46 research outputs found
INTEGRATED NANOSCALE IMAGING AND SPATIAL RECOGNITION OF BIOMOLECULES ON SURFACES
Biomolecules on cell surfaces play critical roles in diverse biological and physiological processes. However, conventional bulk scale techniques are unable to clarify the density and distribution of specific biomolecules in situ on single, living cell surfaces at the micro or nanoscale. In this work, a single cell analysis technique based on Atomic Force Microscopy (AFM) is developed to spatially identify biomolecules and characterize nanomechanical properties on single cell surfaces. The unique advantage of these AFM-based techniques lies in the ability to operate in situ (in a non-destructive fashion) and in real time, under physiological conditions or controlled micro-environments.
First, AFM-based force spectroscopy was developed to study the fundamental biophysics of the heparin/thrombin interaction at the molecular level. Based on force spectroscopy, a force recognition mapping strategy was developed and optimized to spatially detect single protein targets on non-biological surfaces. This platform was then translated to the study of complex living cell surfaces. Specific carbohydrate compositions and changes in their distribution, as well as elasticity change were obtained by monitoring Bacillus cells sporulation process.
The AFM-based force mapping technique was applied to different cellular systems to develop a cell surface biomolecule library. Nanoscale imaging combined with carbohydrate mapping was used to evaluate inactivation methods and growth temperatures effects on Yersinia pestis surface. A strategy to image cells in real time was coupled with hydrophobicity mapping technique to monitor the effect of antimicrobials (antimicrobial polymer and copper) on Escherichia coli and study their killing mechanisms. The single spore hydrophobicity mapping was used to localize the exosporium structure and potentially reconstruct culture media. The descriptions of cell surface DNA on single human epithelial cells potentially form a novel tool for forensic identification.
Overall, these nanoscale tools to detect and assess changes in cell behavior and function over time, either as a result of natural state changes or when perturbed, will further our understanding of fundamental biological processes and lead to novel, robust methods for the analysis of individual cells. Real time analysis of cells can be used for the development of lab-on-chip type assays for drug design and testing or to test the efficacy of antimicrobials
The Turtle Garden: Tan Kah Keeâs last spiritual world
This paper explores the role of diasporic subjects in Chinaâs heritage-making through a case study of the Turtle Garden built by Tan Kah Kee in Xiamen, China. Tan is the first person with Overseas Chinese background who built museums in the P.R. China and has been regarded as a symbol of Overseas Chinese patriotism. This paper argues that the Turtle Garden, conceptualised as a postcolonial âcarnivalesqueâ space, is more than a civic museum for public education. It reflects the ownerâs highly complex and sometimes conflicting museum outlook embedded in his life experience as a migrant, his encounter with (British) colonialism in Malaya, and integrated with his desire and despair about the Chinese Communist Partyâs nation-building project in the 1950s. Rather than a sign of devotion to the socialist motherland as simplistically depicted in Chinaâs discourse, the garden symbolises Tanâs last âspiritual worldâ where he simultaneously engaged with soul-searching as a returned Overseas Chinese and alternative diasporic imagining of Chinese identities and nation. It brings to light the value of heritage-making outside centralised heritage discourses, and offers an invaluable analytical lens to disentangle the contested and ever shifting relationship between diasporic subjects, cultural heritage and nation-(re)building in the Chinese context and beyond
Evaluating interaction forces between BSA and rabbit anti-BSA in sulphathiazole sodium, tylosin and levofloxacin solution by AFM
Protein-protein interactions play crucial roles in numerous biological processes. However, it is still challenging to evaluate the protein-protein interactions, such as antigen and antibody, in the presence of drug molecules in physiological liquid. In this study, the interaction between bovine serum albumin (BSA) and rabbit anti-BSA was investigated using atomic force microscopy (AFM) in the presence of various antimicrobial drugs (sulphathiazole sodium, tylosin and levofloxacin) under physiological condition. The results show that increasing the concentration of tylosin decreased the single-molecule-specific force between BSA and rabbit anti-BSA. As for sulphathiazole sodium, it dramatically decreased the specific force at a certain critical concentration, but increased the nonspecific force as its concentration increasing. In addition, the presence of levofloxacin did not greatly influence either the specific or nonspecific force. Collectively, these results suggest that these three drugs may adopt different mechanisms to affect the interaction force between BSA and rabbit anti-BSA. These findings may enhance our understanding of antigen/antibody binding processes in the presence of drug molecules, and hence indicate that AFM could be helpful in the design and screening of drugs-modulating protein-protein interaction processes
Mechanism of Inhibition of the GluA2 AMPA Receptor Channel Opening by Talampanel and Its Enantiomer: The Stereochemistry of the 4âMethyl Group on the Diazepine Ring of 2,3-Benzodiazepine Derivatives
Stereoselectivity of 2,3-benzodiazepine compounds provides
a unique way for the design of stereoisomers as more selective and
more potent inhibitors as drug candidates for treatment of the neurological
diseases involving excessive activity of AMPA receptors. Here we investigate
a pair of enantiomers known as Talampanel and its (+) counterpart
about their mechanism of inhibition and selectivity toward four AMPA
receptor subunits or GluA1â4. We show that Talampanel is the
eutomer with the endismic ratio being 14 for the closed-channel and
10 for the open-channel state of GluA2. Kinetic evidence supports
that Talampanel is a noncompetitive inhibitor and it binds to the
same site for those 2,3-benzodiazepine compounds with the C-4 methyl
group on the diazepine ring. This site, which we term as the âMâ
site, recognizes preferentially those 2,3-benzodiazepine compounds
with the C-4 methyl group being in the <i>R</i> configuration,
as in the chemical structure of Talampanel. Given that Talampanel
inhibits GluA1 and GluA2, but is virtually ineffective on the GluA3
and GluA4 AMPA receptor subunits, we hypothesize that the âMâ
site(s) on GluA1 and GluA2 to which Talampanel binds is different
from that on GluA3 and GluA4. If the molecular properties of the AMPA
receptors and Talampanel are used for selecting an inhibitor as a
single drug candidate for controlling the activity of all AMPA receptors
in vivo, Talampanel is not ideal. Our results further suggest that
addition of longer acyl groups to the N-3 position should produce
more potent 2,3-benzodiazepine inhibitors for the âMâ
site
Potent and Selective Inhibition of the Open-Channel Conformation of AMPA Receptors by an RNA Aptamer
Mechanism of Inhibition of the GluA1 AMPA Receptor Channel Opening by the 2,3-Benzodiazepine Compound GYKI 52466 and a <i>N</i>âMethyl-Carbamoyl Derivative
2,3-Benzodiazepine derivatives, also
known as GYKI compounds, represent
a group of the most promising synthetic inhibitors of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) receptors. Here we investigate the mechanism of inhibition
of the GluA1 channel opening and the site of inhibition by GYKI 52466
and its N-3 methyl-carbamoyl derivative, which we term as BDZ-<i>f</i>. GluA1 is a key AMPA receptor subunit involved in the
brain function. Excessive activity and elevated expression of GluA1,
however, has been implicated in a number of neurological disorders.
Using a laser-pulse photolysis technique, which provides âŒ60
ÎŒs resolution, we measured the effect of these inhibitors on
the rate of GluA1 channel opening and the amplitude of the glutamate-induced
whole-cell current. We found that both compounds inhibit GluA1 channel
noncompetitively. Addition of an N-3 methyl-carbamoyl group to the
diazepine ring with the azomethine feature (i.e., GYKI 52466) improves
the potency of the resulting compound or BDZ-<i>f</i> without
changing the site of binding. This site, which we previously termed
as the âMâ site on the GluA2 AMPA receptor subunit,
therefore favorably accommodates an N-3 acylating group. On the basis
of the magnitude of the inhibition constants for the same inhibitors
but different receptors, the âMâ sites on GluA1 and
GuA2 are different. Overall, the âMâ site or the binding
environment on GluA2 accommodates the same compounds better, or the
same inhibitors show stronger potency on GluA2, as we have reported
previously [Wang et al. Biochemistry (2011) 50, 7284â7293]. However, acylating
the N-3 position to occupy the N-3 side pocket of the âMâ
site can significantly narrow the difference and improve the potency
of a resulting compound on GluA1