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Mapping and identification of soft corona proteins at nanoparticles and their impact on cellular association

06 Feb 2020-bioRxiv (Cold Spring Harbor Laboratory)-

TL;DR: It is highlighted that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces, and the weakly interacting proteins in the SC are revealed as modulators of nanoparticle-cell association, in spite of their short residence time.
Abstract: The current understanding of the biological identity that nanoparticles may acquire in a given biological milieu is mostly inferred from the hard component of the protein corona (HC). The composition of soft corona (SC) proteins and their biological relevance have remained elusive due to the lack of analytical separation methods. Here, we identified a set of specific corona proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ click-chemistry reaction. We show that these SC proteins are present also in the HC, but are specifically enriched after the capture, suggesting that the main distinction between HC and SC is the differential binding strength of the same proteins. Interestingly, the weakly interacting proteins in the SC are revealed as modulators of nanoparticle-cell association, in spite of their short residence time. We therefore highlight that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces.
Topics: Protein Corona (59%)

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ARTICLE
Mapping and identication of soft corona proteins
at nanoparticles and their impact on cellular
association
Hossein Mohammad-Beigi
1,2
, Yuya Hayashi
3
, Christina Moeslund Zeuthen
1,2
, Hoda Eskandari
1,2
,
Carsten Scavenius
3
, Kristian Juul-Madsen
4
, Thomas Vorup-Jensen
4
, Jan J. Enghild
3
&
Duncan S. Sutherland
1,2
The current understanding of the biological identity that nanoparticles may acquire in a given
biological milieu is mostly inferred from the hard component of the protein corona (HC). The
composition of soft corona (SC) proteins and their biological relevance have remained elusive
due to the lack of analytical separation methods. Here, we identify a set of specic corona
proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ
click-chemistry reaction. We show that these SC proteins are present also in the HC, but are
specically enriched after the capture, suggesting that the main distinction between HC and
SC is the differential binding strength of the same proteins. Interestingly, the weakly inter-
acting proteins are revealed as modulators of nanoparticle-cell association mainly through
their dynamic nature. We therefore highlight that weak interactions of proteins at nano-
particles should be considered when evaluating nano-bio interfaces.
https://doi.org/10.1038/s41467-020-18237-7
OPEN
1
Interdisciplinary Nanoscience Centre (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark.
2
The Centre for Cellular Signal Patterns
(CellPAT), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark.
3
Department of Molecular Biology and Genetics, Aarhus University, Gustav
Wieds Vej 10, 8000 Aarhus C, Denmark.
4
Department of Biomedicine, Faculty of Health, Aarhus University, Høegh-Guldbergs Gade 10, 8000 Aarhus
C, Denmark.
email: duncan@inano.au.dk
NATURE COMMUNICATIONS | (2020) 11:4535 | https://doi.org/10.1038/s41467-020-18237-7 | www.nature.com/naturecommunications 1
1234567890():,;

N
anoparticles (NPs) are promising agents for drug delivery
and visualization in vivo. Upon exposure to biouids, the
NPs acquire a protein corona due to the adherence of
host proteins on the NPs surface. The composition of the corona
is dependent on the types of nanoparticles and the biological
sources
13
, and considered to provide the NPs with a biological
identity
4
that affects stability, circulation time, and cellular
uptake/interactions, and therefore has a strong impact on the
functional role for the NPs
510
.
The formation of protein corona leads to two main con-
sequences that determine how well the nanoparticles associate
with cells. Under a serum-free condition, pristine nanoparticles
spontaneously bind to cell membranes in a nonspecic manner
lowering their surface energy while protein coronas, in general,
reduce this nonspecic interaction as less nanoparticle surface is
exposed
11
. In parallel, nanoparticle-bound proteins provide the
potential for specic interactions during cell association (CA),
including receptor-mediated membrane adhesion and subsequent
uptake
12,13
, and contribute to the resultant biomolecular corona-
dened biological identity of the nanoparticles
14,15
. Therefore, to
predict the biological behavior of nanoparticles, it is essential to
have a combined understanding of the composition and structure
of protein corona.
Kinetic evaluation of protein corona formation and identi-
cation of the proteins forming the corona have become active
research topics aiming to understand the particokinetics, cellular
interactions, and mechanisms of nanoparticle toxicity
16
.Ina
complex and dynamic process, proteins competitively adhere to
the surface of nanoparticles to form a combined Hard (HC) and
Soft corona (SC). HC proteins with a high binding afnity and
low dissociation rate remain tightly bound to the surface, whereas
SC proteins with a high dissociation rate are rapidly exchanged.
At the surface of nanoparticles, proteins can undergo reorienta-
tion and conformational changes
17,18
, presumably leading to at
least a partially denatured state that has a reduced dissociation
rate in a process referred to as hardening
19
. The evolution and
dynamics of HC formation are relatively well studied
14,2022
;HC
is established rapidly, and the evolution of HC over time is only
quantitative with altered relative amounts, rather than the
changes in protein composition expected from the Vroman
effect
12
. The current understanding is that the HC proteinswith
their long residence timegive the nanoparticles a biological
identity by presenting receptor-binding sites for cellular interac-
tions with a biologically relevant timescale
23
. As SC proteins by
denition have a shorter residence time on nanoparticles than
HC proteins making them difcult to isolate from free proteins of
the mother liquid, their potential biological impacts through
specic and/or nonspecic interactions have often been ignored.
Recent work has developed approaches to quantify SC protein
binding and address the potential of soft interactions to modulate
toxicity by localized suldation at the surface of silver nano-
particles
16
. Several methods, such as centrifugation-based
separation techniques together with proteomic characteriza-
tion
24
or multistep centrifugation
25
, are proposed to retain a
larger fraction of the HC proteins for identication during
separation albeit still after long times. In the centrifugation-based
separation technique, by using transmission electron microscopy
technique, it is shown that protein corona is an undened loose
network of proteins; however, in that method, there is a risk to
capture bulk proteins between nanoparticles during centrifuga-
tion. Asymmetric ow eld-ow fractionation (A4f)
26
and sur-
face plasmon resonance (SPR) coupled with mass spectroscopy
27
have been applied to PEGylated nanoparticles to identify weakly
protein-binding proteins in stealth systems. In the later case, SC
and HC proteins are identied in a label-free method, and the SC
proteins are found as the stealth component of the biological
identity. However, it is tested only on liposomes. For the rapidly
exchanging SC proteins, several key open questions remain,
including whether SC proteins are different from HC proteins,
and if there is a role for SC proteins in determining cellular
interactions.
To address these critical questions, a comprehensive picture of
corona composition and residence time for SC proteins is needed.
Here, by developing an experimental approach based on click
chemistry, we capture weakly interacting proteins along with HC
proteins for mass spectrometry-based compositional proling,
and identify proteins that are either new or with increased
amount compared to HC layers as SC proteins. We nd that the
majority of the identied SC proteins are not unique to SC, but
are also present in the HC representing different binding strength
states of the same proteins. On the contrary, only a minor frac-
tion of SC proteins are identied exclusively in the SC. Moreover,
as our method forces SC proteins to stay in place by cross-linking,
such that the SC proteins acquire residence time long enough for
biological interactions, we are able to demonstrate a role for the
SC proteins in cell association of nanoparticles, that are depen-
dent both on the type of cells and nanoparticles. Therefore,
turning off the dynamic nature of dissociation, which is the
modulation of real condition for cell studies, provides us the
possibility to study the effect of the dynamic nature of SC proteins
on cell association.
Results
A click-chemistry method captures SC proteins. Recently, the
catalyst-free strain-promoted alkyne azide cycloaddition
(SPAAC) click chemistry has gained interest in many biological
and medical applications due to its high speed, efciency, speci-
city, and bioorthogonality
2831
. Therefore, we have developed a
SPAAC click reaction between azide-modied HC proteins on
nanoparticles (HC-N
3
) and dibenzocyclooctyne (DBCO)-acti-
vated SC proteins (FBS-D) (Fig. 1a) in order to trap the tran-
siently binding SC proteins on the NPs surface (HC+SC sample).
Sulfo-SASD and DBCO-sulfo-NHS were used for the modica-
tion of proteins to perform the click-chemistry reaction described
in Fig. 1a. The modication occurs through the reaction between
sulfo-NHS moieties on the cross-linkers with primary amines on
proteins. None of the azide or DBCO-reactive groups on these
heterobifunctional cross-linkers react with any of the functional
groups on proteins, which avoid cross-linking of HC or SC
proteins with other HC or SC ones. Moreover, sulfo-SASD con-
tains a dithiol, which provides a possibility to cleave the covalent
bond between proteins by using reducing agents for analysis.
Negatively charged hydrophilic silica nanoparticles (SNPs, 70
nm) and hydrophobic carboxyl-modied polystyrene nano-
particles (PsNPs, 100 nm) were used in this study as model
nanoparticles
13,32,33
. We used four control samples representing
HC and FBS with and without chemical modications (hard
corona (HC), hard corona modied with azide (HC-N
3
), FBS-D
added to HC (D Ctrl), and FBS added to HC-N
3
(N
3
Ctrl)), and
one HC+SC sample that encompasses proteins in both HC and
captured SC states.
We rst optimized the click-reaction conditions. After
formation of HC on SNPs, the concentrations of sulfo-SASD
for modication of HC proteins was optimized. The results show
that all HC proteins on SNPs were modied with azide at
0.55 mM of sulfo-SASD, with at least one azide group per HC
protein (Supplementary Fig. 2). Then, the concentration of
DBCO-sulfo-NHS for labeling free FBS proteins was optimized
by measuring the degree of labeling and the extent of the click
reaction (Supplementary Fig. 3 and Supplementary Table 1).
DBCO-modied proteins were then added to SNPs@HC-N
3
to
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18237-7
2 NATURE COMMUNICATIONS | (2020) 11:4535 | https://doi.org/10.1038/s41467-020-18237-7 | www.nature.com/naturecommunications

capture weakly interacting proteins. SDS-PAGE analysis revealed
a change in protein patterns after the click reaction, which was
further validated by capturing uorescently labeled proteins
(Supplementary Fig. 4). Quanti cation of total protein per
nanoparticle further conrmed the increase in the mass of the
corona proteins (~50 µg ml
1
nanoparticles) after the click
reaction. Extending the reaction time from 2 to 16 h did not
result in a further increase in mass (Fig. 1d). The amount of SC
proteins captured was positively correlated with the amount of
HC proteins bound to the nanoparticles, which increased as a
function of incubation time (Fig. 1e and Supplementary Fig. 5).
Illustrating the applicability of this method to other types of
nanoparticles with different surface chemistry and charge, we
observed comparable BCA and SDS-PAGE results for mixed
charge amine-modied SNPs (SANPs, 75 nm), highly charged
carboxyl-modied SNPs (SCNPs, 75 nm), and PsNPs (Fig. 1f and
Supplementary Fig. 6).
Our click-chemistry approach to capturing SC proteins
maintained a colloidally stable population of nanoparticlecorona
complexes with a slightly increased hydrodynamic size and an
increased particle heterogeneity (Fig. 1gm, Table 1, and
Supplementary Table 2). We further analyzed the formation of
the protein corona by negative staining (TEM), which revealed a
globular appearance of dehydrated proteins on SNPs (Fig. 1ik
µ
µ
a
b
HC-N
3
200
120
85
70
50
60
40
25
30
15
20
2 h
coomassie staining
cd
lkji
Protein corona
formation
Washing and centrifugation:
SC proteins leave NPs
Step 1
Step 2
Step 3
Functionalizetion of HC proteins
with azide (N
3
)
DBCO
N
3
N
3
N
3
N
3
N
3
+N
NaO
3
S
3
Step 5
DBCO-Sulfo-NHS
Sulfo-SASD
Step 4
+
FBS
SPAAC “Click”
50 nm
50 nm
50 nm
50 nm
0.0
0.2
0.4
0.6
0.8
1.0
Normalized Gray value
HC-N3
HC+SC (2h)
HC+SC (16h)
std
1520254050
70
100
120200
10
0
10
1
10
2
10
3
10
4
0
5
10
15
20
25
Hydrodynamic diameter (nm)
10
0
10
1
10
2
10
3
10
4
Hydrodynamic diameter (nm)
Scattering intensity (%)
0
5
10
15
20
25
Scattering intensity (%)
HC+SC
D Ctrl
N Ctrl
HC-N3
HC
pristine
HC-N
HC+SC (2 h)
HC+SC (16 h)
0
50
100
150
200
250
300
350
ns
µg proteins/ mg NPs
h
m
MW
HC-N
3
N Ctrl
HC+SC
g
200
225
250
275
300
325
0.0 0.2 0.4 0.6 0.8
1.0
0 10 20 30 60 120 180 240 300
360
Time (min)
µg proteins/ mg NPs
*
*
*
*
*
16 h
HC+SC
std
Normalized Run length
Incubation times
HC+SC
D Ctrl
N Ctrl
HC-N3
HC
pristine
0
50
100
150
200
250
µg proteins/ mg NPs
n.s.
HC-N3
HC+SC
N Ctrl
PsNPs
Capturing SC proteins
PsNPsSNPs
e
f
kDa
kDa
p=0.006
p=0.016
p=0.025
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18237-7 ARTICLE
NATURE COMMUNICATIONS | (2020) 11:4535 | https://doi.org/10.1038/s41467-020-18237-7 | www.nature.com/naturecommunications 3

and Supplementary Fig. 7), while a more diffuse appearance was
observed for PsNPs (Fig. 1l, m and Supplementary Fig. 8). Image
analysis of the SNPs conrmed a broadened distribution of the
maximum Feret particle diameters with an increase in the mean
size from 72 nm (HC) to 87 nm (HC+SC) (Supplementary
Fig. 7d).
SC/HC show different binding states of the same proteins.
Using the click reaction to x the weakly interacting proteins in
place, we were able to isolate SC proteins along with HC proteins
by centrifugation and subject them to proteomic quantication by
tandem mass spectrometry (LCMS/MS). It should be mentioned
that to avoid the potential for changes in the overall protein
interactions with nanoparticles, e.g., highly modied BSA
34
,we
labeled the HC proteins with N
3
after formation on NPs and with
a relatively low level of labeling. Further centrifugation and
manipulation steps applied in this method, such as N
3
mod-
ication, did not signicantly desorb HC proteins from nano-
particles; however, we believe that the slight effect of
modications on the SC prole is unavoidable.
We rst calculated the copy number of each identied protein
per nanoparticle following quantitation of the total protein mass
(by BCA assays), nanoparticle mass (by uorimetry), and emPAI-
based relative mass percentages of proteins identied in LCMS/
MS. This allows a comparison of different samples without bias
for large-sized proteins or the total protein input. Next, a cluster
of proteins specically enriched in the HC+SC sample (based on
the copy number of corona proteins per nanoparticle) was
identied using bottom-up cluster analysis to construct two-way
dendrograms along with a heatmap (Fig. 2a). In this approach,
having a higher copy number than in the four control HC
samples is not automatically considered to be indicative of an SC
protein because a higher copy number may also be acquired by
random variation. Therefore, the SC protein cluster is restricted
to proteins that had a consistently lower (or zero) copy number in
all of the four control samples without a large variation among
them. The column dendrogram clearly separates the HC+SC
sample from the rest. The row dendrogram reveals a putative SC
cluster (colored in orange) characterized by specic enrichment
of the proteins in HC+SC. For SNPs, 20 proteins were considered
as SC proteins among the total of 80 proteins identied by
LCMS/MS, and only 4 out of the 20 SC proteins were uniquely
captured after the click reaction (i.e., undetected in all HC
controls), while the others were found in the HC controls to some
extent (Fig. 2b). The total copy number of all proteins per NP
increased 1.15-fold after the click reaction (cf. ~1.2-fold increase
in the total protein mass in Fig. 1e), and the increase was mainly
due to higher copy numbers of proteins belonging to the SC
cluster (Fig. 2c), indicating the specic enrichment of these SC
proteins. This can also be explained by the fact that the top 5
abundant proteins, which account for >50% of the total, remained
the same even after the click reaction, whereas SC cluster proteins
were ranked higher than before (e.g., 5.7-fold increase in the
abundance of APOH, Table 2, SNPs and Fig. 2b). Importantly,
the absence of highly abundant serum proteins, such as albumin
in the SC cluster, shows that our click-chemistry method used in
a competitive situation captures only proteins that are resident at
the surface through a weak interaction with HC proteins and/or
NPs surface and not proteins directly from the bulk.
Most of the SC proteins were also found in the HC, leading us
to rethink our initial hypothesis that the SC is formed from
different proteins from those in the HC. Our results rather
indicate that the same proteins could have different binding
constants, and that SC proteins are those capable of both stable
and transient interactions. For simplicity, we describe the SC
proteins as generally having two binding states: hard and soft.
The two binding states are assumed from the default presence of
SC proteins in HC (hard binding) and upon click capturing of
additional SC proteins in HC+SC (soft binding). Accordingly, we
classied SC proteins into three types based on the relative copy
numbers in the hard versus soft binding state: Type-1 SC proteins
have more copies undergoing hard interactions, Type-2 SC
proteins have similar copy numbers in the HC and SC, and
Type-3 SC proteins have more copies undergoing soft interactions.
This classication is visualized in Fig. 2d, e, where only the SC
Fig. 1 SPAAC click-chemistry reaction and characterization of nanoparticlecorona complexes. a Schematic representation of capturing SC proteins.
After protein corona formation (steps 1 and 2), the HC proteins were modied with N
3
by reacting with sulfo-SASD (step 3) followed by a SPAAC click
reaction (step 5) with FBS-D proteins (prepared in step 4). bd Effect of exposure time periods (2 and 16 h) in the click reaction evaluated by coomassie
staining images (b) and densitometry analysis of SDS-PAGE gel (c), and quantication (d) of protein corona recovered from SNPs. The SDS-PAGE analysis
was repeated three times independently with similar results. e Quantication of HC+SC proteins captured by click reaction on HC proteins formed on
SNPs over different incubation times (15 min, 30 min, 1 h, 2 h, and 6 h). The SDS-PAGE image and densitometry analysis of the proteins are shown in
Supplementary Fig. 5. f Quantication of HC+SC proteins captured by click reaction on PsNPs. Quantication data in df represented as the mean ± s.d. of
three independent experiments (n = 3). For the multiple comparison, P value was calculated by one-way ANOVA with Tukey post hoc test
without any adjustment. *P < 0.05; n.s., not signicant (P > 0.05). g, h Hydrodynamic analysis of nanoparticlecorona complexes, SNPs (g), and PsNPs (h).
im Transmission electron microscopy (TEM) analysis of the SNPs@HC (i), SNPs@HC+SC (j, k), PsNPs@HC (l), and PsNPs@HC+SC (m). TEM analysis
was performed three times independently with similar results. Scale bar, 50 nm. FBS-D: FBS proteins modied with DBCO, pristine silica nanoparticles
(SNPs), pristine polystyrene nanoparticles (PsNPs), hard corona (HC), hard corona modied with azide (HC-N
3
), FBS-D added to HC (D Ctrl), FBS added
to HC-N
3
(N
3
Ctrl), FBS-D added to HC-N
3
(HC+SC), HC-coated SNPs (SNPs@HC), and HC-coated PsNPs (PsNPs@HC). Source data are provided as a
Source data le.
Table 1 Characterization of nanoparticlecorona complexes
in buffer.
Nanoparticlecorona
complexes
Zeta
potential ±
SD (mV)
Hydrodynamic
diameter ± SD
(nm) (PDI)
SNPs Pristine 22 ± 3.4 81 ± 5.1 (0.01)
HC 20 ± 4.1 106 ± 5.7 (0.06)
HC-N
3
25 ± 2.4 128 ± 6.3 (0.12)
D Ctrl 23 ± 3.7 133 ± 7.5 (0.08)
N
3
Ctrl 26 ± 3.3 121 ± 10.2 (0.07)
HC+SC 29 ± 2.9 152 ± 12.4 (0.17)
PsNPs Pristine 27 ± 2.1 110 ± 11.2 (0.02)
HC 26.2.3 155 ± 8.3 (0.1)
HC-N
3
25 ± 3.1 168 ± 14.2 (0.15)
D Ctrl 23 ± 2.8 179 ± 13.6 (0.19)
N
3
Ctrl 26 ± 4.3 175 ± 9.2 (0.2)
HC+SC 25 ± 4.2 191 ± 15.3 (0.18)
The average size of nanoparticlecorona complexes was determined using DLS, and the zeta
potential measurement data processing was done by using Smoluchowski model. Zeta potential
measurement was done in 10 mM sodium phosphate buffer, pH 7.4, containing 10 mM NaCl.
Data shown correspond to mean ± s.d. of three independent experiments (n = 3).
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18237-7
4 NATURE COMMUNICATIONS | (2020) 11:4535 | https://doi.org/10.1038/s41467-020-18237-7 | www.nature.com/naturecommunications

cluster proteins are displayed but with a different color code for
each SC type. The total copy number of the SC cluster proteins
increased ~2-fold after the click reaction with Type-3 SC proteins
representing the major SC fraction, which were effectively
captured (Fig. 2d) and thus overall increased in the copy number
(Fig. 2e). In HC samples, the relative contribution of Type-1 SC
proteins (blue) is larger than Type-3 SC proteins (orange), and
vice versa in HC+SC samples. Of particular note, we observed a
tendency for Type-3 SC proteins to have a higher GRAVY score
and instability index, suggesting that these proteins are inherently
less hydrophilic and less stable in serum (Fig. 3a). Neither the
isoelectric point (Fig. 3a) nor multiparametric combinations of the
a
−1 0 1
Zscore
HC+SC
D Ctrl
N Ctrl
HC
HC-N
3
HC+SC
D Ctrl
HC-N
3
SNPs
−1 0 1
Zscore
PsNPs
HC
N Ctrl
Protein cluster
HC (60 unique proteins)
SC (20 unique proteins)
HC
#prot 1341
HC+SC
#prot 1543
APOA1
APOH
ALB (BSA)
SC type
1 2 3
APOH
0
10
20
30
0
5
10
HC HC+SC
# proteins (square root)
HC cluster
SC cluster
0.0
2.5
5.0
# proteins (square root)
Hard
Soft
Type1
SC
Type2
SC
Type3
SC
Binding states
Hard
Soft
Hard
Soft
bc
d
Protein cluster
HC (80 unique proteins)
SC (36 unique proteins)
APOA1
AGT
ALB (BSA)
SC type
1 2 3
0
10
20
30
0
5
10
HC HC+SC
# proteins (square root)
HC cluster
SC cluster
0.0
2.5
5.0
# proteins (square root)
Hard
Soft
Type1
SC
Type2
SC
Type3
SC
Binding states
Hard
Soft
Hard
Soft
fg
e
AGT
HC
#SC prot 80
HC+SC
#SC prot 163
HC
#prot 2076
HC+SC
#prot 2487
HC
#SC prot 247
HC+SC
#SC prot 463
h
i
j
HC HC
HC SC
SC
p=0.0099, n=20
p=0.0081, n=36
n=60
n=80
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Journal ArticleDOI
TL;DR: The ExPASy (the Expert Protein Analysis System) World Wide Web server, provided as a service to the life science community by a multidisciplinary team at the Swiss Institute of Bioinformatics, provides access to a variety of databases and analytical tools dedicated to proteins and proteomics.
Abstract: The ExPASy (the Expert Protein Analysis System) World Wide Web server (http://www.expasy.org), is provided as a service to the life science community by a multidisciplinary team at the Swiss Institute of Bioinformatics (SIB). It provides access to a variety of databases and analytical tools dedicated to proteins and proteomics. ExPASy databases include SWISS-PROT and TrEMBL, SWISS-2DPAGE, PROSITE, ENZYME and the SWISS-MODEL repository. Analysis tools are available for specific tasks relevant to proteomics, similarity searches, pattern and profile searches, post-translational modification prediction, topology prediction, primary, secondary and tertiary structure analysis and sequence alignment. These databases and tools are tightly interlinked: a special emphasis is placed on integration of database entries with related resources developed at the SIB and elsewhere, and the proteomics tools have been designed to read the annotations in SWISS-PROT in order to enhance their predictions. ExPASy started to operate in 1993, as the first WWW server in the field of life sciences. In addition to the main site in Switzerland, seven mirror sites in different continents currently serve the user community.

3,742 citations


Book
01 Sep 1997
TL;DR: Fuzzy Databases: Principles and Applications is comprehensive covering all of the major approaches and models of fuzzy databases that have been developed including coverage of commercial/industrial systems and applications.
Abstract: From the Publisher: This volume presents the results of approximately 15 years of work from researchers around the world on the use of fuzzy set theory to represent imprecision in databases. The maturity of the research in the discipline and the recent developments in commercial/industrial fuzzy databases provided an opportunity to produce this survey. Fuzzy Databases: Principles and Applications is self-contained providing background material on fuzzy sets and database theory. It is comprehensive covering all of the major approaches and models of fuzzy databases that have been developed including coverage of commercial/industrial systems and applications. Background and introductory material are provided in the first two chapters. The major approaches in fuzzy databases comprise the second part of the volume. This includes the use of similarity and proximity measures as the fuzzy techniques used to extend the relational data modeling and the use of possibility theory approaches in the relational model. Coverage includes extensions to the data model, querying approaches, functional dependencies and other topics including implementation issues, information measures, database security, alternative fuzzy data models, the IFO model, and the network data models. A number of object-oriented extensions are also discussed. The use of fuzzy data modeling in geographical information systems (GIS) and use of rough sets in rough and fuzzy rough relational data models are presented. Major emphasis has been given to applications and commercialization of fuzzy databases. Several specific industrial/commercial products and applications are described. These include approaches to developing fuzzy front-end systems and special-purpose systems incorporating fuzziness.

3,065 citations


Journal ArticleDOI
Tommy Cedervall1, Iseult Lynch, Stina Lindman, Tord Berggård  +4 moreInstitutions (1)
TL;DR: The rates of protein association and dissociation are determined using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein–nanoparticle mixtures, and this method is developed into a systematic methodology to isolate nanoparticle-associated proteins.
Abstract: Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. These properties are rapidly revolutionizing many areas of medicine and technology. Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and presentation of the proteins on the surface of the particles leads to an in vivo response. Proteins compete for the nanoparticle "surface," leading to a protein "corona" that largely defines the biological identity of the particle. Thus, knowledge of rates, affinities, and stoichiometries of protein association with, and dissociation from, nanoparticles is important for understanding the nature of the particle surface seen by the functional machinery of cells. Here we develop approaches to study these parameters and apply them to plasma and simple model systems, albumin and fibrinogen. A series of copolymer nanoparticles are used with variation of size and composition (hydrophobicity). We show that isothermal titration calorimetry is suitable for studying the affinity and stoichiometry of protein binding to nanoparticles. We determine the rates of protein association and dissociation using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein-nanoparticle mixtures. This method is less perturbing than centrifugation, and is developed into a systematic methodology to isolate nanoparticle-associated proteins. The kinetic and equilibrium binding properties depend on protein identity as well as particle surface characteristics and size.

2,441 citations


Journal ArticleDOI
TL;DR: The basic concept of the nanoparticle corona is reviewed and its structure and composition is highlighted, and how the properties of the corona may be linked to its biological impacts are highlighted.
Abstract: The search for understanding the interactions of nanosized materials with living organisms is leading to the rapid development of key applications, including improved drug delivery by targeting nanoparticles, and resolution of the potential threat of nanotechnological devices to organisms and the environment. Unless they are specifically designed to avoid it, nanoparticles in contact with biological fluids are rapidly covered by a selected group of biomolecules to form a corona that interacts with biological systems. Here we review the basic concept of the nanoparticle corona and its structure and composition, and highlight how the properties of the corona may be linked to its biological impacts. We conclude with a critical assessment of the key problems that need to be resolved in the near future.

1,909 citations