 mM) before and CD spectra of aspartame aggregation 150 aftersolution (13.5 mM) before 150 and after aggregation 150 100 0h

c

3030

30

-1

-1

g g d

4040 Aggregation of aspartame Aggregation aspartame (33.9 mM) at of different pH(33.9 values

-1

-1

45

60

-1

[]millidegrees.cm2.dmol-1

ff

b

control control 5% seed (w/w) 5% seed (w/w) -1 10 seed (w/w) 1%mg.ml 10 % seed (w/w) 60 60 -1 seed 2%mg.ml 0.4 15 %15 seed (w/w)(w/w) -1 seed (w/w) 4%mg.ml 25 %25seed (w/w) 45 45 0.3

0 0.0 0 30 60 90 120 150 0 Time 30 60 (h) 90 120 150 Time (h) Time (h) Time (h)

ThT signal at 490 nm (A.U.) ThTsignal at 490 nm (A.U.)

Thioflavin Signal ThT signal atT490 nm (A.U.) (A.U.) ThTsignal 490 (A.U.) Thioflavin T450 Signal (A.U.) Absorbance at at nmnm (A.U)

a

0.0

-1

-1

Aspartame 1 mg.ml 3.2 mMAspartame 1 mg.ml 3.2 mMAspartame aggregation at Aspartame 2 mg.ml 2 mg.ml 6.4 mMAspartame 6.4 mM concentrations 60 Aspartame 4 mg.mldifferent 12.8 mM Aspartame 12.8 mM4 mg.ml Aspartame 5 mg.ml 5 mg.ml 16.9 mM Aspartame 16.9 mM Aspartame 10 mg.ml10 mg.ml 33.9 mM Aspartame 33.9 mM

50

2 -1 ]millidegrees.cm CD Signal .dmol

Aspartame (aspartylAspartame (aspartyl-phenylalaninephenylalanine-1Soluble Aggregated Aggregated Soluble 1-methyl ester) aspartame aspartame methyl ester) aspartame aspartame (13.5 mM) (13.5 mM)

0.1 0.1 0.1

ThTThioflavin signal atT490 nm(A.U.) (A.U.) signal

10 days

0.8 mMAspartame Aspartame 0.25mM mg.ml Aspartame aggregation at 0.8 0.25 mg.ml 75 Aspartame 0.5 mM mg.ml0.5 mg.ml 1.6 mMAspartame 1.6 different concentrations

CD Signal

0.2 0.2 0.2

10 days

75 75

ThTsignal atT 490 nm(A.U.) (A.U.) Thioflavin signal

Turbidity atat450 nm Absorbance 450 nm(A.U.) (A.U) Turbidity at 450 nm Absorbance at 450 nm(A.U.) (A.U)

0.3 0.3 0.3

ee

d d75

-1 3.3 mM-1 1 mg.ml 1 mg.ml 3.3 mM -1 6.6 mM-1 2 mg.ml 2 mg.ml 6.6 mM -1 -1 13.5 4 mg.ml 4mM mg.ml 13.5 mM

0.4 0.4 0.4

50

100 Soluble aspartame 0 h 50

0 0 0 30 1010 Aggregation of aspartame pHpH 5.05.0 Aggregation of aspartame -50 -50 Aspartame at different (33.9(33.9 mM) mM) at different -50 150hh pHpH 7.47.4 150 concentration fibrils seed seed concentration 0 0 0 0 0.0 0 200 220 240 260 200 220 240 260 3060 60 60 120 120150 0 90 20 9040 80 150 100 Soluble Aggregated 0 030 9090 90 120 150 150 0 0 30 30 60 60 12060 0 30 120 150 200 220Wavelength 240 (nm)(nm) 260 Wavelength Wavelength (nm) aspartame aspartame Time (h) Time (h) Wavelength (nm) Time (h) Time (h) Time (h) Time (h) Time (h) Wavelength (nm) Time (h)

15 15 0.1

e g h h Aspartame fibrils i ifAspartame fibrilsj j Aspartame oligomers Aspartame fibrils

Aspartame fibrils Aspartame oligomers

pH pHonly 3.03.0 Asp

fibrils khAspartame Aspartame fibrils k

3 m 3 m

0.6 m 0.6 m

Aspartame fibrils Aspartame oligomers Aspartame fibrils Aspartame oligomers

100 nm

100 nm

li

20 nm

50 nm

50 nm

20 nm

500 nm

1 m

500 nm

1 m

n m ThT-stained image of nk fibrils mjSEM aspartame aspartame fibrils

ThT-stained aspartame fibrils

SEM image of aspartame fibrils

2m

v

1

Intramolecular H-bonding Intramolecular H-boningleading leading to -sheet likelike arrangement to -sheet arrangement

Intermolecular H-bonding leading to -sheet like arrangement

37 Å

59 Å

2

3

4 H-bonds donor

H-bond donor

p

1

acceptor

p m

25 10

0

acceptor

10 m 150 kDa 250 100 150 75 100 25 75

2

Intensity intensity

37 Å

o lo

10 m

2m

Self assembly Self-assembly Of of aspartame aspartame

l

59 Å

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cc

bb

aa

Page 4 of 45

1

10

100

1000

RRh(nm) h (nm)

Figure1. 1. Self-assembly of aspartame into amyloid likeamyloid nanostructures under mimicked physiological conditions: a, Figure Self-assembly of aspartame into like nanostructures under mimicked Structure of aspartame (PubChem ID 134601). b, Visualization of soluble aspartame (at 13.5 mM, in PBS buffer at 37°C) of physiological conditions: a, Structure of aspartame (PubChem ID 134601). b, Visualization converting into insoluble aggregates after 10 days of incubation. c, Turbidity data (absorbance at 450 nm) of aspartame at different soluble aspartame (at 13.5 mM, in PBS buffer at 37°C) converting into insoluble aggregates after FINAL April12 concentrations, as labeled. d, Increase in Thioflavin T signal of aspartame sample at different concentrations (0.8mM to 33.9 mM) 10indicating days ofdose incubation. c, Turbidity data as(absorbance atdependent 450 nm) of10%, aspartame different dependent self-assembly process, labeled. e, Dose (5%, 15% and at 25% aspartame seeds w/w) concentrations, as labeled. d, Increase in ThT signal of aspartame sample at different seed-induced aggregation of aspartame (33.9 mM), as labeled. f, Aggregation of aspartame (33.9 mM) at different of pH values concentrations (0.8 mM to 33.9 self-assembly (pH 3, pH 5 and pH 7.4) at 37°C. g, CDmM) spectraindicating of aspartame dose sampledependent (13.5 mM) before (0 h) and after process, aggregationas (150 h), confirming formation of -structures. h, TEM images of aspartame higher order fibrils (from 13.5 mM sample after 150 h). Scale bar 100 nm. i, Magnified view of TEM image showed in h, indicating bundled-assembly of aspartame fibrils. Scale bar 20 nm.4 j, Morphology of spheroidal aspartame oligomers as revealed by TEM images. Scale bar 50 nm. k, AFM images reveal formation of mature fibrils and oligomers due to aspartame self-assembly (from 13.5 mM sample after 150 h), as labeled. l. Molecular ACS Paragon Plus Environment simulation study at 100 ns reveals viable intermolecular interactions during aspartame self-assembly: 1) representative snapshot reveals inter molecular H-bonds between aspartame molecules resembling a typical -sheet assembly; 2) tubular assembled higher

Page 5 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

labeled. e, Dose dependent (5%, 10%, 15% and 25% aspartame seeds, w/w) seed-induced aggregation of aspartame (33.9 mM), as labeled. f, Aggregation of aspartame (33.9 mM) at different of pH values (pH 3, pH 5 and pH 7.4) at 37°C. g, CD spectra of aspartame sample (13.5 mM) before (0 h) and after aggregation (150 h), confirming formation of -structures. h, TEM images of aspartame higher order fibrils (from 13.5 mM sample after 150 h). Scale bar 100 nm. i, Magnified view of TEM image showed in h, indicating bundled-assembly of aspartame fibrils. Scale bar 20 nm. j, Morphology of spheroidal aspartame oligomers as revealed by TEM images. Scale bar 50 nm. k, AFM images reveal formation of mature fibrils and oligomers due to aspartame self-assembly (from 13.5 mM sample after 150 h), as labeled. l. Molecular simulation study at 100 ns reveals viable intermolecular interactions during aspartame self-assembly: 1) representative snapshot reveals inter molecular H-bonds between aspartame molecules resembling a typical -sheet assembly; 2) tubular assembled higher order structures (showing dimensions, ~59 Å and ~37 Å, as labeled) with the packing of water molecules within its inner core; 3) distribution of H-bond donor and acceptor groups within the inner core of the aspartame simulated nanostructure; 4) space filling model of the structure revealing the presence of hydrophobic exterior. m, SEM image of aspartame-fibers (13.5 mM sample after 150 h). Scale bar, 2 m. n, ThT-stained fluorescence images of aspartame fibers (13.5 mM sample after 150 h). Scale bar, 10 m. o, DLS data showing the radius distribution (∼10 -100 nm) of aspartame sample (13.5 mM) at 5 min time point confirm the formation of higher order nanostructures in aspartame solution. p. Self-assembly process of aspartame (13.5 mM, at 0 h and 150 h time points) into higher order structures, as resolved by native PAGE, as labeled. All the experiments related to data shown here are repeated thrice.

phenylalanine and tyrosine molecules to self-assemble into amyloid like entities capable of initiating amyloid cross-seeding in proteins and other metabolites strongly validate the curiosity on the amyloidogenic nature of aspartame molecule. We have sought answers to this critical question by exploring whether aspartame can spontaneously undergo an amyloid-like selfassembly process, and probing what damaging effect such aggregation process would do to the proteins and cells. Here, using different biophysical and computational tools, we describe investigations of the selfassembly of aspartame into amyloid-like nanostructures. Amyloid cross-seeding potential of aspartame fibrils has been studied on Alzheimer’s-linked A1-40 peptide, selected globular proteins, and aromatic metabolites. The toxic effect of aspartame-generated amyloid-like nanofibrils on different cell types including erythrocytes, RAW264.7, SH-SY5Y and mouse bone 5 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 45

marrow cells (BMCs) have been examined. Bioinformatics and computational analysis have been carried out to understand the possible molecular interaction between aspartame nanostructures and the protein species.

RESULTS Self-assembly of aspartame under mimicked physiological conditions of buffer and temperature Aspartame, when incubated under mimicked physiological conditions,1, 4 underwent a concentration-dependent aggregation process, revealing the conversion of soluble aspartame molecules into self-assembled higher order structures (Figure 1b, 1c). Using ThioflavinT (ThT), an amyloid binding dye,12 we confirmed the formation of amyloid-like entities during selfassembly of aspartame (Figure 1d, 1n). The rate of aspartame aggregation was observed to increase with increasing concentration of aspartame in the sample (Figure 1d). To understand whether aggregation of aspartame follows a nucleation growth propagation mechanism, we carried out seed-induced aggregation of aspartame. Our data indicated faster aggregation kinetics of aspartame in the presence of preformed aspartame fibrils (used as seeds) in a dose-dependent manner (Figure 1e). Although we could not find any distinct lag phase either in the control or seed-induced aggregation reactions, the dose-dependent aggregation kinetics data suggest a nucleated growth mechanism for aspartame aggregation. More importantly, such data propose the possibility that aspartame fibrils might also be capable of cross-seeding that stimulate the formation of amyloid fibrils from other molecules, which would have implications for pathology. Next, we explored aggregation of aspartame in acidic conditions because pH values of different kinds of aspartame containing beverages vary between 2.6 to 5.5.13 The obtained data, as shown in Figure 1f, reveals aggregation propensity of aspartame at lower pH 6 ACS Paragon Plus Environment

Page 7 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

values. Circular dichroism (CD) spectroscopy data clearly indicated the formation of fibrils rich in -sheet-like structures after the molecular self-assembly of aspartame (Figure 1g). The ellipticity signal obtained from soluble aspartame before aggregation showed a positive peak at ∼223 nm which is possibly arising from the π−π* transition in the benzene group of phenylalanine component of aspartame.14-16 However, the positive peak at ∼223 nm, obtained from soluble aspartame, changed to a negative peak near ∼218 nm, suggesting the characteristic of β-structures. Transmission electron microscopy (TEM) clearly showed ordered fibrillar morphology (Figure1h, 1i) which resembled bundled assembly of fibrils.17 TEM also detected the presence of spheroidal oligomers (Figure 1j) during aspartame fibril assembly. To further validate the structural properties of aspartame fibrils, we used atomic force microscopy (AFM), scanning electron microscopy (SEM), and fluorescence microscopy tools, which also revealed the formation of regular fibrillar assemblies (Figures 1k, 1m, 1n). The presence of spheroidal oligomers, as detected by TEM, was also observed in our AFM images (Figure 1k). Dynamic light scattering (DLS) data detected that aspartame molecules in solution readily undergo selfassembly within 5 min, resulting in ~10 to ~100 nm size assemblies4 which eventually propagate further, saturating the DLS signal (Figure 1o, S2). Furthermore, native PAGE data displayed characteristic bands indicating the presence of both low and high molecular weight structures1, 4, 18

during the self-assembly of aspartame (Figure 1p). Amyloid nature of the aspartame

aggregates was further validated from the data obtained from Congo red (CR) assay19 (as shown in Figure S36) which showed typical amyloid-specific peak at 541 nm (inset in Figure S36).4, 20


ACS Nano

100 nm

e

60 0

Time (h)

20 0

0

30

bsa

insulin

cyt c

lysozyme

myoglobin

h

15 0

phenylalanine (0.1 mM) kDa

-15

60 90 120 150 180 220 Time (h)200

00 00 240

220

240

i

Phe control 0.1 mM Phe 0.1 mM Phe Phe +Phe 5% seed (w/w) 0.1 mM Phe+aspartame +5 5%%aspartame aspartame seed (w/w) 0.1 mM seed (w/w) Phe + Phe 10% aspartame seed (w/w) 0.1 mM aspartame seed (w/w) 0.1 mM Phe++10 10%% aspartame seed (w/w) Phe +Phe 25% aspartame seed (w/w) 0.1 mM aspartame seed (w/w) 0.1 mM Phe++15 15%% aspartame seed (w/w) 0.1 mM ++25 aspartame seed (w/w) Phe +Phe 15% aspartame seed (w/w) 0.1 mM Phe 25%% aspartame seed (w/w)

1010

protein mixture

10 m

250 lysozyme fibrils Cyt30 c30 fibrils Myoglobin fibrils Coaagretes of mixed proteins 2020 Coaagretes of mixed amino acids

0

200

060 90 120 150 180 0 20 (nm) 40 60 wavelength

f

Aspartame-seed induced aggregation of phenylalanine

10

30 60 60 90 30 90 120 120 150 150 180 180 260 Time (h) Time (h)

mixed proteins [lys+ins+bsa+myo+cyt c] fibrils mixed amino acids [Phe+Tyr+Trp] fibrils

-15

30

Time (h) Morphology of aspartame-seeded protein fibrils (after 72 h )

Aspartame-seeded aggregation of Aspartame-seed induced aggregation of phenylalanine

h

BSA fibrils Insulin fibrils BSA fibrils lysozyme fibrils Insulin fibrils Cyt c fibrils lysozymefibrils fibrils Myoglobin cytochrome c fibrils mixed protein [lys+ins+bsa+myo+cyt c] fibrils myoglobin fibrils[phe+tyr+trp] fibrils mixed aminoacids

15

0

40

0

Wave lengthprotein (nm) CD spectra of aspartame-seeded aggregation revealing formation of -structures 30

0

80

60

2

40

50

Asp fibrils + proteins protein monomers Asp + Mixed Amino acids Asp fibrils + amino acids mixed amino acids

120

120

Protein mixture + 10 % aspatame seed (w/w) 30% aspatame seed (w/w) Protein mixture + 15 Protein mixture + 25 % aspatame seed (w/w)

60

f

Protein mixure [lys+ins+BSA+myo+cyt c], 2M each Amino acid mixture + 15% aspartame seed (w/w) mM each Amino acid mixture [Tyr+Phe+Trp],0.033 0.1mM each 160 Asp aspartame + Proteinsseed (w/w) Protein mixure + 15%

ThT signal after 72 h time point

180

45 BSA 40fibrils Protein mixture [lys+bsa+ins+myo+cyt c], 2M each 40fibrils Insulin Protein mixture + 5 % aspatame seed (w/w)

80

100 nm

ThT signal after 72 h time point

ThT Signal at 490 nm (A.U.)

120

100 nm

ThT Signal at 490 nm (A.U.)

ThT signal at 490 nm (A.U.)

100 nm

20 40 60 80 100 120

180

g 100

j

Time 0(h) 0

[] millidegrees.cm dmol

g


e

25 M Aβ1-40

20 40 60 80 100 120

Thioflavin T Signal (A.U)

0

20

d

100


0

Aβ1-40 fibril showing typical twist


Thioflavin T Signal (A.U.)

20

Aβ1-40 fibrils

c Aspartame-seeded c d

40


40

b Aspartame-seeded b

-1

Thioflavin T Signal (A.U.)

25 M Aβ1-40 + 15 % aspartame seed


a


a

2dmol 2 -1 -1 [] []millidegrees.cm millidegrees.cm dmol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 45

k kDa 250 150 100 75 25

Aspartame-seeded aggregation of tyrosine (0.1 mM) 40

Aspartame seed induced Tyrosine aggregation

30 20 Tyr 0.1 control mM Tyr Tyr + 5% seed (w/w) 0.1 mM Tyraspartame + 5% aspartame seed (w/w) Tyr 10% seed (w/w) 0.1 + mM Tyr +aspartame 10% aspartame seed (w/w) Tyr + 15% seed (w/w) 0.1 mM Tyr +aspartame 15% aspartame seed (w/w) Tyr + 25% seed (w/w) 0.1 mM Tyr +aspartame 25% aspartame seed (w/w)

10 0 0

30

60 90 120 150 180 Time (h) Mature aggregates Higher order oligomers

10

260

wavelength (nm)

Figure 2. Aspartame-induced amyloid cross-seeding in A peptide, different proteins and FINAL April11 amino acids under mimicked physiological conditions (in PBS at pH 7.4 and 37°C): a, Aggressive aggregation of A1-40 peptide (25 M) in the presence of aspartame fibrils (~15%, w/w), as evident from the rapid rise in the ThT signal. b, TEM image of A1-40 fibrils generated in aspartame-seeded aggregation (at 120 h time point). c, TEM image revealing formation typical twisted amyloid fibrils (as pointed by arrows) of A1-40 (at 120 h). d, ThT data revealing the aspartame-seeded (~15% w/w) aggregation of mixed protein monomers [lysozyme + insulin + BSA + myoglobin + cytochrome c, at 2 M each] and mixed amino acids [tyrosine + tryptophan 8 ACS Paragon Plus Environment

Page 9 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

+ phenylalanine, at 0.033 mM each], as labelled. e. Histograms representing the ThT reading of individual protein samples after 72 h confirming amyloid formation stimulated by both soluble aspartame and aggregated aspartame, as labelled. f, ThT stained fluorescence microscopy images of different protein fibrils obtained from aspartame-seeded aggregation after 72 h, as labelled. g, Aggregation of mixed protein monomers [lysozyme + insulin + BSA + myoglobin + cytochrome c, at 2 M each] in the presence of different seed concentrations (5%-25%, w/w) of aspartame fibrils, as labeled. h. Dose dependent aspartame-seeded aggregation of phenylalanine (0.1 mM), as labeled. i. Dose dependent aspartame-seeded aggregation of tyrosine (0.1 mM), as labeled. j, CD spectra of aspartame-seeded protein aggregation showing conversion of native protein structures into β-structured fibrils. k, Native PAGE data revealing aspartame-seeded (~15% w/w) aggregation (after 72 h) of different protein samples [70 M lysozyme, 15 M BSA, 57 M myoglobin, 174 M insulin, 82 M cytochrome and mixed monomer containing 2M of each protein] and mixed amino acids [tyrosine + tryptophan + phenylalanine, at 0.033 mM each], as labeled. All the experiments related to data shown here are repeated thrice.

Molecular interaction within aspartame-generated nanostructures Inspired by the above mentioned data, which confirmed aspartame’s intrinsic ability to form selfassembled nanostructures of amyloid nature, we executed in silico tools to understand how aspartame molecules synergistically engage in promoting a self-assembly process. Packed aspartame molecules were simulated in a cubic box model4 at 37°C by using Discovery Studio 4. The data obtained at 100 ns revealed viable intermolecular interactions between aspartame molecules resulting in well-organized nanospheroidal entities (Figure 1l, S3-S5) made up of relatively hydrophilic interior21 and a hydrophobic exterior with exposed aromatic moieties.4, 22 Aspartame self-assembly was facilitated by viable hydrogen bonds and  interactions (Figures S4, S5). Aspartame fibrils induce amyloid cross-seeding inA peptide, globular proteins and aromatic metabolites Since we found that not only can aspartame self-assemble into amyloid-like fibrils, but also these fibrils exhibit the ability to seed aspartame monomer preparations to give rapid fibril growth, we inquired whether it might also be possible for aspartame fibrils to cross-seed the aggregation of other proteins and peptides 23, 24-a phenomenon of much current interest in research into the 9 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 45

pathobiology of amyloid-associated neurodegenerative diseases. The surprising seeding ability of aspartame fibrils (Figure 1e) prompted us to assess the biological significance of aspartame in inducing amyloid-linked neurodegenerative diseases such as Alzheimer’s. We chose A1-4024, 25 and incubated it with aspartame fibrils (maintaining seed concentration at ~15% w/w). To our surprise, the aspartame fibrils triggered an aggressive aggregation of A1-40 peptide, as evident from the rise in ThT signal, without a lag phase (Figure 2a). Since the lag time of the control aggregation reaction for A1-40 peptide (at the studied concentration) was observed to be ~ 24 h, 24, 26

it is predicted that the specific orientation of the aspartame molecules in the fibrillar entities

can effectively recruit A1-40 monomers and bring the necessary conformational changes to initiate A1-40 peptide’s -sheet assembly process, possibly overcoming the prolonged nucleation event. Morphology of the resultant A1-40 fibrils from the aspartame-induced aggregation process showed typical characteristic structures as seen for A-amyloid fibrils17, 24, 25, 27 ( Figures 2b, 2c). Docking of aspartame with the available monomeric structure of A1-40 (PDB ID:1BA4)28 also predicted aspartame’s strong affinity (Figure S14) for residues spanning from 15-22 position which falls within the aggregation prone region of A1-40.29 The 1BA4 is a micelle associated structure which signifies the presence of hydrophobic interaction. Since the simulated nanostructure of aspartame (Figure 1l) was found to have hydrophobic exterior and hydrophilic interior, 1BA4 becomes an ideal model to understand the interaction between A and aspartame nanostructure, particularly to know if surface hydrophobicity of the nanostructure is favoring its interaction with A. We extended this study to examine whether aspartame fibrils can trigger amyloid cross-seeding in selected globular proteins and aromatic amino acids. We incubated samples containing mixed


Page 11 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

monomers of different globular proteins (lysozyme + bovine serum albumin + insulin + myoglobin + cytochrome c) both in the presence and in the absence of preformed aspartame fibrils (used as seeds) under mimicked physiological conditions (at pH 7.4, T=37°C) in phosphate buffer saline (PBS). The mixed protein monomer sample underwent a rapid aggregation process in the presence of preformed aspartame fibrils (~15% w/w), as clearly evident from the sharp increase in the ThT signal, indicating the conversion of protein monomers into amyloid structures (Figure 2d). For a further validation of the cross-seeding ability of aspartame fibrils, we inspected its effect on individual protein samples and the results (Figures 2e, S6 and S7) indicated substantial rise in the ThT signals, confirming the amyloid formation of individual proteins. ThT stained fluorescence images (Figure 2f) of the resultant fibrils have also confirmed the amyloid nature of protein fibrils. Since aspartame solution was found forming nanostructures once the molecule is dissolved in the solvent (as detected by both DLS and supported by simulation data, Figures 1i, 1l), we have checked whether such initial aspartame aggregates in the solution can trigger aggregation of other proteins. Addition of an aliquot of freshly prepared aspartame solution surprisingly initiated an amyloid aggregation process (Figure 2e, S7) in the sample containing protein monomers. It is important to notice that soluble aspartame also undergoes self-assembly (Figure S7) indicating rise in the ThT signal. It is possible that the on-pathway higher order structures, formed during aspartame self-assembly, are capable of inducing amyloid cross-seeding in globular proteins within the time frame of this study (Figure 2e, S7). Next, we explored the ability of aspartame fibrils to induce aggregation of selected aromatic amino acids (Trp, Phe and Tyr). ThT data as indicated in Figure 2d clearly


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 45

b

a a

c b

Insulin Insulinand and Aspartameaspartame nanostructure nanostructure

dc

ed

Myoglobin Myoglobinand and Aspartameaspartame nanostructure nanostructure

g

e B:HIS5

HIS5 of insulin chain B

A:CYS7

A1-40 and aspartame nanostructure

lysozyme and Lysozyme and Aspartameaspartame nanostructure nanostructure

f

BSA and aspartame nanostructure

Cytochrome c and Cyt C and Aspartamenanostructure aspartame nanostructure

HIS5 of insulin chain B, after docking with aspartame B:HIS5 nanostructure

Aspartame

A:ILE10 A:CYS7 A:ILE10

h

Aspartame TYR14 of insulin chain C, after docking with aspartame nanostructure

TYR14 of insulin chain C

C:SER12 C:TYR14

Aspartame

Aspartame

Aspartame

C:TYR14 C:SER12

Figure 3. Rigid body Z-Dock data between aspartame nanostructure and native conformation of globular proteins: a. Aspartame nanostructure and insulin (PDB ID: 1TRZ); b. Aspartame nanostructure and lysozyme (PDB ID:193L); c. Aspartame nanostructure and A140 (PDB ID: 1BA4). d. Aspartame nanostructure and myoglobin (PDB ID:1DWR). e. Aspartame nanostructure and cytochrome c (PDB ID: 1HRC). f. Aspartame nanostructure with BSA (PDB ID: 4F5S). Detailed lists of the interacting residues are given in the supplementary information 12 ACS Paragon Plus Environment

Page 13 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(Figures S19-S24). g. Analysis of His5 residue of insulin (chain B) before and after its interaction with the aspartame nanostructure reveals formation of two additional H-bonds after the complex formation. h. Analysis of Tyr14 residue of insulin (Chain C) and its interaction with aspartame nanostructure. Data reveal Tyr14 interact via both H-bond and hydrophobic interactions; however, Tyr14 seems to promote more hydrophobic contacts in the docked complex and also it favors  interactions. Furthermore, Tyr14 residue falls in the unstructured region of insulin sequence. Detailed list of all the interactions (shown in panel g, and h) between insulin and aspartame nanostructure is given in the supplementary information (Figure S25).

show amyloid aggregation of the sample containing mixture of amino acids (Trp + Tyr + Phe). We also detected dose-dependent seeding efficacy of aspartame fibrils in triggering aggregation of mixed amino acid sample (Figure S8). Furthermore, we observed aggregation of individual amino acids (Tyr and Phe) in the presence of different concentrations of aspartame seeds (Figure 2h and 2i). Our data suggest that the initial higher order structures of aspartame, while growing into mature fibrils, can effectively cross-seed proteins and aromatic amino acids (Figures 2d, S8). Our control reactions in the absence of aspartame fibrils involving both isolated monomers and mixed monomers of protein samples did not show any indication of substantial aggregation (Figures 2d, 2e, S6-S8). CD data confirmed that the native protein structures are converted into -sheet-rich assemblies during aspartame driven aggregation process (Figure 2j). Our native PAGE data further revealed the formation of intermediate higher order structures during protein aggregation in the presence of aspartame (Figure 2k).

Docking of aspartame nanostructure with native structures of proteins To understand the possible interactions between aspartame generated amyloid-like nanostructures with the native structures of protein species, we carried out docking studies4 using Z-DOCK 3.0 server.30 We examined whether aspartame generated nanostructures, as shown in


ACS Nano

Aspartame fibrils + RBC Aspartame fibrils + RBC

iii

iv

Control Control + soluble aspartame

i

e

f

ii

Control Control + soluble aspartame

i

ii

Soluble aspartame

80 80 60 60 40 40

20 20 00

d

3 m

3 m

c

Aggregated aspartame

percent hemolyisis

3 m

ii

b

Control + aspartame fibrils

iii Control + aspartame fibrils

iii

kDa

100

Cell Viability (%) after 24 h

3 m

iiii

60

40

20

0

Soluble aspartame

50

0

100

Cell Viability (%) after 24 h

ii

Soluble aspartame + RBC

percent hemolyisis

Control RBC

Percentage hemolysis (%) after 24 h

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

Page 14 of 45

Aggregated aspartame

50

0

 and  Spectrin Anion transporter Ankyrin Anion exchanger Anion exchanger Glycophorin A Actin DG3P Stomatin

250 150 100 75 50 37 25

Flotillin Band 8

20

 Globulin

15

Figure 4. Cytotoxicity of aspartame and aspartame generated fibrils: a, SEM images revealing aspartame-induced lysis of RBCs after 24 h: (i) PBS as control; (ii) soluble aspartame (17.5 mM), (iii) aspartame fibrils (17.5 mM) and (iv) lysed RBCs sticking to surface of aspartame fibrils (white arrows). Scale bar 3 m. b, Histograms showing the percentage hemolysis at 24 h in the presence of different concentrations (3.5 mM, 17.5 mM and 28 mM) of aspartame solution, as labelled. c, Optical microscopy images showing the morphology of

FINAL April12


Page 15 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

control (PBS) and aspartame (at 3.6 mM, 24 h) treated RAW264.7 cells: (i) control cells; (ii) cells treated with soluble aspartame; (iii) cells treated with aspartame fibrils. d, MTT assay showing dose dependent cell death of RAW264.7 cells after 24 h in the presence of both soluble aspartame and aspartame fibrils, as labelled. e, Fluorescent micrographs of acridine orange and ethidium bromide double stained RAW264.7 cells indicating apoptosis and necrosis in the presence of aspartame at 3.6 mM (at 24 h): (i) control cells; (ii) cells treated with soluble aspartame; (iii) cells treated with aspartame fibrils. g, Silver stained SDS-PAGE confirms the characteristic bands of membrane proteins released from lysed erythrocytes, as labelled. Deionized water was used as positive control and PBS solution was used as negative control. All the experiments related to data shown here are repeated thrice.

Figure 1l and Figures S3-S5, can preferentially interact with protein species in their native state. The collected data (Figure 3, Figure S19-S24) revealed that aspartame nanostructures can efficiently make non-covalent contacts with the native structures of the proteins. Examination of the docked complex (between aspartame nanostructure and proteins; as shown in Figure 3, Figure S19-S24) predicts that both hydrophobic groups, H-bond promoting moieties and presence of charged side chains31 seem to be crucial for the aspartame nanostructure to recruit proteins in native conformation leading to aggregation.4, 24 Furthermore, to understand the role of amino acids during intermolecular interaction, we have examined selected residues of insulin which make viable contacts with aspartame molecules in its nanostructure. His5 residue of insulin (Chain B) facilitates gaining of two additional H-bonds after docking (Figures 3g, S21, S25a). Similarly, Tyr14 residue of insulin (chain C), a surface located aromatic moiety, was found to interact with aspartame maximally mediated by H-bonds, hydrophobic contacts and - interactions (Figure 3h, Figure S21 and S25b). Intrinsically disordered regions of the protein species have been known to be more flexible and this property becomes one of the important factors that favors protein-protein interaction.32 Interestingly, these interacting residues of insulin (His5 and Tyr14) exist in the unstructured stretches of the protein chain and such an observation suggests that flexibility of the interacting domain of the protein may promote the cross-seeding 15 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 45

potential of aspartame nanostructures. We, additionally, have examined the selected interacting residues of different proteins with aspartame nanostructure before and after docking and the obtained results are given in supplementary Figures S26-S29. Similar docking data on tyrosine generated nanostructure with native protein structures have been recently reported from our group.4 Aspartame generated fibrils cause lysis of red blood cells (RBCs) Having found such a striking amyloid forming propensity of aspartame and the cross seeding ability of aspartame fibrils, we next attempted to unravel whether aspartame is toxic to normal physiology of cells. We carried out hemolysis assays1, 33 on human erythrocytes under physiological conditions. Strong lysis of isolated RBCs was noticed in the presence of aspartame solution (Figures 4a, 4b, S15 and S16). After 24 h, ~60 % lysis was observed in the presence of highest dose of aspartame (28 mM; Figures 4b, S15). Likewise, we observed similar lysis effect when preformed aspartame-fibrils were added to RBC samples (Figures 4b). SEM (Figure 4a) and optical microscopy images (Figure S16) revealed deformed erythrocytes, confirming the aspartame’s ability to damage RBCs. Next, we performed SDS-PAGE of the aspartame treated RBC samples which revealed the release of various surface proteins34 predicted to be released from damaged RBCs, as evident from the characteristic protein bands1, 34-37 (Figure 4f). Normal RBCs display a typical biconcave discoidal shape;38 however, different hemolytic conditions can induce RBC-deformability leading to several clinical complications such as hemolysis39 and hypoxia.40 Subsequently, we examined whether the protein aggregates formed due to aspartameseeded reaction can also trigger lysis of RBCs. The results as shown in supplementary Figure S35 clearly reveal that the resultant aggregates (both from individual proteins and mixed protein sample) are capable of causing hemolysis. Earlier studies have already reported the formation of 16 ACS Paragon Plus Environment

Page 17 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

similar hemolytic protein aggregates generated in a phenylalanine-seeded amyloid cross-seeding reaction.1 Toxic effect of aspartame fibrils on RAW264.7 cells Driven by this result on the toxic effect of aspartame on normal erythrocytes, we were curious whether aspartame is toxic to other cell types as well. RAW264.7 cells are macrophage-like cells which generally show inflammatory response to any external toxic agent. Since aspartame is a chemically synthesized artificial sweetener, that too capable to form toxic nanostructures of amyloid nature, we believed it important to study what is the effect of aspartame aggregates on macrophages. Microscopic images of damaged RAW264.7 cells were detected, suggesting cytotoxic nature of aspartame and preformed aspartame fibrils (Figure 4c). Next, we performed MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on RAW264.7 cell line41 and the results revealed dose-dependent cell death in the presence of both soluble aspartame and preformed aspartame fibrils as well (Figure 4d). Further, our cellular experiments, as evident from Figure 4e, confirmed the apoptosis mediated cell death in the presence of aspartame. Fluorescent micrographs obtained from acridine orange and ethidium bromide double stained RAW264.7 cells (Figure 4e) revealed that aspartame treated cells display both apoptotic (bright green nucleus with condensed or fragmented chromatin)42 and necrotic (uniformly orange-stained nuclei) cells.43 The control untreated RAW264.7 cells remained unaffected (normal green nucleus). It is important to notice that both soluble and aggregated forms of aspartame were able to cause cell damage. We predict that the observed toxicity in the presence of soluble aspartame may be due to the formation of aspartame aggregates during its incubation (24 h).


ACS Nano

a

PBS

5 mM

10 mM

DIC

Control

20 m

20 m

20 m

Magnified View

20 m

b Control

DIC

DAPI

CR Fluorescence

20 m

MERGE

MERGE

20 m

20 m

PBS

20 m

20 m 200

400 600 FSC-H

800

1000 0

200 0

5 mM

0

20 m

20 m

20 m

0 400 200 600 400 800 600 1000800 FSC-H FSC-H

200 0 400 200 600 4000 800 600 200 6001000 4000800 600 1000 0400 1000 200 1000 0800 0 400200 8000 4001000 1000 2001000 800 6000 400800 200 400 200 600 800 600400 1000 0800600 200 1000 20060 FSC-H FSC-H FSC-H FSC-H FSC-H FSC-H FSC-H FSC-H FSC-H

Region Statistics

10 mM

20 m 0

200

c

400 600 FL2-W

800

1000 0

20 m

20 m

Region Events % Gated R1 15894 31.79 R2 28435 56.87 R3 1751 3.50 20 m 20 m R4 2762 5.52 R5 1241 2.48 200 0 400 200 600 400 800 600 1000 800 0 1000 R6 50000 100.00 FL2-W FSC-H R7 48932 97.86

0 400 200 600 400 800 600 1000800 FL2-W FSC-H

200 0

PBS

Control SUB G1 phase cells

G1 phase cells S phase cells G2/M phase cells 0

200

400

600 FL2-A

800

File: ctrl.001 Sample ID: 20 m Tube: Untitled Acquisition Date: 04-May-18 Gated Events: 50000 X Parameter: FL2-W (Linear)

20 m

20 m

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 45

5 mM

Region Statistics

Log Data Units:File: Linear psc.002 Values Patient ID: Sample ID: Panel: Untitled Acquisition Tube: Untitled Tube List Gate: No Gate Acquisition Date: 04-May-18 Total Events: 50000 Gated Events: 50000 Y Parameter: FL2-A X Parameter: (Linear) FL2-W (Linear)

5mm 24h asp.003 Log Data Units:File: Linear Values Sample ID: Patient ID: Untitled Panel: Untitled Tube: Acquisition Tube List Gate: No Gate Acquisition Date: 04-May-18 Gated Events: 50000 Total Events: 50000 X Parameter: Y Parameter: FL2-A (Linear) FL2-W (Linear)

Gated % To X Mean X GeoRegion Mean YEvents Mean Y%Geo Gated Mean% Px,Py Total X Mean X GeoRegion Mean YEvents Mean Y%Geo Mean Px R1 22.60 16560 33.12 5.67 1.31 R1 15.28 13260 26.52 3.63 26.52 6, 0 7.67 1.49 4.02 33. R2 202.39 25882 51.76 98.03 94.52 R2 198.72 25295 50.59 198.41 50.59 6, 0 107.30 99.83 202.08 51. R3 296.63 2266 4.53 176.99 103.32 R3 293.99 2575 291.58 5.15 5.15 6, 0 202.35 109.03 294.18 4. R4 399.54 2901 5.80 242.13 198.23 R4 393.62 5072 10.14 393.17 10.14 6, 0 291.29 233.96 399.07 5. R5 658.53 2508 5.02 268.76 178.63 R5 636.10 3923 616.20 7.85 7.85 6, 0 319.35 169.90 635.71 5. 10000 400 200 600 400 800 6001000 800 200 400 600 800 1000 R6 50000 100.00 83.56 25.77 R6 165.25 50000 100.00 60.19 100.00 6, 0 120.91 37.41 215.13 85.41100. FSC-H FL2-W FSC-H R7 180.45 47991 95.98 97.86 79.45 24.70 R7 154.33 46595 93.19 57.15 93.19 6, 0 106.24 33.58 73.48 95.

% Total 31.79 56.87 3.50 5.52 2.48 200 0 1000 100.00

10 mM

Histogram Statistics

Histogram Statistics

File: ctrl.001 Log Data Units: File: Linear psc.002 Values Log Data Units: File: Linear Values 5mm 24h asp.003 Sample ID: Patient ID: Sample ID: Patient ID: Sample ID: SUB G1 phase cells SUB G1 phase cells SUB G1 phase cells Tube: Untitled Panel: Untitled Acquisition Tube: Untitled Tube List Panel: Untitled Acquisition Tube List Tube: Untitled Acquisition Date: 04-May-18 Gate: G7 Acquisition Date: 04-May-18 Gate: G7 Acquisition Date: 04-May-18 Gated Events: 48979 Total Events: 50000 Gated Events: 46649 Total Events: 50000 Gated Events: 48046 X Parameter: FL2-A (Linear) X Parameter: FL2-A (Linear) X Parameter: FL2-A (Linear) G1 phase cells G1 phase cells G1 phase cells Marker Left, Right Events % Gated % Total Marker Mean Geo Left,Mean Right SD Events CV % Gated Median % Total Peak Mean PeakGeo Ch Marker Left,Mean Right S phase cells S phase cells 97.86 154.33All S phase cells All 0, 1023 48932 100.00 57.15 0, 1023110.09 46595 71.33 100.00193.00 93.19 7340 180.45All 0 0, 73.48 1023 1 SUB G1 phase cells 0, 121 15362 31.39 SUB 30.72 G1 phase 10.88 cells 0, 3.20121 22.19 12409203.93 26.63 SUB 1.00 24.82 734014.38 G1 phase cells 0 0,3.15121 G1 phase cells 121, 240 28968 59.20 57.94 G1 phase 197.66 cells 121, 197.16 240 13.54 26136 6.85 56.09 198.00 52.27 1259 200.43 197 199.75 G1 phase cells 121, 240 G2/M phase cells G2/M phase cells G2/M phase cells S phase cells 240, 360 1774 3.63 3.55 S phase 294.84 cells 240, 292.38360 38.27 2598 12.985.58290.00 5.20 40 297.19 294.70 S phase cells 240 240, 360 G2/M phase cells 360, 481 2908 5.94 G2/M 5.82 phase 397.34 cells 360, 396.69481 23.13 5539 5.82 11.89394.00 11.08phase 76 404.53 403.83 G2/M cells 387 360, 481 1000 1000 200 400 600 800 1000 10000 1000 0 200 0 400 200 600 400 800 600 1000800 0 200 0 400 200 600 400 800 6001000 800 0 200 0 400 200 600 400 800 600 1000 800 FSC-H FSC-H FSC-H FSC-H FL2-A FL2-A FL2-A

Propidium Iodide

Figure 5. Cytotoxic effect of aspartame fibrils on mouse bone marrow cells (BMCs): a. Gate Statistics Gate Statistics Differential interference contrast (DIC) microscopic visualization of50000 bone marrowALLcells treated ALL EVENT 50000 ALL EVENT ALL EVENT 50000 EVENT 50000 R7 R7 File: ctrl.001 Linear psc.002 Values Log Data Units:File: Linear Values R7 Log Data Units:File: R7 5mm 24h asp.003 with different doses of aspartame fibrils (5 mM and 10 mM) at Lower panels Sample ID:24 h, as labeled. Patient ID: Sample ID: Patient ID: Sample ID: R6 R6 R6 R6 R1

R3 R2

0

200

R1

R5

R2

R4

400

600 FL2-A

R3

800

1000 0

200 0

R5

R1

R3

Tube: Untitled Acquisition Date: 04-May-18 Gated Events: 50000 X Parameter: FL2-W (Linear) R1

R5 Gate Events % Gated G1 15894 31.79 R2 R4 G2 28435 56.87 G3 1751 3.50 G4 2762 5.52 G5 1241 2.48 1000 0 200 0 400 200 600 400 800 600 1000 800 G6FSC-H 50000 100.00 FL2-A

ACS Paragon Plus Environment R4

400 200 600 400 800 600 1000800 0 FL2-A FSC-H

Panel: Untitled Acquisition Tube: Untitled Tube List Gate: No Gate Acquisition Date: 04-May-18 Total Events: 50000 Gated Events: 50000 Y Parameter: FL2-A X Parameter: (Linear) FL2-W (Linear) R3 R5 % Total Gate Events % Gated % Total 31.79 G1 13260 26.52 26.52 R2 R4 56.87 G2 25295 50.59 50.59 3.50 G3 2575 5.15 5.15 5.52 G4 5072 10.14 10.14 2.48 G5 3923 7.85 7.85 1000 200 0 400 200 600 400 800 6001000 800 10000 100.00 G6FSC-H 50000 100.00 100.00 FL2-A

Gate S

Panel: Untitled Tube: Acquisition Tube List Untitled Gate: No Gate Acquisition Date: 04-May-18 Total Events: 50000 Gated Events: 50000 Y Parameter: FL2-A (Linear) FL2-W (Linear) X Parameter:

18

200

Gate Events % Gated % Tota G1 16560 33.12 33.12 G2 25882 51.76 51.76 G3 2266 4.53 4.53 G4 2901 5.80 5.80 G5 2508 5.02 5.02 400 600 800 1000 G6 50000 100.00 100.00 FSC-H

Page 19 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

show the magnified view of the region marked by rectangles in the images of upper panel confirming the presence of deformed cells. Scale bar, 20 μm. b. Congo red (CR) stained BMCs treated with aspartame fibrils (5 mM and 10 mM) as observed for 24 h, as labeled. The magnified view of the region marked by rectangles in merged (DAPI + Congo red) images is shown on far right column which reveals presence of amyloid aggregates in the cytoplasm. c. Cell cycle analysis (24 h, after treatment) by FACS of bone marrow cells under control, PBS treated and aspartame fibril treated (5 mM and 10 mM) conditions shows respective histograms revealing Sub-G1, G1, S and G2/M phases in the cells under study. All microscopic images were captured at x60 magnification. All the experiments related to data shown here were repeated thrice. Effect of aspartame fibrils on bone marrow cells (BMCs) Further, we extended the study to explore possible cytotoxic effect of aspartame fibrils on bone marrow cells. Bone marrow cells were isolated from the central cavity of femora and tibia (from 7-8 weeks old C57BL/J6 mice) and this sample is known to contain diverse types of cells including monocytes, macrophages, endothelial cells, fibroblasts, adipocytes, and stem cells.44, 45 Using the trypan blue assay46 and microscopic tools, we confirmed the yield and viability of the bone marrow cells used for this study. We examined both the morphology of aspartame treated cells and the appearance of cellular aggregates after aspartame treatment (24 h). Both control cells and cells in PBS buffer showed spherical or round shaped morphology (Figure 5a). However, cells treated with aspartame fibrils showed altered shape and size47 (Figures 5a, S17). Next, we performed Congo red (CR) staining to detect the amyloid aggregates in aspartame treated bone marrow cells. Congo red dye is known for its interaction with amyloid structures and such interaction results in the appearance of apple green signal under polarized light.48-50 We could detect the presence of cellular aggregates in the bone marrow cells treated with aspartame fibrils (Figures 5b, S17) by Congo red staining. More importantly, the enhanced magnitude of the appearance of cellular aggregates was found to correlate well with the rise in the amount of aspartame aggregates (5 mM and 10 mM; Figures 5b, S17). Obtained results, as shown in Figure 5b, revealed that bone marrow cells in the presence of aspartame fibrils have typical apple green 19 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 45

clumps50 whose intensity appears to increase in a dose-dependent manner. Longer incubation time (48 h) also resulted in increased green clumps (Figures 5b, S17) as evident from our quantitative analysis of CR fluorescence intensity data (Figure S18d). This observation predicts the occurrence of cytoplasmic aggregation, possibly mediated through the amyloid cross-seeding effect after cellular uptake of aspartame fibrils (Figures 5b, S17 and S18d). In the next step, we performed fluorescence-activated cell sorting (FACS) for the cell cycle analysis of BMCs treated with aspartame fibrils at 24 h (see materials and methods). We used Propidium iodide (PI) staining assay, which is most commonly used for DNA content/cell cycle analysis,51 and such staining assay detects the differentiation of cells in Sub-G1, G1, S and G2/M phase, as well as identification of aneuploid populations (Figure S18, panel b). The results, as shown in panel c of Figure S18, clearly show suppression of G1 phase in cells treated with aspartame fibrils, indicating about ~40% reduction in the presence of 10 mM aspartame aggregates. Aspartame treated cells, however, showed the higher percentage of Sub-G1 phase cells (Figures 5c and S18c). Since the cells lacking intact DNA content are considered as sub-G1 cells, our data suggest the DNA-damaging potential of aspartame aggregates. Finally, the effect of aspartame fibrils on cell viability of BMCs was measured by flow cytometry. Bone marrow cells isolated from mouse were exposed to different doses of aspartame fibrils and the cell viability was examined after 24 h using FACS. Using PI, a highly polar and fluorescent substance that stains only the damaged cells, quantitation of viable and non-viable cells was examined. Exposure to different concentrations of aspartame fibrils (5 mM, and 10 mM) resulted in the occurrence of lesser number of viable cells (Figures S18a and S18b), suggesting cytotoxicity of aspartame fibrils on bone marrow cells.


Page 21 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Cytotoxicity of aspartame and aspartame-seeded aggregates of proteins and amino acids on human neuroblastoma cells Since aspartame consumption has been reported to be linked to neurophysiological and neurobehavioral health issues,6, 7 we intended to examine the toxicity of aspartame on human neuroblastoma cell line (SH-SY5Y cells). We investigated the change in the morphology of SHSY5Y cells after aspartame treatment for 24 h (methods in supplementary information). Cells treated with soluble aspartame showed deformity in cell shape (Figure S30a) while the untreated control cells retained normal characteristic neuronal morphology (Figure S30a). Subsequently, we performed Congo red (CR) staining to detect the presence of amyloid aggregates in SHSY5Y cells treated with soluble aspartame and the results revealed increasing intensity of typical apple green clumps in the presence of aspartame solution in a dose-dependent manner (Figure S30b). Next, we examined the effect of aggregated aspartame on SH-SY5Y cells and our results indicated dose-dependent damaging effect (Figure S31) similar to the results obtained for soluble aspartame (Figure S30). Aspartame has the intrinsic property to undergo spontaneous aggregation process even at 0.8 mM (Figure 1d). Therefore, the observed toxicity in the presence of soluble aspartame after 24 h may be due to the formation of aspartame aggregates during the incubation period. Our MTT assay clearly revealed dose-dependent cytotoxic effect of both soluble and aggregated forms of aspartame (Figures S32). Additionally, we examined the toxicity of aspartame-seeded aggregates of proteins and amino acids on SH-SY5Y cells. The results (as shown in Figures S33 and S34) reveal that the resultant aggregates (both from individual and mixed protein samples) cause cell death. Similarly, the aggregates generated from the amino acid samples were also found to be toxic to SH-SY5Y cells (Figure S33).


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 45

DISCUSSION Our findings reveal that aspartame is inherently amyloidogenic and it can readily self-assemble into cytotoxic entities of amyloid nature and can effectively drive amyloid-cross-seeding in other proteins and metabolites. Moreover, amyloid cross-seeding efficacy of the aspartame fibrils is sufficient to recruit diverse globular proteins (in their native state) and metabolites initiating a toxic aggregation pathway. Facilitated by strong intermolecular H-bonds and  interactions,4, 5, 52, 53

aspartame molecules appear to engage in building an architecture that in some ways

resembles β-sheet amyloid structures4 (Figures 1l, 1g and S3-S5 ), in spite of the absence of an extended covalent polypeptide backbone, which is a usual requirement of a β-sheet structure, or indeed any protein secondary structure. Surprisingly, the specific arrangement of aspartame molecules within fibrils seems to be capable of inducing fibril formation (i.e., “cross-seeding”) by Aβ monomers. Typically, seeding and cross-seeding of amyloid formation has been thought to be depending largely on the presence of unsatisfied H-bond acceptors and donors on the edge strands at the fibril ends. Thus, the ability of L-polyQ fibrils to cross-seed amyloid formation by D-polyQ

was rationalized by the geometric feasibility of a D, L anti-parallel β-sheet motif that

exists in spite of radically different side chain topologies.54 Likewise, the inability of many globular proteins55 and mutated amyloidogenic proteins56 to support β-sheet mediated aggregation has been rationalized by the presence of various kinds of “β-breaker” modifications either in the exposed polypeptide chain or in neighboring structures that prevent free access to the chain. Given the absence of a contiguous polypeptide chain and its array of H-bond donors and acceptors, how can we rationalize the ability of aspartame fibrils to act as seeds for amyloid formation by aspartame itself as well as other amyloidogenic proteins? One possible explanation comes from thinking about possible mechanisms of secondary nucleation of protein aggregates, 22 ACS Paragon Plus Environment

Page 23 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

in which it is speculated that monomers may bind to lateral sites on the fibril structure, which by some mechanism enhances the ability of the bound monomer to initiate the formation of a new fibril.57 It is also possible that upon binding to aspartame fibrils, the protein species may undergo a conformational change towards an aggregation prone state triggering the self-assembly process.54, 58, 59 Recent studies have shown that amyloid aggregates of one type of protein can effectively trigger amyloid aggregation in other proteins as well, irrespective of their sequence similarity. 1, 23, 24, 54, 60 Moreover, amyloid structures generated through self-assembly of phenylalanine were found to induce amyloid cross-seeding by driving aggregation process in diverse species of proteins and amino acids.1 Likewise, tyrosine generated nanostructures of amyloid nature were found to drive amyloid cross-seeding by recruiting soluble native proteins into an aggregation pathway.4 The same study has also proposed changes in the intramolecular interactions within the protein’s native conformation when they interact with the tyrosinenanostructure.4 It is possible that aspartame generated amyloid-like entities can interact with native protein structures and such contacts may induce the required nucleation event58, 61 possibly through the necessary conformational changes in the native structure, converting them to aggregation prone conformers. This hypothesis has been proposed for infectious prion amyloids which can induce amyloid aggregation in non-amyloidogenic species by inducing an aggregation prone conformation, making them into an amyloid prone species.62 Even feeding food products containing amyloid entities has been known to trigger systemic amyloidosis in the subjects.50 Consumption of aspartame has also been known to predispose nephrotoxic effects,63 and many studies have also revealed occurrence of kidney dysfunction due to amyloidosis.64-66 These reports certainly signify the possible relevance of both the amyloidogenic nature and the cross-


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 45

seeding efficacy of aspartame molecule to the mechanistic understanding of diverse metabolic disorders related to aspartame consumption.

Research investigations have confirmed that higher dose of aspartame (in the range of 10 to 20 mM) can cause multiple defects in cervical carcinoma cells such as alterations in cell shape,47 loss of cell-adherence and conversion of normal cells to granular cells with multiple nuclei.67 The same study has also detected cell shrinkage, formation of apoptotic bodies and chromatin condensation after aspartame treatment.67 As shown in Figures 5, S17, S30 and S31, our study confirms similar morphological alterations such as deformity in cell shape and size47 of BMCs and SH-SY5Y cells in the presence of aspartame aggregates. Here, in our investigation, Congo red staining of bone marrow cells and SH-SY5Y cells treated with aspartame fibrils displayed dense apple-green clumps50 which suggest the presence of cellular aggregates of amyloid nature. We have observed substantial increase in the intensity of these green clumps48, 50 (Figure S17) with longer incubation time. Aspartame aggregates in the cytosol may possibly induce amyloid cross-seeding resulting in the formation of cytosolic protein aggregates, as evident from our comparison of Congo red-stained cells imaged at 24 h and 48 h (Figure 5, S17). This hypothesis is consistent with in vitro observation of striking amyloid cross-seeding ability of aspartame fibrils that induce aggressive aggregation of diverse cytoplasmic proteins and metabolites (Figure 2). Presence of aspartame is also known to trigger DNA damage in both time-dependent and dose-dependent manner.67, 68 Interestingly, our data revealed that aspartame treated cells show higher percentage of Sub-G1 phase cells and it confirms DNA-damaging effect of aspartame amyloid fibrils (Figures 5c, S18).67,69, 70 The toxic effects on the BMCs may affect the


Page 25 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

bone marrow stem cells, and subsequently the cells of blood and immune system that are derived from the bone marrow stem cells.

Our results also confirmed the potential of aspartame generated amyloid-like nanostructures to cause lysis of RBCs. Amyloid-RBC interaction is known to cause deformability and oxidative reduction in RBCs.1, 40, 71, 72 Furthermore, membrane damaging effects of aspartame on RBCs have been reported.73, 74 Presence of both hydrophobic exterior and inner hydrophilic core regions in the amyloid structures has been proposed to intervene RBC bilayer75 and to induce ion-leakage mediated cellular damage.76 Time dependent structural evolution during aspartame self-assembly (as observed between 10-100 ns) showed preferable minor readjustment of aspartame’s molecular orientation, facilitating precise distribution of its charged and aromatic moieties that eventually contribute to the surface hydrophobicity (Figures 1l, S3-S5). Hence, it is possible that aspartame generated amyloid entities with outer hydrophobic surface can effectively interact with the crucial components of RBC bilayer,40, 77 preferably through spontaneous hydrophobic-hydrophobic interactions.78 Moreover, we have observed the striking tendency of the lysed-deformed RBCs to adhere to the surface of aspartame fibrils (Figures 3a, S16). Metabolic process (including aspartame’s hydrolysis) after aspartame consumption in our body is known to yield free aspartic acid, free phenylalanine and methanol.79-82 Rise in the concentration of aspartame may trigger its amyloid formation and even if its hydrolysis begins in our body,80 such process would certainly promote local accumulation of Phe,82, 83 a molecule that is inherently amyloidogenic.1, 2 Furthermore, the cross-seeding ability of aspartame fibrils to trap soluble Phe and to drive its aggregation (Figure 2h) would be substantially favoured by


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 45

accumulation of Phe residues (resultant of aspartame hydrolysis) in the local environment that surrounds aspartame’s amyloid entities. The results of our study have revealed that aspartameseeded aggregates of proteins and aromatic amino acids are cytotoxic (Figure S33-S35). Such a situation compels us to believe that aspartame hydrolysis machinery in our body may not be efficient enough to prevent aspartame mediated toxic complications. Another byproduct from aspartame hydrolysis is methanol and its diverse toxic effects63, 81, 84 such as its direct link to AD pathology79, 85 have already been reported.

Regarding the concentration of aspartame in food consumption, we have explored different table-top sweetener products which have aspartame as a major ingredient. If one tablet from such products is dissolved in 100 ml solvent (a cup of water, tea, milk etc.), it would maintain approximately 1 mM of aspartame in solution. Similarly, the minimum concentration of aspartame remains approximately 0.5 mM in most of the soft drinks. In our study, using ThT assay, the results indicate in vitro aggregation of aspartame even at 0.8 mM (Figure 1d). Furthermore, we observed cytotoxic effect of aspartame at ~0.2 mM concentration (Figure 4d). Hence, it is possible that formation of amyloid-like nanostructures of aspartame could happen during the storage of such artificially sweetened food products prior to their consumption.

Recent reports postulate that aspartame can be absorbed into the mucosal cells prior to its hydrolysis,11 and our findings further validate the biological significance of the interplay between aspartame intake and multiple defects in cellular metabolism. Though our cellular metabolism may be efficient in hydrolysing single aspartame molecule,80 there remains serious explanatory vacuum in predicting how our body would respond to aspartame generated toxic


Page 27 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

amyloid-like nanostructures. Recently, Raynes et al. (2014) have already discussed the presence of protein nanostructures in food and possible risks associated with their consumption.86 More importantly, it has been proposed that the consumption of amyloid-like nanostructures may result in serious health complications, as amyloid toxicity is believed to promote the onset of many neuronal and non-neuronal pathologies. This risk factor would certainly increase when individuals already suffering from amyloid-linked diseases are exposed to the food products containing amyloid nanostructures. Strikingly, Solomon et al. (2007) have provided experimental evidence which reveals that an amyloid-containing food (Foie gras) product can substantially hasten the development of protein amyloidosis in a susceptible mice population.50 Further, research reports have also indicated that amyloid deposits present in the cattle can also cause amyloidosis in mouse model.86, 87 Considering the results revealed by these studies, the aspartame consumption seems to be capable of predisposing serious consequences both directly and indirectly. Direct defects can arise from the formation of cytotoxic aspartame fibrils and the formation of toxic amyloids of other essential proteins and peptides due to aspartame-driven amyloid cross-seeding. Loss of essential proteins and metabolites caused by amyloid-crossseeding mediated aggregation may have indirect toxic consequences because of the imbalance and deficiency of such vital biomolecules. Since aromatic residues such as tyrosine and tryptophan have been considered as vital precursors to many neuromodulators,88 loss of these precursor metabolites would indirectly affect normal neurophysiology by altering the homeostasis of vital neuromodulators. Further, formation of aspartame driven tyrosine fibrils as well as phenylalanine fibrils would be dangerous to normal physiology, as these are known to trigger cell death and hemolysis.1, 2, 4 The knowledge on the toxic consequences mediated through Phe-fibrils2 and Tyr-fibrils4 also suggests possible indirect effect of aspartame


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 45

consumption, because aspartame-fibrils can trigger amyloid cross-seeding in these aromatic metabolites (Figures 2h and 2i).

CONCLUSIONS As amyloid formation has been recognized as a foundational event to many devastating health issues including both neuronal and non-neuronal complications, consumption of amyloid-prone aspartame molecule is predicted to have a plethora of possible direct and indirect toxic consequences including cognitive-related pathologies.7, 79, 85 Since aspartame is inherently amyloidogenic, capable of forming cytotoxic nanostructures of amyloid nature and used in numerous food products, it is urgent to investigate whether amyloid-linked complications can appear from the dietary uses of aspartame.89

MATERIALS AND METHODS Reagents All the chemicals, reagents, proteins, peptide and amino acids used in this study were procured from Sigma-Aldrich, St. Louis, USA. Protein concentrations were measured on a UV1800 Shimadzu spectrophotometer. Extinction coefficient values used are as follows: 43824 M-1cm-1 at 280 nm for BSA, 6080 M-1cm-1 at 278 nm for insulin, 36 mM.cm-1 at 280 nm for lysozyme, 12.8 mM-1cm-1 at 550 nm for myoglobin and 28 mM-1cm-1 at 550 nm for cytochrome c. Aggregation studies of aspartame and amyloid cross-seeding studies ThioflavinT (ThT), a fluorescent dye, specifically binds to the cross-beta structure was used to study the aggregation.90 Aggregation kinetic studies of aspartame, aspartame induced aggregation of proteins, peptides as well as co-aggregation of proteins and a mixture of amino 28 ACS Paragon Plus Environment

Page 29 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

acids were performed at physiological conditions of temperature and buffer (phosphate buffer saline, pH 7.4 at 37 °C without agitation). ThT concentration was maintained at 30 μM and the fluorescence intensity of ThT at different time points was recorded under ambient conditions, using a Shimadzu fluorescence spectrophotometer (RF-5300, Japan). The rise in ThT emission signal was detected at 490 nm by exciting ThT molecule at 440 nm. The pathlength of the cuvette used was 10 mm. Aggregation of aspartame was studied at a concentration of ~35 mM. Aggregation reactions for all the protein samples were carried out at ~15 μM for BSA, ~174 μM for insulin, ~70 μM for lysozyme, ~57 μM for myoglobin, ~82 μM for cytochrome c in the presence and absence of soluble aspartame and preformed aspartame fibrils. For the sample containing mixture of protein monomers, equimolar concentration was maintained at ~2 μM for all proteins. In case of aspartame seed induced aggregation, aspartame fibrils of~15% w/w were used as seeds for conducting all reactions. Aspartame induced co aggregation of amino acids like proline, tryptophan, tyrosine, glutamine, alanine and arginine were also studied in the presence and absence of Aspartame seeds at an equimolar concentration of 0.1 mM. Aggregation studies of A-40 in the presence of aspartame One milligram of A1-40 from a lyophilized stock was suspended in 1 ml of trifluoro acetic acid, followed by a sonication for 10 minutes and the resultant solution was dried under argon steam. Then this peptide was dissolved in 1 ml hexafluoro isopropanol and incubated for 1 h at 37°C. The solution after incubation was transferred into 0.05% TFA and dried under stream of nitrogen. The dried film was again re dissolved in 2 ml hexafluoro isopropanol and dried under vacuum for 30-60 min to ensure the removal of trifluoro acetic acid and hexafluoro isopropanol from the peptide. Then 0.5 ml of freshly prepared 2 mM sodium hydroxide was added gently to the glass vial and kept undisturbed for few minutes. Finally, 0.5 ml of 2x PBS with 0.1% sodium 29 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 45

azide was added to the peptide solution. Then the peptide solution was centrifuged at 386,000g overnight at 4°C27. The supernatant containing the peptide was further used for our aggregation studies. The effect of aspartame on A1-40 peptide was studied by incubating aspartame seeds (~15% w/w) with ~25 M of peptide at 10 mM phosphate buffer saline, pH 7.4 at 37 °C27.

Circular dichroism (CD) The secondary structural changes in aspartame as well as the protein samples in the presence of aspartame aggregates were analyzed by CD spectroscopy. Ellipticity signals from CD experiments were recorded on a Chirascan™ qCD instrument with attached Peltier temperature controller. The changes in the secondary structures of the protein samples in the presence of aspartame aggregates were analyzed by monitoring the ellipticity values of the samples taken from the aggregation reactions. An aliquot of approximately 700 μl of the sample was taken in a CD cuvette with a pathlength of 2 mm. The reference solution was the buffer (PBS) that was used in the aggregation reactions. All the measurements were recorded at room temperature and each reported CD curve was the average representative curve of three acquisitions (between 200 and 260 nm).

Dynamic light scattering (DLS) The self-association of aspartame molecules were detected experimentally by dynamic light scattering approach. The DLS measurement was performed by spectrosize300 from Nano Bio chem Technology, Hamburg equipped, with an inbuilt Peltier controller unit. Samples before analysis were filtered through a ~0.1 μm pre-rinsed filter.


Page 31 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Fluorescence microscopy The fluorescence microscopic images of ThT stained aggregates were imaged by using fluorescence microscope (Nikon) at 10x magnification. The diluted aggregated samples were smeared over glass slide, dried and then stained with ThT and then imaged. Atomic force microscopy (AFM) The surface morphology of these aggregated samples were visualized in air by conventional atomic force microscopy measurements, by using WITec AFM system, Germany. The samples were diluted (10 folds) in ultrapure water and then 20 l aliquot of the diluted sample was smeared on the freshly cleaved mica and then air dried. The dried samples were then washed thoroughly with ultrapure water and air dried again. Images were taken immediately using tapping mode (NC-AFM) with a resonance frequency of 300 Hz. All AFM images were captured under ambient condition. Scanning electron microscopy (SEM) The amyloid fibrils generated were visualized in a Carl Zeiss EVO18 SEM. Samples were drop casted over carbon stubs, sputtered with gold/ palladium for 180 seconds. The prepared samples were then imaged in SEM at a constant voltage of 20 kV. Transmission electron microscopy (TEM) Transmission electron microscope (HR-TEM JEOL 2100F) was used to examine the morphology of amyloid fibrils. Amyloid fibril samples were spotted on a grid for ~2 min and the samples were then washed with water. The samples were then stained with 2% (w/v) aqueous uranyl acetate solution27 for ~10 min followed by another washing step. Air-dried grids were then examined at an accelerating voltage of 120 kV.


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 45

Native PAGE Native (non- denaturing) polyacrylamide gel electrophoresis for the aggregated samples was performed in a 12.5% the acrylamide gel which was run at a constant voltage of 10 Amp in a mini-PROTEIN II Bio-Rad electrophoresis system using a Tris-HCl polyacrylamide gel at 4 ºC. The main objective of this experiment was to check the formation of higher order entities but not the molecular weight. The gels were then developed by silver staining. The stained gels were visualized by Bio-Rad gel documentation unit, CA, USA and the images were processed by image lab software (Bio-Rad). Molecular dynamics on aspartame assembly The molecular dynamics studies were performed with Discovery Studio 4.0 (DS4) on a 16 core Dell Precision 5610 Workstation. The ninety nine aspartame molecules were initially processed and packed in a cubic box model along with 1500 water molecules by using PACKMOL91. Mixture model algorithm, a predefined algorithm was selected to pack these above molecules2 inside a cubic box. The Charmm36 force field was applied to the solvated cubic box and simulations were performed at 310 K. The obtained solvated molecules were further simulated by DS4. A steepest-descent algorithm was performed to minimize the energy of each system and to relax the solvent molecules this minimization step was done twice. The obtained molecules were then re-equilibrated for 100, 000 picoseconds from 273 K to a target temperature of 310 K. Leap frog verlet algorithm with shake constraints are used to fix the chemical bonds between these atoms. The temperature and pressure for various components were maintained by langevien dynamics and Berendsen pressure with MOLLY and Impulse algorithm. After successful simulation, the simulated outputs were downloaded, the final structure of the


Page 33 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

simulated molecules and the intermolecular interactions were visualized and analyzed by discovery studio visualizer and chimera tool 1.11.2. In vitro hemolysis assay Human blood samples were collected from healthy donor. All the experimental protocols related to this study were approved by the Ethics Committee of Indian Institute of Technology Jodhpur (Letter no. IITJ/EC/2016/02-D). Informed consent was obtained from the subject prior to conducting experiments. Erythrocytes were separated out from blood by rapidly spinning it at 1500rpm for 10 minutes. Thus, the obtained cell pellet was dispersed in PBS (pH 7.4) and then continuously washed to remove other components in blood. From 25 % v/v of diluted RBC’s, a 100μl of RBC was added to the artificial sweetener in both aggregated as well as soluble state. RBC’s incubated with deionized water and PBS was used as positive and negative controls respectively. All the samples were incubated at 37°C for 24 h and were vortexed regularly. After 24 h of incubation, the samples were vortexed again, centrifuged at 1600 rpm for 10 min at 4°C. The obtained pellets were saved for microscopic observation. Small aliquot of the supernatants was carefully separated out to check the absorbance values for calculating percentage of lysis. All the spectroscopic measurements for hemolysis assay were recorded using Shimadzu-UV1800 spectrophotometer. Percentage of hemolysis was calculated following the established protocols.1 We did SDS gel electrophoresis to identify the released proteins during hemolysis. The supernatant obtained after the hemolysis assay was centrifuged at 15000 rpm for 20 min at 4°C, after which the resultant pellet was washed thrice in sterile PBS buffer. Thus, the resultant final pellet which has the released proteins was resuspended in PBS before loading. The protein samples were then mixed with laemmli buffer and loaded on a 12.5% polyacrylamide gel at ~ 30 Amp. Silver stained gels were then visualized by Bio-Rad gel documentation unit. 33 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 45

MTT assay to examine the cytotoxic effect of aspartame Raw 264.7(Macrophage) were maintained in Dulbecos modified Eagles medium (DMEM) at 37ºC with 5% CO2. Once cells reach the confluency, 5x104 cells/well were seeded in 96 well tissue culture plates and incubated at 37ºC with 5% CO2. After 24 hours of incubation, the medium was removed to this aspartame aggregates and soluble aspartame were added. The cells were then supplemented with 5% minimal essential media and the experiments were triplicated. The plates were then incubated at 37ºC with 5% CO2 for 24 hours. The control used in this experiment was PBS. After 24 hours of treatment the wells were observed in an inverted phase contrast tissue culture microscope (Olympus, JAPAN with Optika Pro 5 Camera) and images were captured. Any detectable changes in the morphology of the cells, such as rounding or shrinking of cells, granulation and vacuolization in the cytoplasm of the cells were considered as indicators of cytotoxicity. Then 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium Bromide (MTT)41(34) was added to all test and cell control wells and the plates were shaken gently and incubated at 37ºC for 4 hours. After the incubation, the supernatant was removed and 100 µL of MTT solution was added to wells followed by addition of Dimethyl sulfoxide (DMSO) and the samples were mixed gently in order to solubilize the formazan crystals. The absorbance values were measured by using microplate reader ERBA, GERMANY at a wavelength of 540 nm. Study of effect of aspartame fibrils on mouse bone marrow cells (BMCs) Bone marrow cells (BMCs) were isolated from the bones of 7-8 weeks old male C57BL/J6 mice as described earlier,51, 92-95 with certain modifications in the methods. The animal experiments were approved by the Institutional Animal Ethics Committee (IAEC). Isolated bone marrow cells were maintained in complete medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics (penicillin-streptomycin). Bone marrow cells were counted on hemocytometer 34 ACS Paragon Plus Environment

Page 35 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

with trypan blue solution to assess the cell number and cell viability. One million BMCs were seeded on glass coverslips in 2 ml complete medium in each well of 6 well plate and cells were allowed to settle for 2 h at 37°C in 5% CO2 cell culture incubator. After 2h, BMCs were treated with different doses of aspartame amyloid aggregates (5 mM and 10 mM) and the plates were placed at 37°C in 5% CO2 for 24 h. Congo red staining Congo red positive-BMCs were studied by fluorescence microscopy. Cells were seeded on the glass coverslips per well in 6-well plates from the control, PBS and aspartame treated cells (5 mM and 10 mM) and the samples were incubated in the CO2 incubator for 30 min at 37°C. The cells (after 24 h and 48 h time points) were washed with 2 ml PBS followed by fixation of the cells with 2 ml of 4% paraformaldehyde for 30 min at RT. After that, paraformaldehyde was aspirated, and cells were washed thrice with 2 ml PBS. Cells were permeabilized with 2 ml, 0.1% Triton X-100 (in PBS) for 15 min at RT, followed by cleaning the cells twice with PBS. After this, cells were then stained with Congo-red for 2 min. Next, the cells were mounted with Vectasheild with DAPI (Vector Labs H-1200). Images were captured under polarized light by a Nikon TiE fluorescence confocal microscope,51 analyzed by inbuilt NIS4.00.00 software and total intensity of the cells was measured. Cell viability of bone marrow cells. Bone marrow cells treated with aspartame fibrils were harvested after 24 h. Cell samples (in media, non-attached) were collected in separate polypropylene culture tubes (~15 ml) simply by transferring the media to culture tubes. Next, cells attached to the plastic surface of plate were collected by trypsinization. For trypsinization, adherent cells were trypsinized with prewarmed (37°C) 2 ml of 0.25% trypsin/1.0 mM Ethylenediaminetetraacetic acid (EDTA) for 5-10 min at 35 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 45

37°C. Trypsinization was stopped by adding 1 ml FBS. Then, the attached cells were collected and mixed with non-attached cells present in 15 ml polypropylene tube. The pool of cell suspension was centrifuged at 300 x g for 5 min. Cell pellet was then resuspended in 200 μl PBS, and later the cell suspension was incubated on ice for 1 min after adding propidium iodide (10 μg/μl) and analyzed to record 20,000 events per sample by using FL1 and FL2 channels in a FACS Calibur machine (BD). The obtained data were then analyzed by the BD CellQuest Pro software, and the FSC and SSC gating excluded the dead cells. As described earlier, flow cytometric analysis of cell cycle with propidium iodide was performed.51 BMCs were collected after aspartame treatment and analyzed to record 50,000 events per sample by using FL1 and FL2 channels in a FACS Calibur machine (BD). Molecular Docking Biomolecular affinity of aspartame towards proteins and peptides can be analyzed with the help of molecular docking protocol, using discovery studio software. In this platform, the aspartame molecule, CID 134601, a structure obtained from PubChem was used for the study. The aspartame molecule was prepared by using ‘prepare ligand wizard’ of discovery studio 4.0 (DS4). The ligand was then processed with a pH based ionization method (pH 6.5 to 8.5). Similarly, the X-ray crystal structure of proteins and peptide96 viz. BSA (PDB ID: 4F5S), insulin (PDB ID: 1TRZ), lysozyme (193L) cytochrome C (1HRC) and Myoglobin (1DWR) and A 1-40 (PDB ID:1BA4 ) were obtained from Protein Data Bank (PDB). The protein molecules prior to molecular docking studies were prepared through ‘prepare protein wizard’ of DS4. The protein/peptide structures were primarily cleaned in silico by removing water and heteroatoms, leaving them behind as nascent protein/peptide molecules. These nascent molecules were then protonated and preprocessed with a predefined ionic strength of 0.145 and at pH 7.4 with 36 ACS Paragon Plus Environment

Page 37 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

CHARMM force fields.97 The solvent was handled by an explicit method in each case. The processed protein and peptide molecules were downloaded and used for docking studies. The ligand was docked with the processed protein/peptide molecules using C-DOCKER docking tool. C-DOCKER is a powerful CHARMM based docking method which can generate highly accurate docked poses.98 During docking the ligand molecule was set to be in dynamic state (100 ligand poses with a pose cluster of 0.1Å-predefined in C-DOCKER protocol with 100 dynamic steps, 100 ligand orientation having 2000 heating and 5000 cooling steps while docking with the nascent protein structure). Each orientation is subjected to simulated annealing molecular dynamics protocol. After successful docking, the C-DOCKER ranks the poses based on CHARMM energy, considering more negative as the most favorable binding. Molecular docking of aspartame nanostructure and native protein structures The affinity of aspartame nanostructure to the native structures of proteins was analyzed by using ZDOCK 3.0 tool.30 The simulated aspartame molecules at 10 ns yielded a nanosphere resembling morphology and such aspartame generated entities were selected for the docking study. The protein structures selected were of BSA (PDB ID: 4F5S), lysozyme (PDB ID: 193L), myoglobin (PDB ID: 1DWR) and A1-40 peptide (PDB ID: 1BA4), downloaded from Protein database. The molecules were then docked individually with the aspartame nanostructure, using ZDOCK program. Three rotational angles were tested with 6° spacing and three translational degrees of freedom were sampled with a 1.2 Å spacing. The top 10 docked poses were downloaded from the ZDOCK server and were examined thoroughly and the best pose was selected using Discovery Studio 4.0 visualizer.


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 45

ASSOCIATED CONTENT Supporting Information: Figures S1-S36, Table S1, and Materials and Methods. The Supporting Information is available free of charge on the ACS Publications website. Data processing Error bars are standard deviations from analysis in either duplicate or triplicate. The kinetic data points were fit in Origin v2015 software (Origin Lab). All the aggregation data as shown in figures were connected through the b-spline line using the ORIGIN program. Discovery studio DS4.0 was used for obtaining computational data. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] and [email protected] ORCID Karunakar Kar: 0000-0001-7047-6539

Conflict of interest: The Authors declare no conflicts of interests with the contents of this article. Author contributions. BGA, KPP, DSS, KJG, KD, PCR, NA and KK designed and conducted experiments, analyzed the data and wrote the manuscript. ACKNOWLEDGEMENTS Authors are grateful to Prof. R. Wetzel for his valuable suggestions to improve this manuscript. The authors thank Jawaharlal Nehru University for providing the required facilities (AIRF-JNU 38 ACS Paragon Plus Environment

Page 39 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

and CIF-School of Life Sciences). The authors thank Prof. Gourinath for his help with the DLS facility. The authors thank Dr. G. Bagler for his help with computational study. Authors thank DST-PURSE II, UGC-RN, UGC-DRS SAP-I, UGC-BSR Startup grant, UPOE II JNU (377),

BRNS EMR grant (37-1-/14/38/2014-BRNS) and DST-SERB (EMR/2017/005000) for financial support. K.P.P. thanks CSIR for fellowship support.

REFERENCES: 1. Anand, B. G.; Dubey, K.; Shekhawat, D. S.; Kar, K. Intrinsic Property of Phenylalanine to Trigger Protein Aggregation and Hemolysis has a Direct Relevance to Phenylketonuria. Sci. Rep. 2017, 7, 11146. 2. Adler-Abramovich, L.; Vaks, L.; Carny, O.; Trudler, D.; Magno, A.; Caflisch, A.; Frenkel, D.; Gazit, E. Phenylalanine Assembly into Toxic Fibrils Suggests Amyloid Etiology in Phenylketonuria. Nat. Chem. Biol. 2012, 8, 701-706. 3. Shaham-Niv, S.; Adler-Abramovich, L.; Schnaider, L.; Gazit, E. Extension of the Generic Amyloid Hypothesis to Nonproteinaceous Metabolite Assemblies. Sci. Adv. 2015, 1, e1500137. 4. Anand, B. G.; Prajapati, K. P.; Shekhawat, D. S.; Kar, K. Tyrosine Generated Nanostructures Initiate Amyloid Cross-Seeding in Proteins Leading to Lethal Aggregation Trap. Biochemistry 2018, 56, 5202-5209. 5. Makin, O. S.; Atkins, E.; Sikorski, P.; Johansson, J.; Serpell, L. C. Molecular Basis for Amyloid Fibril Formation and Stability. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 315-320. 6. Lindseth, G. N.; Coolahan, S. E.; Petros, T. V.; Lindseth, P. D. Neurobehavioral Effects of Aspartame Consumption. Res. Nurs. Health 2014, 37, 185-193. 7. Choudhary, A. K.; Lee, Y. Y. Neurophysiological Symptoms and Aspartame: What is the Connection? Nutr. Neurosci. 2018, 21, 306-316. 8. Suez, J.; Korem, T.; Zeevi, D.; Zilberman-Schapira, G.; Thaiss, C. A.; Maza, O.; Israeli, D.; Zmora, N.; Gilad, S.; Weinberger, A.; Kuperman, Y.; Harmelin, A.; Kolodkin-Gal, I.; Shapiro, H.; Halpern, Z.; Segal, E.; Elinav, E. Artificial Sweeteners Induce Glucose Intolerance by Altering the Gut Microbiota. Nature 2014, 514, 181-186. 9. Belpoggi, F.; Soffritti, M.; Padovani, M.; Degli Esposti, D.; Lauriola, M.; Minardi, F. Results of Long-Term Carcinogenicity Bioassay on Sprague-Dawley Rats Exposed to Aspartame Administered in Feed. Ann. N. Y. Acad. Sci. 2006, 1076, 559-577. 10. Soffritti, M.; Belpoggi, F.; Degli Esposti, D.; Lambertini, L.; Tibaldi, E.; Rigano, A. First Experimental Demonstration of the Multipotential Carcinogenic Effects of Aspartame Administered in the Feed to Sprague-Dawley Rats. Environ. Health Perspect. 2006, 114, 379-385. 11. Magnuson, B. A.; Burdock, G. A.; Doull, J.; Kroes, R. M.; Marsh, G. M.; Pariza, M. W.; Spencer, P. S.; Waddell, W. J.; Walker, R.; Williams, G. M. Aspartame: A Safety Evaluation Based on Current Use Levels, Regulations, and Toxicological and Epidemiological Studies. Crit. Rev. Toxicol. 2007, 37, 629-727. 12. Lee, J.; Culyba, E. K.; Powers, E. T.; Kelly, J. W. Amyloid-Beta Forms Fibrils by Nucleated Conformational Conversion of Oligomers. Nat. Chem. Biol. 2011, 7, 602-609. 13. Reddy, A.; Norris, D. F.; Momeni, S. S.; Waldo, B.; Ruby, J. D. The pH of Beverages in the United States. J. Am. Dent. Assoc. 2016, 147, 255-263. 39 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 45

14. Krysmann, M. J.; Castelletto, V.; Kelarakis, A.; Hamley, I. W.; Hule, R. A.; Pochan, D. J. SelfAssembly and Hydrogelation of an Amyloid Peptide Fragment. Biochemistry 2008, 47, 4597-4605. 15. Takahashi, J.; Shinojima, H.; Seyama, M.; Ueno, Y.; Kaneko, T.; Kobayashi, K.; Mita, H.; Adachi, M.; Hosaka, M.; Katoh, M. Chirality Emergence in Thin Solid Films of Amino Acids by Polarized Light from Synchrotron Radiation and Free Electron Laser. Int. J. Mol. Sci. 2009, 10, 3044-3064. 16. Perween, S.; Chandanshive, B.; Kotamarthi, H. C.; Khushalani, D. Single Amino Acid Based SelfAssembled Structure. Soft Matter 2013, 9, 10141-10145. 17. Luhrs, T.; Ritter, C.; Adrian, M.; Riek-Loher, D.; Bohrmann, B.; Dobeli, H.; Schubert, D.; Riek, R. 3D Structure of Alzheimer's Amyloid-Beta(1-42) Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 1734217347. 18. Lorenzen, N.; Nielsen, S. B.; Buell, A. K.; Kaspersen, J. D.; Arosio, P.; Vad, B. S.; Paslawski, W.; Christiansen, G.; Valnickova-Hansen, Z.; Andreasen, M.; Enghild, J. J.; Pedersen, J. S.; Dobson, C. M.; Knowles, T. P.; Otzen, D. E. The Role of Stable Alpha-Synuclein Oligomers in the Molecular Events Underlying Amyloid Formation. J. Am. Chem. Soc. 2014, 136, 3859-3868. 19. Klunk, W. E.; Jacob, R. F.; Mason, R. P. Quantifying Amyloid by Congo Red Spectral Shift Assay. Methods Enzymol. 1999, 309, 285-305. 20. Kar, K.; Kishore, N. Enhancement of Thermal Stability and Inhibition of Protein Aggregation by Osmolytic Effect of Hydroxyproline. Biopolymers 2007, 87, 339-351. 21. Perutz, M. F.; Finch, J. T.; Berriman, J.; Lesk, A. Amyloid Fibers are Water-Filled Nanotubes. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5591-5591. 22. Mossou, E.; Teixeira, S. C. M.; Mitchell, E. P.; Mason, S. A.; Adler-Abramovich, L.; Gazit, E.; Forsyth, V. T. The Self-Assembling Zwitterionic Form of L-Phenylalanine at Neutral pH. Acta Crystallogr. Sect. C Struct. Chem. 2014, 70, 326-331. 23. Dubey, K.; Anand, B. G.; Temgire, M. K.; Kar, K. Evidence of Rapid Coaggregation of Globular Proteins During Amyloid Formation. Biochemistry 2014, 53, 8001-8004. 24. Anand, B. G.; Prajapati, K. P.; Kar, K. Abeta1-40 Mediated Aggregation of Proteins and Metabolites Unveils the Relevance of Amyloid Cross-Seeding in Amyloidogenesis. Biochem. Biophys. Res. Commun. 2018, 501, 158-164. 25. Schmidt, M.; Sachse, C.; Richter, W.; Xu, C.; Fandrich, M.; Grigorieff, N. Comparison of Alzheimer Abeta(1-40) and Abeta(1-42) Amyloid Fibrils Reveals Similar Protofilament Structures. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 19813-19818. 26. Xiao, Y.; Ma, B.; McElheny, D.; Parthasarathy, S.; Long, F.; Hoshi, M.; Nussinov, R.; Ishii, Y. Abeta(1-42) Fibril Structure Illuminates Self-Recognition and Replication of Amyloid in Alzheimer's Disease. Nat. Struct. Mol. Biol. 2015, 22, 499-505. 27. Kodali, R.; Williams, A. D.; Chemuru, S.; Wetzel, R. Abeta(1-40) Forms Five Distinct Amyloid Structures Whose Beta-Sheet Contents and Fibril Stabilities are Correlated. J. Mol. Biol. 2010, 401, 503517. 28. Coles, M.; Bicknell, W.; Watson, A. A.; Fairlie, D. P.; Craik, D. J. Solution Structure of Amyloid Beta-Peptide(1-40) in a Water-Micelle Environment. Is the Membrane-Spanning Domain Where We Think It is? Biochemistry 1998, 37, 11064-11077. 29. Liu, R.; McAllister, C.; Lyubchenko, Y.; Sierks, M. R. Residues 17-20 and 30-35 of Beta-Amyloid Play Critical Roles in Aggregation. J. Neurosci. Res. 2004, 75, 162-171. 30. Pierce, B. G.; Wiehe, K.; Hwang, H.; Kim, B. H.; Vreven, T.; Weng, Z. ZDOCK Server: Interactive Docking Prediction of Protein-Protein Complexes and Symmetric Multimers. Bioinformatics 2014, 30, 1771-1773. 31. Oki, S.; Iwashita, K.; Kimura, M.; Kano, H.; Shiraki, K. Mechanism of Co-Aggregation in a Protein Mixture with Small Additives. Int. J. Biol. Macromol. 2018, 107, 1428-1437. 32. Tompa, P. Intrinsically Unstructured Proteins. Trends Biochem. Sci. 2002, 27, 527-533. 40 ACS Paragon Plus Environment

Page 41 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

33. Zhao, Y.; Sun, X.; Zhang, G.; Trewyn, B. G.; Slowing, II; Lin, V. S. Interaction of Mesoporous Silica Nanoparticles with Human Red Blood Cell Membranes: Size and Surface Effects. ACS Nano 2011, 5, 1366-1375. 34. Murphy, S. C.; Samuel, B. U.; Harrison, T.; Speicher, K. D.; Speicher, D. W.; Reid, M. E.; Prohaska, R.; Low, P. S.; Tanner, M. J.; Mohandas, N.; Haldar, K. Erythrocyte Detergent-Resistant Membrane Proteins: Their Characterization and Selective Uptake During Malarial Infection. Blood 2004, 103, 19201928. 35. Chu, C.; Woods, N.; Sawasdee, N.; Guizouarn, H.; Pellissier, B.; Borgese, F.; Yenchitsomanus, P. T.; Gowrishankar, M.; Cordat, E. Band 3 Edmonton I, A Novel Mutant of the Anion Exchanger 1 Causing Spherocytosis and Distal Renal Tubular Acidosis. Biochem. J. 2010, 426, 379-388. 36. Olszewska, M.; Wiatrow, J.; Bober, J.; Stachowska, E.; Golembiewska, E.; Jakubowska, K.; Stanczyk-Dunaj, M.; Pietrzak-Nowacka, M. Oxidative Stress Modulates the Organization of Erythrocyte Membrane Cytoskeleton. Postepy Hig. Med. Dosw. (Online) 2012, 66, 534-542. 37. Foretz, M.; Hebrard, S.; Guihard, S.; Leclerc, J.; Do Cruzeiro, M.; Hamard, G.; Niedergang, F.; Gaudry, M.; Viollet, B. The Ampkgamma1 Subunit Plays an Essential Role in Erythrocyte Membrane Elasticity, and Its Genetic Inactivation Induces Splenomegaly and Anemia. FASEB J. 2011, 25, 337-347. 38. Toumey, C. Compare and Contrast as Microscopes Get up Close and Personal. Nat. Nanotechnol. 2011, 6, 191-193. 39. Rother, R. P.; Rollins, S. A.; Mojcik, C. F.; Brodsky, R. A.; Bell, L. Discovery and Development of the Complement Inhibitor Eculizumab for the Treatment of Paroxysmal Nocturnal Hemoglobinuria. Nat. Nanotechnol. 2007, 25, 1256-1264. 40. Nakagawa, K.; Kiko, T.; Miyazawa, T.; Sookwong, P.; Tsuduki, T.; Satoh, A. Amyloid Beta-Induced Erythrocytic Damage and Its Attenuation by Carotenoids. FEBS Lett. 2011, 585, 1249-1254. 41. Xue, W. F.; Hellewell, A. L.; Gosal, W. S.; Homans, S. W.; Hewitt, E. W.; Radford, S. E. Fibril Fragmentation Enhances Amyloid Cytotoxicity. J. Biol. Chem. 2009, 284, 34272-34282. 42. Horio, Y.; Sun, Y.; Liu, C.; Saito, T.; Kurasaki, M. Aspartame-Induced Apoptosis in PC12 Cells. Environ. Toxicol. Pharmacol. 2014, 37, 158-165. 43. Ribble, D.; Goldstein, N. B.; Norris, D. A.; Shellman, Y. G. A Simple Technique for Quantifying Apoptosis in 96-Well Plates. BMC Biotechnol. 2005, 5, 12. 44. Pleyer, L.; Valent, P.; Greil, R. Mesenchymal Stem and Progenitor Cells in Normal and Dysplastic Hematopoiesis-Masters of Survival and Clonality? Int. J. Mol. Sci. 2016, 17. E1009. 45. Zhao, E.; Xu, H.; Wang, L.; Kryczek, I.; Wu, K.; Hu, Y.; Wang, G.; Zou, W. Bone Marrow and the Control of Immunity. Cell. Mol. Immunol. 2012, 9, 11-19. 46. Strober, W. Trypan Blue Exclusion Test of Cell Viability. Curr. Protoc. Immunol. 2001, Appendix 3, Appendix 3B. 47. van Eyk, A. D. The Effect of Five Artificial Sweeteners on Caco-2, HT-29 and HEK-293 Cells. Drug Chem. Toxicol. 2015, 38, 318-327. 48. Clement, C. G.; Truong, L. D. An Evaluation of Congo Red Fluorescence for the Diagnosis of Amyloidosis. Hum. Pathol. 2014, 45, 1766-1772. 49. Khurana, R.; Uversky, V. N.; Nielsen, L.; Fink, A. L. Is Congo Red an Amyloid-Specific Dye? J. Biol. Chem. 2001, 276, 22715-22721. 50. Solomon, A.; Richey, T.; Murphy, C. L.; Weiss, D. T.; Wall, J. S.; Westermark, G. T.; Westermark, P. Amyloidogenic Potential of Foie Gras. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 10998-11001. 51. Chaudhary, J. K.; Rath, P. C. Microgrooved-Surface Topography Enhances Cellular Division and Proliferation of Mouse Bone Marrow-Derived Mesenchymal Stem Cells. PLoS One 2017, 12, e0182128. 52. Banik, D.; Dutta, R.; Banerjee, P.; Kundu, S.; Sarkar, N. Inhibition of Fibrillar Assemblies of LPhenylalanine by Crown Ethers: A Potential Approach toward Phenylketonuria. J. Phys. Chem. B 2016, 120, 7662-7670. 41 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 45

53. Gazit, E. A Possible Role for Pi-Stacking in the Self-Assembly of Amyloid Fibrils. FASEB J. 2002, 16, 77-83. 54. Kar, K.; Arduini, I.; Drombosky, K. W.; van der Wel, P. C.; Wetzel, R. D-Polyglutamine Amyloid Recruits L-Polyglutamine Monomers and Kills Cells. J. Mol. Biol. 2014, 426, 816-829. 55. Richardson, J. S.; Richardson, D. C. Natural Beta-Sheet Proteins Use Negative Design to Avoid Edge-to-Edge Aggregation. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 2754-2759. 56. Kar, K.; Baker, M. A.; Lengyel, G. A.; Hoop, C. L.; Kodali, R.; Byeon, I. J.; Horne, W. S.; van der Wel, P. C.; Wetzel, R. Backbone Engineering within a Latent Beta-Hairpin Structure to Design Inhibitors of Polyglutamine Amyloid Formation. J. Mol. Biol. 2017, 429, 308-323. 57. Rotter, M.; Yosmanovich, D.; Briehl, R. W.; Kwong, S.; Ferrone, F. A. Nucleation of Sickle Hemoglobin Mixed with Hemoglobin A: Experimental and Theoretical Studies of Hybrid-Forming Mixtures. Biophys. J. 2011, 101, 2790-2797. 58. Kar, K.; Hoop, C. L.; Drombosky, K. W.; Baker, M. A.; Kodali, R.; Arduini, I.; van der Wel, P. C.; Horne, W. S.; Wetzel, R. Beta-Hairpin-Mediated Nucleation of Polyglutamine Amyloid Formation. J. Mol. Biol. 2013, 425, 1183-1197. 59. Fink, A. L. Protein Aggregation: Folding Aggregates, Inclusion Bodies and Amyloid. Folding & Design 1998, 3, R9-23. 60. Raynes, J. K.; Day, L.; Crepin, P.; Horrocks, M. H.; Carver, J. A. Coaggregation of Kappa-Casein and Beta-Lactoglobulin Produces Morphologically Distinct Amyloid Fibrils. Small 2017, 13 . 1603591 61. Kar, K.; Jayaraman, M.; Sahoo, B.; Kodali, R.; Wetzel, R. Critical Nucleus Size for Disease-Related Polyglutamine Aggregation is Repeat-Length Dependent. Nat. Struct. Mol. Biol. 2011, 18, 328-336. 62. Prusiner, S. B. Cell biology. A Unifying Role for Prions in Neurodegenerative Diseases. Science 2012, 336, 1511-1513. 63. Ardalan, M. R.; Tabibi, H.; Ebrahimzadeh Attari, V.; Malek Mahdavi, A. Nephrotoxic Effect of Aspartame as an Artificial Sweetener: a Brief Review. Iran J. Kidney Dis. 2017, 11, 339-343. 64. Dember, L. M. Amyloidosis-Associated Kidney Disease. J. Am. Soc. Nephrol. 2006, 17, 3458-3471. 65. de Mello, R. A.; Marques Santos, D. S.; Freitas-Silva, M. P.; Andrade, J. A. Renal Failure due to Primary Amyloidosis: A Case Report and Literature Review. Sao Paulo Med. J. 2011, 129, 176-180. 66. Khalighi, M. A.; Dean Wallace, W.; Palma-Diaz, M. F. Amyloid Nephropathy. Clin. Kidney J. 2014, 7, 97-106. 67. Pandurangan, M.; Enkhtaivan, G.; Kim, D. H. Cytotoxic Effects of Aspartame on Human Cervical Carcinoma Cells. Toxicol. Res. (Cambridge, U. K.) 2016, 5, 45-52. 68. Kirkland, D.; Gatehouse, D. "Aspartame: A Review of Genotoxicity Data". Food Chem. Toxicol. 2015, 84, 161-168. 69. Yilmaz, S.; Ucar, A. A Review of the Genotoxic and Carcinogenic Effects of Aspartame: Does It Safe or Not. Cytotechnology 2014, 66, 875-881. 70. Sahoo, B.; Arduini, I.; Drombosky, K. W.; Kodali, R.; Sanders, L. H.; Greenamyre, J. T.; Wetzel, R. Folding Landscape of Mutant Huntingtin Exon1: Diffusible Multimers, Oligomers and Fibrils, and No Detectable Monomer. PloS One 2016, 11, e0155747 71. Jayakumar, R.; Kusiak, J. W.; Chrest, F. J.; Demehin, A. A.; Murali, J.; Wersto, R. P.; Nagababu, E.; Ravi, L.; Rifkind, J. M. Red Cell Perturbations by Amyloid Beta-Protein. Biochim. Biophys. Acta 2003, 1622, 20-28. 72. Mattson, M. P.; Begley, J. G.; Mark, R. J.; Furukawa, K. Abeta25-35 Induces Rapid Lysis of Red Blood Cells: Contrast with Abeta1-42 and Examination of Underlying Mechanisms. Brain Res. 1997, 771, 147-153. 73. Schulpis, K. H.; Papassotiriou, I.; Parthimos, T.; Tsakiris, T.; Tsakiris, S. The Effect of L-Cysteine and Glutathione on Inhibition of Na+, K+-Atpase Activity by Aspartame Metabolites in Human Erythrocyte Membrane. Eur. J. Clin. Nutr. 2006, 60, 593-597. 42 ACS Paragon Plus Environment

Page 43 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

74. Tsakiris, S.; Giannoulia-Karantana, A.; Simintzi, I.; Schulpis, K. H. The Effect of Aspartame Metabolites on Human Erythrocyte Membrane Acetylcholinesterase Activity. Pharmacol. Res. 2006, 53, 1-5. 75. Pillot, T.; Goethals, M.; Vanloo, B.; Talussot, C.; Brasseur, R.; Vandekerckhove, J.; Rosseneu, M.; Lins, L. Fusogenic Properties of the C-Terminal Domain of the Alzheimer Beta-Amyloid Peptide. J. Biol. Chem. 1996, 271, 28757-28765. 76. Do, T. D.; Kincannon, W. M.; Bowers, M. T. Phenylalanine Oligomers and Fibrils: The Mechanism of Assembly and the Importance of Tetramers and Counterions. J. Am. Chem. Soc. 2015, 137, 1008010083. 77. Ulrich, A. S.; Tichelaar, W.; Forster, G.; Zschornig, O.; Weinkauf, S.; Meyer, H. W. Ultrastructural Characterization of Peptide-Induced Membrane Fusion and Peptide Self-Assembly in the Lipid Bilayer. Biophys. J. 1999, 77, 829-841. 78. German, H. W.; Uyaver, S.; Hansmann, U. H. Self-Assembly of Phenylalanine-Based Molecules. J. Phys. Chem. A 2015, 119, 1609-1615. 79. Stegink, F. Aspartame: Physiology and Biochemistry. CRC Press: 1984; Vol. 12. 80. Ranney, R. E.; Oppermann, J. A.; Muldoon, E.; McMahon, F. G. Comparative Metabolism of Aspartame in Experimental Animals and Humans. J. Toxicol. Environ. Health 1976, 2, 441-451. 81. Gonzalez-Quevedo, A.; Obregon, F.; Urbina, M.; Rousso, T.; Lima, L. Effect of Chronic Methanol Administration on Amino Acids and Monoamines in Retina, Optic Nerve, and Brain of the Rat. Toxicol. Appl. Pharmacol. 2002, 185, 77-84. 82. Filer, L. J., Jr.; Stegink, L. D. Aspartame Metabolism in Normal Adults, Phenylketonuric Heterozygotes, and Diabetic Subjects. Diabetes Care 1989, 12, 67-74. 83. Burgert, S. L.; Andersen, D. W.; Stegink, L. D.; Takeuchi, H.; Schedl, H. P. Metabolism of Aspartame and Its L-Phenylalanine Methyl Ester Decomposition Product by the Porcine Gut. Metab., Clin. Exp. 1991, 40, 612-618. 84. Stegink, L. D.; Brummel, M. C.; McMartin, K.; Martin-Amat, G.; Filer, L. J., Jr.; Baker, G. L.; Tephly, T. R. Blood Methanol Concentrations in Normal Adult Subjects Administered Abuse Doses of Aspartame. J. Toxicol. Environ. Health 1981, 7, 281-290. 85. Yang, M.; Lu, J.; Miao, J.; Rizak, J.; Yang, J.; Zhai, R.; Zhou, J.; Qu, J.; Wang, J.; Yang, S.; Ma, Y.; Hu, X.; He, R. Alzheimer's Disease and Methanol Toxicity (Part 1): Chronic Methanol Feeding Led to Memory Impairments and Tau Hyperphosphorylation in Mice. J. Alzheimer's Dis. 2014, 41, 1117-1129. 86. Raynes, J. K.; Carver, J. A.; Gras, S. L.; Gerrard, J. A. Protein Nanostructures in Food – Should We be Worried? Trends Food Sci. Technol. 2014, 37, 42-50. 87. Greger, M. Amyloid Fibrils: Potential Food Safety Implications. Int. J. Food Safety, Nutr. Public Heal. 2008, 1, 103-115. 88. Poos, M. I., Costello, R., Carlson-Newberry S.J. The Role of Protein and Amino Acids in Sustaining and Enhancing Performance. In Committee on Military Nutrition Research: Activity Report:, National Academics Press, USA: Washington (DC), 1999. 89. Smith, R. J. Aspartame Approved Despite Risks. Science 1981, 213, 986-987. 90. Wetzel, R.; Chemuru, S.; Misra, P.; Kodali, R.; Mukherjee, S.; Kar, K. An Aggregate WeightNormalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates. Methods Mol. Biol. 2018, 1777, 121-144. 91. Kochman, A.; Koska, C.; Metodiewa, D. Submolecular Adventures of Brain Tyrosine: What are We Searching for Now? Amino Acids 2002, 23, 95-101. 92. Dominici, M.; Le Blanc, K.; Mueller, I.; Slaper-Cortenbach, I.; Marini, F.; Krause, D.; Deans, R.; Keating, A.; Prockop, D.; Horwitz, E. Minimal Criteria for Defining Multipotent Mesenchymal Stromal Cells. The International Society For Cellular Therapy Position Statement. Cytotherapy 2006, 8, 315-317. 43 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 45

93. Soleimani, M.; Nadri, S. A Protocol for Isolation and Culture of Mesenchymal Stem Cells from Mouse Bone Marrow. Nat. Protoc. 2009, 4, 102-106. 94. Zhu, H.; Guo, Z. K.; Jiang, X. X.; Li, H.; Wang, X. Y.; Yao, H. Y.; Zhang, Y.; Mao, N. A Protocol for Isolation and Culture of Mesenchymal Stem Cells from Mouse Compact Bone. Nat. Protoc. 2010, 5, 550560. 95. Ahamad, N.; Rath, P. C. Expression of Interferon Regulatory Factors (IRF-1 and IRF-2) During Radiation-Induced Damage and Regeneration of Bone Marrow by Transplantation in Mouse. Mol. Biol. Rep. 2019, 46, 551-567. 96. Pechkova, E.; Bragazzi, N.; Bozdaganyan, M.; Belmonte, L.; Nicolini, C. A Review of the Strategies for Obtaining High-Quality Crystals Utilizing Nanotechnologies and Microgravity. Crit. Rev. Eukaryotic Gene Expression 2014, 24, 325-339. 97. Brooks, B. R.; Brooks, C. L., 3rd; Mackerell, A. D., Jr.; Nilsson, L.; Petrella, R. J.; Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S.; Caflisch, A.; Caves, L.; Cui, Q.; Dinner, A. R.; Feig, M.; Fischer, S.; Gao, J.; Hodoscek, M.; Im, W.; Kuczera, K.;et al. CHARMM: the Biomolecular Simulation Program. J. Comput. Chem. 2009, 30, 1545-1614. 98. Wu, G.; Robertson, D. H.; Brooks, C. L., 3rd; Vieth, M. Detailed Analysis of Grid-Based Molecular Docking: A Case Study of CDOCKER-A CHARMm-Based MD Docking Algorithm. J. Comput. Chem. 2003, 24, 1549-1562.


Page 45 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

128x92mm (150 x 150 DPI)

ACS Paragon Plus Environment

Self-Assembly of Artificial Sweetener Aspartame ... - ACS Publications

Recommend Documents