Salivary proteomics of healthy dogs: An in depth catalog
Salivary proteomics of healthy dogs: An in depth catalog
Sheila M. F. Torres 1 2
Eva Furrow 1 2
Clarissa P. Souza 1 2
Jennifer L. Granick 1 2
Ebbing P. de Jong 0 2
Timothy J. Griffin 0 2
Xiong Wang 2
0 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota , Minneapolis , Minnesota, United States of America, 4 Department of Biochemistry and Molecular Biochemistry, SUNY Upstate Medical University , Syracuse , New York, United States of America, 5 Department of Veterinary Biomedical Sciences, University of Minnesota , Saint Paul , Minnesota, United States of America, 6 Minnesota Department of Health , Saint Paul, Minnesota , United States of America
1 Veterinary Clinical Sciences Department, College of Veterinary Medicine, University of Minnesota , Saint Paul , Minnesota, United States of America, 2 Clinical Sciences Department, College of Veterinary Medicine and Biomedical Sciences, Colorado State University , Fort Collins, Colorado , United States of America
2 Editor: Xuejiang Guo, Nanjing Medical University , CHINA
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The work was self-funded by the
corresponding author. Partial funding for E.F. was
provided by the Office of the Director, National
Institute of Health (NIH) under award number
K01OD019912. The funder had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
To provide an in-depth catalog of the salivary proteome and endogenous peptidome of
healthy dogs, evaluate proteins and peptides with antimicrobial properties, and compare the
most common salivary proteins and peptides between different breed phylogeny groups.
36 healthy dogs without evidence of periodontal disease representing four breed phylogeny
groups, based upon single nucleotide polymorphism haplotypes (ancient,
herding/sighthound, and two miscellaneous groups). Saliva collected from dogs was pooled by
phylogeny group and analyzed using nanoscale liquid chromatography-tandem mass
spectrometry. Resulting tandem mass spectra were compared to databases for
identification of endogenous peptides and inferred proteins.
2,491 proteins and endogenous peptides were found in the saliva of healthy dogs with no
periodontal disease. All dog phylogeny groups' saliva was rich in proteins and peptides with
antimicrobial functions. The ancient breeds group was distinct in that it contained unique
proteins and was missing many proteins and peptides present in the other groups.
Conclusions and clinical relevance
Using a sophisticated nanoscale liquid chromatography-tandem mass spectrometry, we
were able to identify 10-fold more salivary proteins than previously reported in dogs. Seven
of the top 10 most abundant proteins or peptides serve immune functions and many more
with various antimicrobial mechanisms were found. This is the most comprehensive
analysis of healthy canine saliva to date, and will provide the groundwork for future studies
analyzing salivary proteins and endogenous peptides in disease states.
Saliva is composed of a complex mixture of enzymes, glycoproteins, immunoglobulins,
peptides, inorganic substances, white blood cells, epithelial cells, and microflora, in addition to
water. The substances in saliva originate primarily from salivary glands but also blood and
nasal-bronchial secretions [1±2]. Food digestion and lubrication are well-recognized functions
of saliva; however, this complex fluid also protects the oral cavity against pathogens, maintains
the mouth pH and has a role in taste [3±4].
For many decades saliva has been considered the reflection of health and disease states of
the oral cavity in addition to the whole body [
]. The relatively easy and non-invasive access to
saliva samples compounded with the remarkable advances in the technology to investigate
proteinsÐa major saliva componentÐhave spiked researchers' interest in looking at the
composition of this biological fluid in healthy individuals with the ultimate goal of identifying
biomarkers of diseases [4±9].
Various mass spectrometry methods are currently available and have been widely used to
study the salivary proteins, and smaller, endogenous peptides. However, despite extensive
investigation in humans [2,5,8,10±26], much less work has been done in other species [3,27±
34]. Recently, the protein components of dog saliva were examined using SDS-PAGE-LC
coupled to tandem mass spectrometry (MS/MS) [
], but only one dog sample was analyzed.
Hence, a more comprehensive characterization of the salivary proteome and endogenous
peptidome of healthy dogs is needed and can provide a valuable groundwork for future studies
searching for specific changes in salivary protein composition associated with oral and
The primary study aim was to provide an in-depth catalog of the salivary endogenous
peptidome and proteome of healthy dogs. Additional aims included an evaluation of proteins and
peptides with antimicrobial properties and comparison of the most common salivary proteins
between different breed phylogeny groups.
Materials and methods
A summary of the study design and methodology used is shown in Fig 1.
The study was approved by the Institutional Animal Care and Use Committee of the
University of Minnesota. Thirty-six clinically healthy dogs owned by faculty, staff and students at the
University of Minnesota, College of Veterinary Medicine, were selected for the study. The
dogs were carefully examined by a veterinarian with experience in dental disease to assure the
absence of any dental abnormalities, especially periodontal disease. Sixteen dogs were males
(11 intact and 5 neutered) and 20 were females (16 spayed and 4 intact). Their age ranged
from 4 to 148 months (mean = 40.58 months; median = 25 months). The following breeds
were represented: Hound cross (n = 6), Labrador Retriever (5), Alaskan Malamute (3), Bernese
Mountain Dog (3), Siberian Husky (3), Golden Retriever (2), German Shepherd cross (2),
American Staffordshire Terrier cross (1), Australian Cattle Dog (1), Belgian Tervuren (1),
Boxer cross (1), French Bulldog (1), German Shorthair Pointer (1), German Wirehair Pointer
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Fig 1. Flow chart summarizing the study design.
(1), Irish Wolfhound (1), Jack Russell Terrier (1), Mixed breed dog (1), Newfoundland (1),
and Scottish Deerhound (1) (Fig 1).
To try to investigate the potential effect of genetic lineage on the salivary proteomics and
peptidomics, the dogs were initially selected to represent the four lineages that cluster based on
structure analysis of microsatellite markers: asian/ancient, herding, hunting, and mastiff [
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However, more recent analysis of single nucleotide polymorphism haplotypes divides dog
breeds into 10 clusters [
]. Given this newer classification and the inclusion of some dogs
with incomplete parentage information (crossbred dogs), the four groups were renamed as
follows: ancient, herding-sighthound, and miscellaneous 1 and 2 (Fig 1). Saliva was pooled for
dogs within each group.
Whole saliva was collected without previous stimulation using saliva collection kits
(SalivaBio kit; Salimetrics1, State College, PA) according to the manufacturer's recommendation.
Briefly, dog owners were asked to withhold food and water from their pets for at least 1
hour before sample collection. A cotton swab of 125mm length and 8mm diameter was
placed in each dog's cheek pouches for 45±60 seconds, with the collector gently holding the
dog's muzzle to prevent swallowing. Upon removal, the swab was placed in a special tube
(Swab Storage Tube) and the saliva extracted by centrifugation at 685 × g for 15 minutes.
The swab was removed from the tube and the saliva immediately stored at -80ÊC freezer
until analysis [
Sample preparation (offline high pH reverse phase-liquid chromatography
Saliva samples were thawed on ice and cleared of cells and debris by centrifugation at 3000 × g
for 10 minutes and 16,100 × g for 1 minute at 4ÊC. Protein concentrations of the supernatants
were determined by the bicinchoninic acid (BCA) assay. All samples were qualitatively
analyzed by SDS-PAGE prior to being used in the study.
Pools of saliva from each group were prepared for proteomic analysis by combining equal
protein amounts for each sample within a category to a total protein amount of 200 μg per
pool. The pools were digested using the FASP protocol [
] using 10kDa filters (Pall Nanosep
10kDa filters; VWR, OD010C34) and the resulting peptides desalted using silica-based sorbent
cartridges (Sep-Pak tC18 cartridges; Waters, WAT054925).
Endogenous peptides (naturally occurring salivary peptides below 10kDa) were collected
from the flow-through after the initial centrifugation step using the 10kDa filters (prior to
protein alkylation). These peptides were separately reduced using 5 mM tris (2-carboxyethyl)
phosphine (TCEP) and alkylated using 50 mM iodoacetamide. Endogenous peptide samples
were cleaned up on mixed-mode polymeric sorbent cartridges (Oasis MCX cartridges; Waters,
The trypsin-digested protein samples were fractionated using high-pH reversed phase with
subsequent concatenation similar to the protocol of Wang et al [
]. The samples were
dissolved in 200 mM ammonium formate, pH 10 containing 2% acetonitrile (ACN) and loaded
onto a C18 column (Phenomenex Kinetek C18 column; 2.6 μm, 2.1 x 100 mm). Solvents A
and B were 20 mM ammonium formate, pH 10, containing 2 and 90% ACN respectively. A
gradient was run at 200 μL/min with the following steps: 0 min, 2% B; 5 min, 2% B; 5.5 min,
5%B; 28 min, 30% B; 31 min, 60% B; 33 min, 90% B; 40 min, 90% B; 41 min, 2% B; 45 min, 2%
B. The column was heated to 55ÊC with a heated sleeve (Analytical Sales & Service, Inc.,
HSI25L). Fractions were collected every minute, and were concatenated by combining a volume
equivalent to 15 mAU from fractions 7 and 21, 8 and 22, etc., until fractions 20 and 34 to
produce 14 concatenated fractions. These were dried in a speed-vac, and re-dissolved in 37.5 μL of
0.1% trifluoroacetic acid (TFA) in 2% ACN load solvent.
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Nano liquid chromatography±tandem mass spectrometry (LC-MS/MS)
Analysis of the concatenated fractions from the digest proteins, as well as the endogenous
peptides, was performed on a mass spectrometer (Orbitrap Fusion Tribrid with Easy-nLC
autosampler and LC; Thermo Scientific, Waltham, MA) equipped with an autosampler and LC system.
For protein samples, 2.5 μL of each concatenated fraction was injected directly onto an in-house
packed, 10 cm x 75 μm column packed with 3 μm C18 particles. Separation was achieved by a
gradient from 2±30% B over 50 min, followed by a 2 min ramp to 90% B and 8 min hold at 90%
B. The flow rate was 200 nL/min. The MS operated in a top speed, data-dependent mode with a
cycle time of 3 s. MS1 scans were performed in the Orbitrap at 120k resolution from 400±1500
m/z with an AGC target of 4E5. Percursor isolation took place in the quadrupole with an
isolation width of 1.6 m/z. CID was performed at 35% NCE and MS2 spectra were collected in the
ion trap. Dynamic exclusion used a repeat count of 1 for a duration of 30 s.
Sequence database search for proteins and peptides
The data were searched against a RefSeq Canis familiaris database with common contaminant
proteins, containing 47336 entries, using protein analysis software (Sequest HT node in
Proteome Discoverer 2.0; Thermo Scientific, Waltham, MA). Search parameters used included
trypsin enzyme specificity with 2 missed cleavages for analysis of intact proteins,
carbamidomethyl as a fixed modification on cysteine and variable modification of methionine oxidation
and protein N-terminal acetylation. Precursor and product ion mass tolerances of 35 ppm and
0.6 Da were used. For identification of endogenous peptides, all parameters were the same as
above, except that no enzyme was specified.
Criteria for protein identification
MS/MS based peptide and inferred protein identifications were validated using proteomic
analysis software (Scaffold; version Scaffold 4.6.1; Proteome Software Inc., Portland, OR). For the
analysis of intact salivary proteins, peptide identifications were accepted if they could be established
at greater than 92.0% probability by the Scaffold Local FDR algorithm. Peptide identifications
were also required to exceed specific database search engine thresholds. Sequest identifications
required at least deltaCn scores of greater than 0.0 and XCorr scores of greater than 1.8, 2.2, 2.5
and 3.5 for singly, doubly, triply and quadruply charged peptides. Protein identifications were
accepted if they could be established at greater than 5.0% probability to achieve an estimated
FDR less than or equal to 1.0% and contained at least 1 identified peptide. Protein probabilities
were assigned by the Protein Prophet algorithm [
]. Proteins that contained similar peptides
and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the
principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.
Collectively, the above criteria resulted in an estimated peptide FDR of 0.2% and an estimated
protein FDR of 1.0%, both estimated using the target-decoy method. S1 Appendix contains all
information on identified proteins and peptides for the analysis of intact salivary proteins.
For the identification of endogenous salivary peptides, accepted peptide identifications
were stringently filtered to an estimated FDR level of 0.0% using the target-decoy method for
estimation. S2 Appendix contains all information on identified endogenous peptides.
The semi-quantitative protein data from the dog saliva was measured via protein spectral
counts from the MS-based proteomics data. Normalized spectral counts were assigned to each
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identified protein using the ªQuantitative Valueº assignment tool within the Scaffold software
used for organizing protein identifications and comparing spectral counts across samples.
Quantitative values for identified proteins and endogenous peptides were compared across the
To be included in the analyses each specific protein and peptide had to meet at least one of
the following criteria: (i) an assigned normalized spectral count value of 5 or higher in at least
one group or (ii) a normalized spectral count value of 1 or more in at least two groups. After
application of the criteria in the dataset, the gene symbols of included proteins were imported
into a web-based program (Venny 2.1) for construction of Venn's diagram [
] with the goal
of comparing the protein and peptide content among groups. A two dimensional Principle
Component Analysis (PCA) and Heatmap were also generated using a web tool for visualizing
multivariate data (ClustVis) [
] to further evaluate similarities and differences in the salivary
proteomics and peptidomics among the four groups.
Search of proteins and peptides with immune functions
Papers on salivary proteomics/peptidomics were reviewed to identify proteins and peptides
with antimicrobial functions that are reported to be abundant in human saliva (specific
proteins and references are provided in the results); our database was then searched for these
proteins and peptides.
Dog saliva proteomic and peptidomic profile
Using nanoscale LC MS/MS 2,491 proteins and endogenous peptides were identified in the
dog saliva (S3 Appendix), and 1,588 of those met the defined quantitative criteria for further
analysis. The top 10 most abundant proteins and their function are provided in Table 1; 7 of
the 10 have immune functions. Table 2 additionally shows salivary proteins and peptides with
various antimicrobial properties that have been reported to be abundant in previous studies.
Most of these proteins and peptides were present in all four groups.
Gene name Total normalized
Binds to IgG on mucosal surfaces [
Transports IgA across epithelial cells [
Key components of the innate immune response against Gram-negative bacteria [
Key components of the innate immune response against Gram-negative bacteria [
Serum-derived protein believed to passively enter saliva. Saliva-specific functions include
binding to hydroxyapatite and lubrication of oral tissues [
GO: serine-type endopeptidase inhibitor activity
Gel-forming mucin that lubricates saliva and plays a role in reducing adherence and
increasing clearance of bacteria [
Cytoskeletal protein with multiple functions in the defense against intracellular
Component of immunoglobulin light chains
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The N-terminal cationic domain has an antibacterial, antifungal and antiviral
Agglutinates bacteria (e.g. Streptococcus mutans) [50±52]
Key components of the innate immune response against Gram-negative
Key components of the innate immune response against Gram-negative
Binds to Staphylococcus aureus 
Antibacterial and antifungal effects by disruption of cell membrane. It also
binds and neutralizes lipopolysaccharide from Gram-negative bacteria
Cystatins block the action of bacterial proteases [
Known as salivary agglutinin and is identical to Gp-340 expressed in lungs.
Binds to a wide variety of microorganisms [
Kills Gram-negative and Gram-positive bacteria [
Agglutinates bacteria and prevents its adhesion to oral surfaces [
Binds to Staphylococcus aureus 
Bacteriostatic due to its iron-depriving effects [
Catalysis the formation of bactericidic compounds [
Defense response to bacterium; regulation of macrophage activation; lysis
bacteria cell wall polysaccharides; activates bacterial autolysins [
Modulates the microbial colonization of oral epithelial surfaces [
Binds to a variety of bacteria [49,50,59±61]
Gel-forming mucin that lubricates saliva and plays a role in reducing adherence
and increasing clearance of bacteria [
Catalyses the hydrogen peroxide oxidation of thiocynate ions which forms the
bactericidal product, hypothiocyanite [
It binds to the bacterial cell wall peptidoglycans to exert the bactericidal effect,
but do not permeabilize bacterial membranes. They are bactericidal for
Grampositive bacteria and bacteriostatic for Gram-negative bacteria [
Polymeric immunoglobulin receptor
Serpin B10 (predicted)
Transports IgA across epithelial cells [
Positive regulation of defense response to virus by host
Also known as calgranulin A (S100-A8) and B (S100-A9). The dimer of
calgranulin A and B is called calprotectin is expressed in neutrophils,
macrophages and keratinocytes cytosols. They inhibit bacterial growth by
scavenging divalent cation [
Zymogen granule protein 16 homolog B
Binds to Staphylococcus aureus 
Groups are sequentially as follows: Ancient, Herding-Sighthound, Miscellaneous-1,Miscellaneous-2
The salivary proteomic and peptidomic content of the Ancient group
stands out from other breed groups
Comparative analyses using a Venn diagram showed that the four groups shared 614 proteins
and peptides representing 38.7% of the analyzed proteins/peptides (Fig 2). In contrast to the
other three groups, unique proteins (SPTBN2 (normalized spectral count = 7), TMOD3 (6)
and PSMC4 (5)) were identified only in the Ancient group. In addition, there were 245
PLOS ONE | https://doi.org/10.1371/journal.pone.0191307
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Fig 2. Venn diagram displaying the overlapped and unique proteins among the four groups. H-S =
(15.5%) proteins that were shared by the Herding-Sighthound and Miscellaneous 1 and 2
groups, but not the Ancient group (Fig 2).
The unique protein profile in the Ancient group was also reflected in the PCA analysis (Fig
3). The Ancient group nested distant from the other three groups at the far end of PC1
(Xaxis), which explained 47.6% of the total variance. When taking into consideration PC2
(Yaxis), which explained 26.8% of total variance, Herding-Sighthound and Miscellaneous group
2 clustered more closely than Miscellaneous group 1 and Ancient group.
The intergroup relationship in the PCA analysis is also confirmed by the dendrogram in
Fig 4, which shows the relationship of the four groups. The predominance of blue color in the
Ancient group indicates relatively less abundance of specific proteins and peptides in this
group compared to the other groups.
Using nanoscale LC±MS/MS we identified 2,491 proteins and peptides in the saliva of healthy
dogs with no periodontal diseases. In contrast, a recent study described only 244 proteins in
dog saliva [
]. The substantially higher number of proteins identified in this study could be
partially explained by differences in sample collection but a main contributor is most likely the
mass spectrometer instrumentation used. For our study, we used a sophisticated mass
spectrometer (Orbitrap Fusion Tribrid with Easy-nLC autosampler and LC (Thermo Scientific,
Waltham, MA) for LC-MS/MS analysis which provides some of the highest sensitivity
currently available for analysis of complex protein mixtures [
]. The prior study by de
Sousa-Pereira and colleagues utilized an older generation MALDI-TOF/TOF instrument [
most likely explains the order of magnitude difference in proteins identified. It also highlights
the depth of our study in terms of proteins and endogenous peptides identified, which
provides a much more comprehensive view of the salivary proteome and peptidome in healthy
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Fig 3. Two dimensional principal coordinate analysis showing the salivary protein and peptide profile relationship among the groups.
One of the most important functions of saliva is to protect the oral cavity and indirectly
other organs against infections. In this study, 7 of the top 10 most abundant proteins have
immune functions. Additionally, we identified 26 peptides and proteins (as well as some
isoforms) that have been reported to have antimicrobial functions in human saliva; 4 of these
were also in the top 10 most abundant in canine saliva. Six of the 26 proteins and peptides
were not present in all four breed groups indicating the variability among individual dogs or
dog breeds. There are likely many additional proteins and peptides with antimicrobial
functions in the 2,491 identified in the study.
Antimicrobial peptides (AMP) are small molecular weight, typically cationic peptides that
have a broad spectrum of action against bacteria, fungi, parasites and some viruses [
are an important part of the innate immune response of almost all living organisms including
plants, invertebrates and vertebrates and generally function by forming holes in the
microorganisms' cell membrane. Various AMP such as, alfa and beta defensins, cathelicidin,
adrenomedullin, histatins, elafin, secretory leukocyte protease inhibitor (SLPI) and lysozyme have
been found in human saliva [
]. Of these, we identified precursors of elafin, SLPI,
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Fig 4. Heatmap showing the relative abundance (color) and relationship (dendogram) of salivary proteins and peptides among the groups.
cathelicidin and lysozyme and, de Sousa-Pereira and collaborators listed lysozyme in the saliva
of the dog included in their study [
]. β-defensins are expressed in many epithelial tissues and
have been identified in the skin of healthy dogs [68±70]. The absence of this category of AMP
in the dog saliva was, to some extent, unexpected but, defensins were not reported in the saliva
of dog, cattle, sheep, horse, rabbit and rat in a recent study [
]. Moreover, the lack of
identification could be explained by their presence below the limit of detection of the method used.
Differences in the expression of proteins and peptides in the saliva of humans and dogs could be
partially explained by phylogenetic and dietary variations between these species. However,
additional studies including a large number of dogs will be needed to corroborate our
The dogs in this study were selected to represent diverse ancestral lineages both to provide
a comprehensive dataset of the canine salivary proteome and peptidome and to determine if
there were clear differences between breed groups. While only two of the four groups
ultimately represented distinct genetic clusters based on the most recent canine genomics data,
differences were evident, with only 38.7% of the analyzed proteins and peptides shared by all
groups. The Ancient group, which included Siberian Huskies and Alaskan Malamutes, was the
most distinct, and the only one with unique proteins. This parallels genetic differences in the
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breed groups; the breeds within the ancient group have a high level of divergence from other
breeds. It cannot be determined from this data whether the unique proteins identified in the
Ancient group are specifically characteristic of the northern group (the clade comprising the
Siberian Husky and Alaskan Malamute) or if they are also a feature of saliva from other ancient
]. Future studies including a larger breed representation in the various phylogenic
groups and a larger number of dogs per breed will help answer this question. Interestingly, a
recent study showed unique proteins in the saliva of Korean people when compared to a
comprehensive database of human salivary proteins indicating ethnic differences in the human
saliva proteome [
In addition to genetic differences, other factors not investigated in this study could have
also played a role in variations noted between the dog groups. Age, diurnal variation, health
status and individual variation have all been shown to influence the composition of proteins in
the saliva in humans [72±78]. These variables, and possibly others, most likely also impact the
salivary protein and peptide profiles of dogs and need to be carefully and urgently investigated
before we can obtain accurate information on changes in saliva protein and peptide
components in disease states.
This study provides a comprehensive catalog of the proteins and endogenous peptides
present in canine saliva. We included 36 dogs and divided them in groups based on breed
phylogeny which revealed differences that parallel genetic clusters. Samples were pooled within each
group, and this could have masked any inter-individual or gender variances in the salivary
protein composition of the dogs. An important next step is to evaluate any possible influence
of age, gender, breed and individual in the composition of proteins and peptides of the dog
S1 Appendix. Detailed information on identified proteins and peptides for the analysis of
intact canine salivary proteins.
S2 Appendix. Detailed information on identified endogenous peptides in canine saliva.
S3 Appendix. Comprehensive list of protein and peptide genes identified in the saliva of
dogs in each of the four groups.
The authors thank Dr. Kevin Stepaniuk for helping with sample collection. The authors also
thank the Center for Mass Spectrometry and Proteomics at the University of Minnesota for
assistance with data generation and analysis, and maintenance of required instrumentation.
Conceptualization: Sheila M. F. Torres, Timothy J. Griffin.
Data curation: Eva Furrow, Clarissa P. Souza, Jennifer L. Granick, Ebbing P. de Jong, Timothy
J. Griffin, Xiong Wang.
Formal analysis: Sheila M. F. Torres, Eva Furrow, Ebbing P. de Jong, Timothy J. Griffin,
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Funding acquisition: Sheila M. F. Torres.
Investigation: Clarissa P. Souza.
Methodology: Sheila M. F. Torres, Eva Furrow, Clarissa P. Souza, Ebbing P. de Jong, Timothy
J. Griffin, Xiong Wang.
Project administration: Sheila M. F. Torres, Jennifer L. Granick.
Resources: Ebbing P. de Jong, Timothy J. Griffin.
Software: Jennifer L. Granick, Ebbing P. de Jong, Timothy J. Griffin, Xiong Wang.
Supervision: Sheila M. F. Torres.
Validation: Ebbing P. de Jong, Xiong Wang.
Visualization: Jennifer L. Granick.
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