Cyanine-Dyad Molecular Probe for the Simultaneous Profiling of the Evolution of Multiple Radical Species During Bacterial Infections

Article abstract of DOI:10.1002/anie.202104100

Real-time monitoring of the evolution of bacterial infection-associated multiple radical species is critical to accurately profile the pathogenesis and host-defense mechanisms. Here, we present a unique dual wavelength near-infrared (NIR) cyanine-dyad molecular probe (HCy5-Cy7) for simultaneous monitoring of reactive oxygen and nitrogen species (RONS) variations both in vitro and in vivo. HCy5-Cy7 specifically turns on its fluorescence at 660 nm via superoxide or hydroxyl radical (O₂^.?, ^.OH)-mediated oxidation of reduced HCy5 moiety to Cy5, while peroxynitrite or hypochlorous species (ONOO^?, ClO^?)-induced Cy7 structural degradation causes the emission turn-off at 800 nm. Such multispectral but reverse signal responses allow multiplex manifestation of in situ oxidative and nitrosative stress events during the pathogenic and defensive processes in both bacteria-infected macrophage cells and living mice. Most importantly, this study may also provide new perspectives for understanding the bacterial pathogenesis and advancing the precision medicine against infectious diseases.

Full text of DOI:10.1002/anie.202104100

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Accepted Article
Title: Cyanine-Dyad Molecular Probe for Simultaneous Profiling of
Multiple Radical Species Evolution in Bacterial Infection
Authors: Bengang Xing, Zhimin Wang, Thang Cong Do, Wenbin
Zhong, Jun Wei Lau, Germain Kwek, and Mary B. Chan-Park
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202104100
Link to VoR: https://doi.org/10.1002/anie.202104100

10.1002/anie.202104100
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COMMUNICATION
Cyanine-Dyad Molecular Probe for Simultaneous Profiling of
Multiple Radical Species Evolution in Bacterial Infection
Zhimin Wang+, Thang Do Cong+, Wenbin Zhong, Jun Wei Lau, Germain Kwek, Mary B. Chan-Park and
Bengang Xing*
Abstract: Real-time sensing the evolution of bacterial infection-
associated multiple radical species is critical to accurately profile
the pathogenesis and host-defense mechanisms, however, only
a few currently existing techniques are capable of fully achieving
the demanded accuracy in complex living conditions. Here, we
present a unique dual wavelength near-infrared (NIR) cyanine-
dyad molecular probe (HCy5-Cy7) for simultaneous monitoring
of reactive oxygen and nitrogen species (RONS) variations both
in vitro and in vivo. HCy5-Cy7 specifically turns on its
pathological evolution of infections. The accessibility of the
traditional strategies have also been impeded due to the
processes of invasiveness, time consumption and risk of
complications.[5] Therefore, simple, safe and effective methods
are highly desired that would provide novel perspectives on
precise diagnosis and therapeutic interventions.
As compared to EPA analyses, optical imaging techniques
demonstrate the advantages of highly sensitive, noninvasive and
time/cost-effective capacities for broad biomedical detections
from the cellular (or molecular) level up to whole-body sections
in living animals.[6] Furthermore, the diverse fluorescence
sensors are emerging as powerful tools to investigate
contagious diseases through specific targeting or responses of
infection-associated bio-indicators including toxins, enzymes,
inflammatory cytokines and other factors.[7] Particularly, some
•-
fluorescence at 660 nm via superoxide or hydroxyl radical (O2 ,
•OH)-mediated oxidation of reduced HCy5 moiety to Cy5, while
peroxynitrite or hypochlorous species (ONOO-, ClO-)-induced
Cy7 structural degradation causes the emission turn-off at 800
nm. Such multispectral but reverse signal responses allow
multiplex manifestation of in situ oxidative and nitrosative stress
events during the pathogenic and defensive processes in both
bacteria-infected macrophage cells and living mice. Most
importantly, this study may also provide new perspectives for
understanding the bacterial pathogenesis and advancing the
precision medicine against infectious diseases.
•-
radical species-activated small molecular probes towards O2 ,
•OH, NO•, ClO-, ONOO- etc. have been developed and readily
utilized in fluorescence imaging of bacterial infection with
superior specificity.[8] In spite of the great success for
understanding the substantive relevance between those radical
species and infections, detailed mechanisms of multi-radical
variations in bacterial pathogenesis and host defence have not
yet been exploited, mainly due to lack of multi-responsive
imaging platforms. Recent studies have attempted to directly
measure infection-induced oxidative and nitrosative stress status
through bio-engineered redox-sensitive GFPs or the
combination of different fluorescent probes,[9] however, these
approaches are still limited by the inadequate multiplexing
capacity, tissue penetration and unified sensing outcomes for
real-time and simultaneous recognition of RONS dynamics
during the pathological infection progression in vivo.
Bacterial infections remain the worldwide challenge in recent
decades, mainly due to the evolving pathogenicity, emergence
of foreign pathogens and spread of antibiotic resistance.[1]
Comprehensive efforts have been devoted to identify the
infections pathogenesis, host defense and antibacterial
mechanisms, which comprise the dynamic inflammatory
response, immune activation as well as lethal actions.[2] Among
the various bioactive factors in reference to infectious diseases,
phagocyte-derived free radical species are widely implicated to
play essential roles in the regulation of bacteria-induced
inflammatory cascade and triggering intrinsic bactericidal
effects.[3] Furthermore, several investigations have also
demonstrated the imperative contributions of multiple radical
mediators including reactive oxygen and nitrogen species
Here, we report a unique dual wavelength near-infrared
(NIR) cyanine-dyad fluorescent molecular probe (HCy5-Cy7) for
simultaneously monitoring of multiple radical species variations
in vitro and in living mice. As illustrated in Scheme 1, upon the
bacterial stimulation, HCy5-Cy7 specifically turns on its
fluorescence at 660 nm due to the local overproduction of
•-
(RONS, e.g. O2 , •OH, H2O2, NO, ONOO−, ClO− etc.) towards
4]
the infection pathological development.[3a,
However,
•-
inflamation-associated O2 or •OH that triggers the oxidation of
conventional studies primarily relying on end-point analysis
(EPA) such as histology and biochemical assays could not real-
time monitor the complex interplay between radical status and
reduced cyanine moiety (HCy5) to conjungated Cy5, while the
burst of ONOO- or ClO- specie induces Cy7 structural
degradation, thereby leading to the emission depletion at 800
nm.[10] Such unique multispectral signal responses allow HCy5-
Cy7 to investigate the dynamic oxidative/nitrosative stress and
can thus correlate their evolutions with the pathogenic and
defensive processes in bacterial infection both in vitro and in
vivo.
[1]
†Z. Wang, †T.D. Cong, J. Lau, G. Kwek, Prof. B. Xing
Division of Chemistry and Biological Chemistry, School of Physical
& Mathematical Sciences, Nanyang Technological University, 21
Nanyang link, 637371, Singapore..
E-mail: Bengang@ntu.edu.sg
[2]
[3]
Prof. B. Xing
We prepared the probe molecule HCy5-Cy7 by coupling
hydroreductive Cy5 (e.g. HCy5) with Cy7 according to Scheme
S1 (Supporting Information). Briefly, the dual carboxylic-
substituted Cy5 was firstly synthesized, which was then reduced
by NaBH4 to obtain HCy5. In parallel, Cy7 fluorophore was
modified with a cyclohexanediamine linker, thereby enabling its
covalent link with the as-synthesized HCy5. The final molecule
HCy5-Cy7 was purified with HPLC and characterized by NMR
and mass spectroscopy (see the Supporting Information for
School of Chemical and Biomedical Engineering, Nanyang
Technological University, 70 Nanyang Drive, 637459, Singapore
Dr. W. Zhong, Prof. M. Chan-Park
School of Chemical and Biomedical Engineering, Nanyang
Technological University, 62 Nanyang Drive, 637459, Singapore.
These authors contributed equally to this work.
[+]
Supporting information for this article is given via a link at the end
of the document.
1
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10.1002/anie.202104100
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COMMUNICATION
Scheme 1. Illustration of the pathogenesis and host defence of bacterial infections (left). Design and principle of HCy5-Cy7 for multiple radical species
imaging (right): ROS oxidation of HCy5 to turn on the emission at 660 nm; RNS degradation of Cy7 to diminish the fluorescence at 800 nm. Ex1/Em1: 620/660
nm, Ex2/Em2: 740/800 nm.
details). Although cyanine dyes have been used for the sensitive
detection of various biological radical species,[11] our rational
design comes from the judicious integration of the O2 and •OH
responsive HCy5 with ONOO--sensitive Cy7 that triggers the
opposite fluoresence changes under two different spectral
channels (e.g. at 660 nm and 800 nm), thus introducing the
multiplexed recognitions of both ROS and RNS simultaneously.
expected, HCy5-Cy7 demonstrated superb sensing specificity
and multiplexed capability towards different radical species
(Figure 1c). The fluorescence analysis exihibited opposite signal
•-
•-
changes that HCy5-Cy7 treated with ROS (e.g. O2 or •OH)
resulted in a positive enhancement of the emission at 660 nm,
whereas no obvious increase was observed upon the reaction
with any other radical species. Meanwhile, fluorescence
variations at 800 nm demonstrated a reverse trend after the
reaction with RNS (e.g. ONOO- or ClO-). In addition, both the
fluorescence intensities at 660, 800 nm were linearly dependent
We envision that such
a smart and multi-sensitive NIR
fluorescent probe (HCy5-Cy7) could be particularly suitable for
the arduous task of real-time multiple reactive species profiling,
attribute to its synchronized biological distribution, metabolism
as well as responsiveness.
•-
with the concentrations of O2 and ONOO- (Figure 1d), in which
the limits of detection (LODs) were determined to be 0.34 and
0.082 μM, respectively. Such dual-channel and reversed
fluorescent signal readout enabled HCy5-Cy7 as a multi-
responsive reporter for simultaneous detection of multiple radical
Firstly, we tested the stability of HCy5-Cy7 in different
environments such as PBS, PBS with 10% fetal bovine serum
(FBS) or 10% human serum, and even cell culture DMEM
(Dulbecco’s Modified Eagle’s-Medium) under dark or room light
exposure. The measurements of absorption and fluorescence
intensities at 660, 800 nm in Figure S1 proved that there is no
obvious self-degradation of the Cy7 moiety or the auto-oxidation
of the HCy5 part of our probe molecule. To validate the sensing
ability of multiple radicals, the absorption and fluorescence
spectra of HCy5-Cy7 were tested in PBS buffer with various
radical species treatments. As shown in Figure 1a, the probe
molecule only showed a major absorption peak at ~780 nm
•-
attributing to the Cy7 chromophore. Upon O2 reaction, a new
peak was appeared at ~ 640 nm, suggesting the structural
conversion from HCy5-Cy7 to π-conjugation regenerated Cy5-
Cy7 (Scheme 1). Differently, the addition of ONOO- led to a
remarkable decrease of the absorption at ~780 nm, which could
be ascribed to the disruption of HCy5-Cy7 framework and the
formation of truncated HCy5-DCy7. Furthermore, fluorescence
responses of this probe with or without representative radical
•-
species (e.g. O2 and ONOO-) treatment were tested upon 620
and 740 nm excitations, respectively (Figure 1b). In comparison
•-
to HCy5-Cy7 alone, addition of O2 induced the emission
occurance at ~ 660 nm while ONOO- incubation significantly
reduced the fluorescence signal at ~ 800 nm, which were
consistent with the absorption changes observed. In addition,
the comparative studies of the photophysical properties between
probe HCy5-Cy7 and the reference molecule Cy5-Cy7 further
confirmed the specific probe structural conversion and related
fluorescence responses (Figure S2). Although O2•- could cause a
slight fluorescence decrease at 800 nm, the obvious dual-
spectral responses were still able to identify ROS and RNS. As
Figure 1. Multiple radical species detection via HCy5-Cy7. a) UV-vis
absorption and b) fluorescence spectra of HCy5-Cy7 (20 μM) in the
•-
absence or presence of O2 (100 μM) or ONOO- (50 μM). c) The sensing
specificity of HCy5-Cy7 towards different radical species (50 μM), including
peroxynitrite (ONOO−), hypochlorite (−OCl), nitric oxide (NO), tert-butoxy
radical (•OtBu), H2O2, tert-butyl hydroperoxide (TBHP), hydroxyl radical
(•OH), and superoxide radical (O2•-); ΔF = F1 – F0, where F1 indicates the
flurescence intensity of HCy5-Cy7 after RONS reaction, F0 indicates the
flurescence intensity of HCy5-Cy7 alone. d) The fluorescence intensity
fitting of HCy5-Cy7 as a function of ONOO- (or O2•-, inset) concentration. y
= -5.83x + 278 (blue); y = 0.378x + 2.88 (red).
2
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10.1002/anie.202104100
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COMMUNICATION
(iNOS),[13] our cell stimulation
studies
together
with
the
experiments treated with RNS
scavenger clearly evidenced the
•-
coexistence and correlation of O2
and ONOO- in the inflammatory
process, which illustrated the
importance
probes in
of
the
multi-responsive
profiling of
pathophysiology in complex and
dynamic conditions. Besides, the
HCy5-Cy7 probe molecule has also
been proved to be able to target
mitochondria and therefore enabled
its possibility to precisely monitor in
situ oxidation, nitration as well as
subsequent
cellular
damages
induced by oxidative stress (Figure
2b). Moreover, in light with the
negligible cytotoxicity of the probe
(Figure S3), such unique multi-
sensitive and compelling dual
channel readout capabilities could
also
make
HCy5-Cy7
more
conducive
to
simultaneously
Figure 2. In vitro multiple radical species sensing. a) Fluorescence imaging of endogenous radical variations
with HCy5-Cy7 (10 μM) in the RAW264.7 macrophage cells stimulated by PMA (0.2 μg ml-1) or LPS/IFN-
γ/PMA (2/0.05/0.01 μg ml-1) accordingly. Blue: Hoechst 33342 (Ex: 405 nm, Em: 460/30 nm); Red: Cy5
channel (Ex: 641 nm, Em: 630/30 nm); violet: Cy7 channel (Ex: 786 nm, Em: 800/30 nm). Scale bars: 50 μm.
b) Confocal analysis of HCy5-Cy7 cellular localization in PMA-stimulated RAW264.7 cells. Green:
Mitochondria tracker (Ex: 488 nm, Em: 520/30 nm). Scale bar: 50 μm. c, d) The fluorescence intensity
changes of HCy5-Cy7 (10 μM) incubated with RAW264.7 after gram-positive and gram-negative bacterial
stimulations. ΔF = Fbacteria - Fcontrol, where Fbacteria indicates the flurescence intensity of HCy5-Cy7 incubated
with RAW264.7 and bacterial cells, Fcontrol indicates the flurescence intensity of HCy5-Cy7 incubated with
RAW264.7 alone. F0 indicates the fluorescence intensity of HCy5-Cy7 with RAW264.7/bacterial co-incubation
at time t0. Antibiotics: penicillin, 500 I. U. mL-1 and streptomycin, 500 μg mL-1. The fluorescence intensities at
660 and 800 nm were measured with the excitation wavelength at 620 and 740 nm, respectively.
manifest biological multi-radical
variations in comparison with
previously reported peroxynitrite- or
superoxide-selective probes with
their signal readout only under
single spectral channel.[12, 14]
Encouraged by the effective
RONS imaging performance, HCy5-
Cy7 was further applied to
investigate
the
possibility
of
sensitive recognition multiple radical
species in living system.
species evolutions in the host-defense process triggered by the
invasion of bacterial pathogens. As proof of concept, we
established the bacterial infection model through the typical
stimulations of gram-positive (e.g. S. aureus) and gram-negative
(e.g. E. coli) bacteria in the RAW264.7 macrophage cells.[9]
Upon the incubation with bacteria, HCy5-Cy7 was applied to
monitor the oxidative stress in macrophage cells. The dual
wavelength fluorescence changes induced by bacteria infection
at the different time duration in the comparison with control
groups without bacteria were represented in Figure 2c and 2d.
Generally, the fluorescence intensities at 660 nm in either S.
aureus or E. coli incubated macrophages showed the similar
time-dependent enhancement, demonstrating the obvious ROS
production during the infection process. However, only slight
fluorescence changes at 800 nm could be observed in both S.
aureus and E. coli incubated cells, which suggested the
possibility of the less RNS response along the bacteria
contagion. To better understand whether the oxidative stress
status was closely related to the interaction of bacteria with
macrophage cells, we monitored the dynamic phagocytosis
processes by using green fluorescent protein (GFP) labeled
bacterial strains (Figure S4). To our surprise, the clear
differences were observed that the phagocytosis of GFP/S.
aureus was in a time-dependent manner, while GFP/E. coli
showed less efficient uptake by RAW264.7 probably due to the
distinct bacterial cell wall components. Such a phenomenon
provided the evidence that the slight higher ROS enhancement
We then validated the feasibility of HCy5-Cy7 to monitor
the endogenous multiple radical correlations in murine
RAW264.7
macrophage
cells.
The
specific
cellular
•-
overproductions of O2 and ONOO- were induced by chemical
stimulations with phorbol 12-myristate 13-acetate (PMA) and
lipopolysaccharide
(LPS)/interferon-γ
(IFN-γ)/PMA,
respectively.[10a, 11-12] Thereafter, the treated cells were incubated
with the probe molecule for fluorescence confocal imaging
analysis. As indicated in Figure 2a, the control cells treated with
HCy5-Cy7 alone showed very weak fluorescence in the Cy5
channel (red) but strong fluorescence in the Cy7 channel (violet),
clearly suggesting a low level RONS under normal conditions.
However, a dramatic increase of red fluorescence can be
observed in the PMA treated group and the violet fluorescence
signal remained unchanged. These results demonstrated that
HCy5-Cy7 could specifically detect cellular O2•- production, which
was further confirmed by a negative control study with ROS
scavenger pretreatment in the cells. Moreover, LPS/IFN-γ/PMA
costimulated cells with HCy5-Cy7 addition exhibited a significant
Cy5 flurescence enhancement while the Cy7 flurescence
intensity was largely decreased with the signal close to the
•-
background, implying that both O2 and ONOO- could be
endogenously generated and detected in live cells. Although
several studies proposed that LPS/IFN-γ/PMA stimulated
macrophages could mostly induce ONOO- upregulation through
the activation of NADPH oxidase and nitric oxide synthase
3
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10.1002/anie.202104100
Angewandte Chemie International Edition
COMMUNICATION
appeared along with the
bacterial pathogenesis, we
injected HCy5-Cy7 after
prolonged bacterial infection
(e.g. 5 days). The results in
figure 3b and 3c clearly
indicated the significant
increase of the fluorescence
signal
demonstrating
at
660
nm,
the
occurrence of oxidative
stress at the long-term
infection. However, only the
minimum
fluorescence
change at 800 nm were
observed. These results
therefore evidence the ROS
stress
predominant
pathogenesis in the process
of the bacterial infection in
the current model. In
parallel, we also performed
a
clinical
inflammation
diagnostic test through the
standard procalcitonin (PCT)
method using mice blood
samples to investigate the
correlations between the
status of bacterial infection
Figure 3. Multiple radical species profiling in living mice after bacterial infections. a, b) In vivo fluorescence signals of
short-term (2 h) and long-term (5 days) mice infections post typical gram-negative and gram-positive bacteria
injections, respectively. The probe molecule HCy5-Cy7 (0.05 mg/mL in saline, 200 μL) was injected via tail vein. Cy5
channel (Ex: 640/680 nm); Cy7 channel (Ex/Em: 740/820 nm). c) The ratiometric fluorescence signal changes plot at
Cy5 and Cy7 channels after the probe injection. Where ΔF = Fbacteria – F0, Fbacteria indicates the total fluorescence
intensity (ventral and dorsal) in the infected mouse, F0 indicates the total fluorescence intensity (ventral and dorsal) in
the control mouse. d) Serum procalcitonin tests (PCT) of short-term (2 h) and long-term (5 days) bacteria infected mice
(n = 3).
and
oxidative
stress
changes.[16] As expected,
the serum procalcitonin
level showed upward trends
in S. aureus infected microphages could be likely ascribed to the
continuous stimulation of phagocytosis, yet E. coli-induced
infection was more possibly due to endotoxin lipopolysaccharide
(LPS)-mediated inflammatory stress.[15] Moreover, it should be
noted that the antibiotics treatment of bacteria-infected
macrophages slightly enhanced the ROS level in E. coli
incubated cells, while antibiotics addition in S. aureus infected
microphages cells triggered a small production of RNS radicals.
These results implied that both ROS and RNS radical species
played essential roles in the bacterial infection and antibiotics
response,[9a] but the detailed biochemical mechanisms regarding
the correlations of redox biology varying from bacterial strains,
intracellular and intrabacterial redox-related signaling in
infections still need to be further investigated.
in the animals with prolonged infection of E.coli and S. aureus
bacterial strains (Figure 3d), which was consistent with the
results observed in the radical species-mediated fluorescence
change in Figure 3c, clearly suggesting the good
correspondence of pathological process with oxidative stress
changes. There was no obvious procalcitonin enhancement
observed in the short-term (e.g., 2h) bacterial infections,
probably originated from the relatively weak bacteria-
pathological
responses.
Comparatively,
HCy5-Cy7
demonstrated obvious fluorescent signal changes in the living
mice along with bacterial infection, which could thus provide
more sensitive and detailed outcomes for accurately profiling the
pathology behind the bacterial infection progressions, especially
for early detection of infectious diseases.
Moreover, we futher hypothesized that HCy5-Cy7 could
also demonstrate a potential to study the multiplex redox biology
of bacteria infection in living animals. Accordingly, the mice were
intraperitoneally (i.p.) injected with living bacteria E. coli and S.
aureus, respectively to induce the animal infections. After 2 h
bacterial stimulation, the probe molecule was injected via tail
vein (i.v.) and animal imaging was carried out over the time in an
IVIS system (Figure 3 and Figure S5). As shown in Figure 3a
and 3c, the fluorescence at 660 nm channel in the infected mice
showed a significant enhancement as comparison with saline-
treated controls, such ROS enhancement under this spectral
window reached the maximum response upon 2 h post probe
injection. At this time point, only E. coli-injected mice showed a
slight decrease of the fluorescence at 800 nm, indicating the
possibility of a low level of RNS production in the early-stage
bacterial infection. To explore whether the nitrosative stress was
In conclusion, we have developed a unique NIR cyanine-
dyad molecular probe HCy5-Cy7 that simultaneously reacted
with radical species and triggered multispectral signal variations,
which was further applied for multiplex manifestation of oxidative
and nitrosative stress evolutions during the bacterial infections in
live cells and living mice. The in vitro and in vivo results clearly
disclosed the complex interplays of RONS during bacterial
pathogenesis and the host-defense response. Overall, this study
provides new perspectives for understanding the intrinsic
correlations of oxidative/nitrosative stress and bacterial
pathogenesis, which may contribute to current imaging-guided
precision medicine in the fight against infectious diseases.
Acknowledgements
4
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10.1002/anie.202104100
Angewandte Chemie International Edition
COMMUNICATION
The authors sincerely thank Dr. John Chen and Dr. Yuan Qiao
for the generous sharing of GFP-labeled bacterial strains for the
phagecytosis study. B.X. acknowledges the financial supports
from Tier 1 RG6/20, MOE 2017-T2-2-110, A*Star SERC
A1983c0028, A20E5c0090, awarded in Nanyang Technological
University (NTU), and National Natural Science Foundation of
China (NSFC) (No. 51929201). M.B.C and W.Z. acknowledge
the financial supports from ASTAR RIE2020 Advanced
Manufacturing and Engineering (AME) IAP-PP Specialty
Chemicals Programme Grant (No. A1786a0032) and MOE Tier
3 Grant (MOE2018-T3-1-003).
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Conflict of interest
The authors declare no conflict of interest.
Keywords: small-molecule probe • NIR fluorescence imaging •
multiple radical dynamics • bacterial infection
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10.1002/anie.202104100
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
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Zhimin Wang , Thang Do
+
Cong , Wenbin Zhong, Jun
Wei Lau, Germain Kwek,
Mary B. Chan-Park and
Bengang Xing*
Page No. – Page No.
Cyanine-Dyad
Probe for Simultaneous
Profiling of Multiple
Radical Species Evolution
in Bacterial Infection
Molecular
The cyanine-dyad molecular probe, HCy5-Cy7 specifically turns on its fluorescence at 660 nm
via superoxide or hydroxyl radical (O2 , •OH)-mediated oxidation of reduced HCy5 moiety to
Cy5, while peroxynitrite or hypochlorous species (ONOO-, ClO-)-induced Cy7 structural
degradation leads to the emission turn-off at 800 nm. Such multispectral signal responses allow
multiplex manifestation of in situ oxidative and nitrosative stress events during the pathogenic
and defensive processes of bacterial infection in both macrophage cells and living mice.
•-
6
This article is protected by copyright. All rights reserved.