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CNSI- UCLA Speakers
Debalina Chatterjee
Student, Bioengineering
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"Proteomics
with Digital Microfluidics"
Debalina
Chatterjee1, Anders Jimmy Ytterberg2, Sang Uk Son2 and
Robin L. Garrell1, 2
1Biomedical Engineering Interdepartmental Program,
University of California Los Angeles
2Chemistry
and Biochemistry Department, University of California Los Angeles and
California NanoSystems Institute
We have developed a droplet-based (digital) microfluidic
platform for preparing and purifying samples for matrix assisted laser
desorption/ionization mass spectrometry (MALDI-MS). Here we demonstrate
integration of three protein processing steps prior to MALDI-MS analysis:
reduction, alkylation and enzymatic digestion, followed by crystallization with
the MALDI matrix. This method is faster and results in lower reagent
consumption and sample loss than conventional techniques for proteomics sample
preparation. Droplets containing the analyte (bovine/human serum albumin,
insulin or lysozyme) and the reducing agent TCEP (tris 2-carboxyethyl phosphine
hydrochloride) were merged and allowed to react at room temperature for 5 min.
A third droplet containing the alkylating reagent, NEM (N-ethylmaleimide) was
transported to the reduced analyte and allowed to react for 5 min. A fourth
droplet containing a proteolytic enzyme trypsin was transported to the reduced,
alkylated analyte and allowed to react for 4 h in an incubator. Finally, a
droplet containing the matrix, dihydrobenzoic acid or sinnapinnic acid, was
dispensed into the processed analyte droplet and allowed to dry. The device was affixed to a
custom MALDI target and inserted into a Voyager DE STR MALDI mass spectrometer.
MALDI spectra were collected in reflector mode,
and calibrated against standard peptide spectra. The peptide coverage in the
MALDI spectra of the analytes was found to be ~ 90%.
*Poster Presenter
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Sarah Cross
Student, Chemistry and Biochemistry
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"Application of AFM to Nano-Medicine
and- Dentistry: Biomechanical and Structural Properties of Various Cell
Systems"
Sarah E. Cross1, 2, Qing-Yi Lu3, Yu-Sheng Jin4, Renate
Lux5, Wenyuan Shi5, 6, JianYu Rao4 and James K. Gimzewski1, 2
1Chemistry and
Biochemistry Department and 2California
NanoSystems Institute, University of California, Los Angeles, CA
3Center for
Human Nutrition and Department of 4Pathology
and Laboratory Medicine, University of California, Los Angeles, CA
5School of
Dentistry and 6Molecular
Biology Institute, University of California, Los Angeles, CA
The connections between single-cell
structure and biomechanics and disease states have been the subject of
considerable scientific research in the past decade1. With a growth
in interest in biomechanical, biophysical and structure-function properties of
cells, application of non-traditional approaches to study these biological and
physiological problems has increased. In particular, atomic force microscopy
(AFM) has garnered much interest in recent years for its ability to probe the
structure, function and cellular nanomechanics inherent to specific biological
systems.
Here we use AFM to probe the
important structure-function relationships of the bacterium Streptococcus
mutans. S. mutans is the primary etiological agent in human dental carries
(tooth decay) worldwide, and is of medical importance due to the virulence
properties of these cells in biofilm initiation and formation, leading to
increased tolerance of antibiotics. We have used AFM to characterize unique
surface structures of distinct mutants of S. mutans. Many of these
mutations are programmed in specific genes that encode surface proteins, AFM
enables resolution of characteristic surface features for mutant strains
compared to the wild-type. These surface structures of the different bacterial
strains provide a unique prototype for analyzing the communal behavior of S.
mutans associated with biofilm formation.
Ultimately, our characterization of surface morphology shows distinct
differences in the local properties displayed by various S. mutans strains on the
nanoscale, imperative for understanding the collective properties of these
cells in biofilm formation.
In addition, we have used atomic
force microscopy (AFM) to probe nanomechanical properties, such as cell
adhesion and stiffness, as a method for elucidating various cellular events
associated with both cultured cell lines and clinical patient samples.
Investigation of cell stiffness, adhesion and motility are pivotal in our
understanding of changes in cytoarchitecture, which are characteristic of
cellular dedifferentiation, malignant transformation, growth activation and
spreading. This study uses AFM to probe the potential mechanisms of the
anticancer effect of green tea extract (GTE) and identify potential biomarkers
for GTE based chemoprevention trials via monitoring associated nanomechanical
properties. We identify and probe a particular actin binding protein (ABP),
annexin-I, as the protein target responsible for the actin remodeling effect.
Using AFM we show nanomechanical responses of GTE induced annexin-I expression
on actin regulation in human lung and prostate adenocarcinoma cells in vivo and further hypothesize that these protein targets might be
used as potential surrogate end point markers for GTE based chemoprevention
trials. Moreover, we have applied this high resolution technique to study the
associated cell stiffness and surface adhesion of cells taken from body fluid
(pleural fluid) samples collected from patients thought to have metastatic
adenocarcinoma of the lung. We show the ability of AFM to detect changes in the
local mechanics inherent to both "normal (mesothelial cells)" and "tumor" cells
under analogous conditions. AFM was able to accurately perform cytological
diagnosis of cancer cells initially undetected by routine laboratory techniques
used for cancer detection. Our findings indicate that AFM may serve as an
excellent tool for assessing functional surrogate markers for cancer cell
detection and evaluation, and may potentially revolutionize the cytomorphology
analysis for cancer diagnosis
References:
Suresh, S.
Biomechanics and biophysics of cancer cells. Acta Biomaterialia 2007; 3:413-438.
*Poster Presenter
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James Gimzewski
Professor, Chemistry and Biochemistry,
Physical Chemistry,
Member, NanoBiotechnology and Biomaterials,
NanoElectronics, Photonics, Architectonics, NanoMechanical and Nanofluidic
Systems, California NanoSystems Institute,
Institute for Cell Mimetic Space
Exploration (CMISE)
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"Nanomechanics
of Human Metastatic Cancer Cells in Clinical Pleural Effusions"
Sarah E.
Cross1,2, Yu-Sheng Jin3, JianYu Rao3** & James K. Gimzewski1,2**
1Department
of Chemistry and Biochemistry, and 2California NanoSystems Institute,
University of California, Los Angeles, CA 90095, USA.
3Department
of Pathology and Laboratory Medicine, University of California, Los Angeles, CA
90095, USA.
*These
authors contributed equally on this project
Change in
cell stiffness, a newly recognized phenotypic event of cancer cells, is crucial
in cancer cell spreading. Despite several studies on cytoarchitectural changes
of cultured cell lines, no reported ex vivo mechanical analyses of cancer cells
have been obtained from patients. We report on the stiffness of live metastatic
cancer cells taken from body(pleural) fluid effusions of patients with
suspected metastatic adenocarcinoma from the lung, breast, and pancreas. Using
atomic force microscopy we find that cell stiffness of metastatic cancer cells
is >70% softer than benign mesothelial cells present in the same sample,
with a standard deviation 5 times narrower than that for the normal mesothelial
cells. Our experiments indicate a common modulus in different cancer types.
Mechanical analysis demonstrates the ability to distinguish cell-type even when
undetected by cytomorphology alone. The results suggest that
nanomechanical-based analysis is a novel functional biomarker for the detection
and evaluation of cancer.
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Mark Henle
Student, Chemistry and Biochemistry
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"Effects of Topology and Curvature on the Hydrodynamics
of Membranes and Interfaces"
Authors: Mark L.
Henle (UCLA), Ryan McGorty (UMass Amherst, Harvard), Anthony Dinsmore (UMass
Amherst), and Alex J. Levine (UCLA).
Molecular transport in membranes and fluid interfaces is
vital for a variety of biological systems and technological applications.
Within the cell membrane, for example, the diffusion of transmembrane proteins
is essential for cell-cell signaling. From a technological standpoint, a
fluid/fluid interface can provide a template for the creation of
two-dimensional colloidal structures. Such materials can be used to create
selectively permeable capsules ("colloidosomes") designed for the encapsulation
and delivery of drugs, proteins, living cells, and other ingredients. In many
of these technological applications and biological systems, membrane transport
occurs in compact, highly curved membranes.In this poster, we present theoretical calculations and
experimental measurements of the interfacial fluid velocity field around a
moving colloidal rod bound to the crowded interface of a spherical water-in-oil
droplet. By using different droplet sizes, membrane viscosities, and rod
lengths, we show that both the curvature and the topology of the interface can
have a significant effect on the dynamics of the rod.We find that our theoretical description agrees
quantitatively with the experimental results;specifically, the viscosity mismatch between the interior
and exterior fluids leads to a suppression of the fluid flow on small droplets
that cannot be captured by the flat interface predictions.
*Poster Presenter
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Kendall Houk
Professor, Chemistry and Biochemistry
Member, NanoElectronics, Photonics, Architectonics,
NanoMechanical and Nanofluidic Systems, California NanoSystems Institute
CNSI-CNBI Executive Meeting
Chemistry Research in the CNSI
Department of Chemistry and Biochemistry
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NanoSystems research in the Department of Chemistry and
Biochemistry will be reviewed and summarized.The broad areas of interest are:
I. Reticular
Materials (Omar Yaghi)
II. Nanomachines,
Imaging, Solid States, and Colloids (Miguel
Garcia-Garibay, Jeffrey Zink, James Gimzewski, Ric Kaner, Thomas Mason, Benjamin Schwartz)
III. Microfluidics,
Single Molecule Spectroscopy, Bioprobes (Shimon
Weiss and Robin Garrell)
IV. Proteins
and BioNano Materials (David Eisenberg,
Todd O. Yeates, William Gelbart)
V. Polymers,
Oligomers, Nanomaterials (Tim Deming
(Chair, Bioengineering), Sarah Tolbert, Heather Maynard)
VI. Theory
of NanoSystems (Kendall Houk, Daniel
Neuhauser, Alexander Levine)
VII. Organic
Synthesis (Michael Jung )
*This talk will take place during the Executive Meeting on
October 31st, 2007.
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Steve Huyn
Student, Molecular and Medical
Pharmacology
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"Development of a Targeted Gene
Therapy Vector for the Treatment and Diagnosis of Metastatic Breast Cancer"
Steven T Huyn, BS1,
Makoto Sato, PhD2, Jeremy Burton, BS, MS1 and Lily Wu, MD, PhD1,2. 1Molecular
and Medical Pharmacology, University of California at Los Angeles, Los Angeles,
CA, United States, 90095 and 2Urology, University of California at
Los Angeles, Los Angeles, CA, United States, 90095
In the past, our group has demonstrated that an adenoviral mediated two-step
transcriptional amplification (TSTA) system can greatly augment the activity of
weak promoters, while still maintaining tissue specificity. This system has
shown to be successful in a prostate cancer model. We therefore believe that
this scheme holds great potential for use in a gene therapy vector targeted
against metastatic breast cancer.We have successfully incorporated both breast, and cancer-specific
promoters into the TSTA system.Studies carried out in vitro have
demonstrated that the activity of all promoters evaluated within the TSTA
system were able to maintain their tissue specificity within the context of
this amplification scheme, while at the same time achieving efficiency of up to
250 fold over non amplified promoters. A breast tumor specific, Mucin-1
promoter driven TSTA construct expressing firefly luciferase has been
incorporated into adenoviral vectors, and is currently being evaluated in
vivo.This vector has shown very low systemic toxicity following
intravenous injections in non tumor bearing mice.Furthermore,Xenograft studies utilizing various breast cancer cell lines, in
combination with optical imaging have shown this vector to be very specific and
sensitive for breast tumor cells.
A critical aspect of this project
will be the incorporation of cytotoxic genes for therapeutic studies. At the
same time, the ability to express reporter genes for bioluminescent imaging
studies would also be desirable, and would allow for efficient, non-invasive
monitoring of gene expression. To address this issue, we successfully developed
a MUC-1 driven TSTA amplification scheme able to express and amplify two
separate transgenes. This bi-directional reporter TSTA construct is currently
being evaluated for use in adenoviral vectors. Through use of the TSTA system
we aim to develop a gene therapy vector that can achieve robust targeted gene
expression. We believe that this approach holds great promise for the treatment
and diagnosis of metastatic breast cancer.
*Poster Presenter
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Shuwen Koh
Student, Bioengineering
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"Modulating
the Transduction Efficiency of Polypeptide-Coated Adenoviral Gene Delivery
Vectors"
S.B. KOH1, T.J. DEMING1,
L. WU2
Department of Bioengineering, University of California, Los Angeles,
USA
2 Department of
Urology, University of California, Los Angeles, USA
In treating prostate cancer using gene therapy, the specific
targeting of malignant tissue is necessary in order not to cause significant
harm to normal tissues or host as a whole. Ideally,
the effect of gene therapy must be systemic to allow the therapeutic effect to
access distant sites of malignancy, especially those not detectable by
conventional diagnostic methods.
We have adopted a novel two-prong
strategy of combining biomaterials technology for transductional targeting with
prostate-specific regulation of transcription for gene therapy applications.
The two-step transcriptional amplification (TSTA) system incorporated in an
adenovirus amplifies prostate-specific transgene expression with retention of
proper hormonal regulation. The molecular imaging and therapeutic capabilities
of the TSTA system enables visualization of site-specific delivery and gene
expression in living animals. In addition, we
have synthesized a polypeptide that acts as a stealth coating to shield the
viral vector from antibody neutralization and degradation in host circulation, increasing
its availability for uptake by target cells and subsequent transgene
expression.
The cationic polypeptides
synthesized in this project have been shown to interact with negatively charged
Ad particles and the resulting hybrid complexes have been shown to
significantly enhance transduction efficiency in vitro. PEGylated polypeptides have been shown to modulate the zeta
potential and aggregation characteristics of the hybrid complexes. Preliminary
results also indicate the involvement of heparan sulfate glycosaminoglycans (HS
GAGs) as receptors in addition to the coxsackie adenovirus receptor (CAR) and avb integrins found on many cell surfaces. The results of these
experiments are significant because gene therapy applications require maximum
gene expression with minimum cytotoxicity and immune response. Being able to
enhance the transduction efficiency of Ad may allow the use of lower dose of Ad
as well as cationic polypeptide to be administered for therapeutic effects.
Subsequent ligand-directed targeting of the vector to malignant tissues will
add an additional level of safety. A reduction in Ad-associated immune response
would be favorable for clinical translation.
*Poster Presenter
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Mike Kovochich
Student, NanoMedicine
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"Correlating Toxicity of
Nanomaterial with Physical Characterization Using an Oxidative Stress Model"
Michael Kovochich, Tian Xia, Andre
E. Nel
Nanotechnology is rapidly
expanding and some estimates have predicted a $1 trillion market by 2015.
Manufactured nanomaterial (NM) are already in use through products such as
cosmetics, sunscreens, sporting goods, tires and some future medical applications
include imaging, diagnosis, and drug delivery. The unique physico-chemical
properties of engineered NM are attributable to their small size, large surface
area, durability, chemical composition, crystallinity, electronic properties,
surface reactivity, surface groups, surface coatings, solubility, shape and
aggregation. These novel properties of NM raise the possibility that they could
interact with cellular tissue and cause damage to biological systems. With this
in mind I aim to further develop a system whereby NM toxicity can be classified
utilizing the Hierarchical Oxidative Stress Model. Due to the large number of
new NM being produced each year it would be impossible from a time and cost
perspective to test each on its own. For these reasons it is one of my aims to
correlate the biological outcome of the tested NM with its physico-chemical
properties with the ultimate goal of understanding basic nano/bio principles
which may predict future toxic outcomes.
In recent
work we have classified several NM on their ability to generate Reactive Oxygen
Species (ROS) in a macrophage cell line using air pollutant particles as a
positive control. Interestingly, TiO2 , Carbon Black and Fullerol
were void of toxicity while the NH2 modified polystyrene NP tested
positive in cytotoxicity as compared to the COOH or unmodified versions. The
mechanism for this toxic outcome has been correlated to its small size and
positive charge. Furthermore, this nano/bio interaction is cell specific when
tested in 5 different cell lines and can be related to its mechanisms of
cellular uptake. Taken together these data have helped establish a method of
toxic detection utilizing the Hierarchical Oxidative Stress model for future
NM. I will further use this system to categorize a set of Fullerene derivatives
which is a good candidate due to their high volume of production and
conflicting literature data about the potential toxicity. As nanotechnology
develops, it is essential that the toxicological approach also evolves and
stays up to date.This will
provide an important safeguard for the continued expansion of the
nanotechnology industry.
*Poster Presenter
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Tatiana Kuriabova
Student, Chemistry and Biochemistry
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"Nanorheology of viscoelastic shells: Applications to viral
capsids"
We study the microrheology of nanoparticle shells [Dinsmore
et al.
Science 298,
1006 (2002)] and viral capsids[Ivanovska et al. PNAS 101, 7600 (2004)] by computing the mechanical
responsefunction and thermal
fluctuationspectrum of a
viscoelastic spherical shell that is permeable to thesurrounding solvent. We determine analytically the damped
dynamics ofthe shear, bend, and
compression modes of the shell coupled to the solventboth inside and outside the sphere in the zero Reynolds
number limit.
We identify
fundamental length and time scales in the system,and compute the thermal correlation function of
displacements of antipodalpoints
on the sphere and the mechanical response to pinching forcesapplied at these points. We describe
how such a frequency-dependentantipodal correlation and/or response function, which should be
measurablein new AFM-based
microrheologyexperiments, can
probe the viscoelasticity of these synthetic andbiological shells constructed of nanoparticles.
*Poster Presenter
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Zhibo Li
Student, Bioengineering
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"Hydrogels Assembled from Amphiphilic Pentablock
Copolypeptides"
Zhibo Li and Timothy J. Deming*
Department of Bioengineering, University of California, Los
Angeles
Los Angeles, California 90095
Using the transition-metal mediated
polymerization from protected α-amino acid N-carboxyanhydrides (NCA), we
synthesized a series of pentablock copolypeptides with alternatively connected
hydrophilic poly(L-lysineHBr) (K) blocks and hydrophobic poly(L-leucine) (L)
domains. The living polymerization allows a great deal of control on the
architecture and composition of the resulting copolypeptides. Similar to
previous KL diblock amphiphiles, the pentablock copolypeptides self-assemble
into moderately strong hydrogels at relatively low concentrations. In contrast
to KL diblock hydrogels, which is mainly composed of fibrous networks, the
pentablock copolypeptides tend to form membrane-like structures.
*Poster Presenter
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James Liao
Professor, Chemical Engineering
Member, NanoBiotechnology and Biomaterials, California
NanoSystems Institute
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*This talk will take place during the Executive Meeting on
October 31st, 2007.
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Jie Lu
Student, Microbiology, Immunology,
Molecular Genetics (MIMG)
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"Controlled delivery of anticancer drugs using nanomachine equipped nanoparticles"
Targeted delivery and on-demand
release of chemotherapeutic agents by nanoparticles provide a promising approach for cancer therapy. The primary goal of this
study is to utilize mesoporous silica nanoparticles and nanovalves to achieve targeted and controlled
release of chemotherapeutic agents to human cancer cells, thereby leading to the tumor-specific chemotherapy while sparing the
normal tissues. We have already
successfully used the mesoporous silica nanoparticles to overcome the problem
of hydrophobicity of anticancer drugs which is one of major obstacles for
chemotherapy. We demonstrated that the uptake of silica nanoparticles by cancer
cells was through energy and temperature-dependent endocytosis. We also
synthesized mesoporous silica nanoparticles derivatized with azobenzene
nanoimpellers inside of the pores and
used them as an effective delivery system that only releases drugs inside cancer cells upon light-irradiation at a
specific wavelength and on a time and power-dependent manner. This is
the first step towards a novel platform for a new generation of
nanotherapeutics with both spatial and temporal external control.Our nanoparticles provide a new cancer-targeting vehicle that
achieves controllable means for cancer therapy.
*Poster Presenter
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Edward McCabe
Director, Mattel Children's Hospital; Executive Chair Department of Pediatrics
Professor, Departments of Pediatrics and Human Genetics, and
Mattel Executive Endowed Chair in Pediatrics, David Geffen School of Medicine
at UCLA; Professor, Department of Bioengineering, Henry Samueli School of
Engineering and Applied Science; Physician-in-Chief, Mattel Children's Hospital
UCLA; and Co-Director, UCLA Center for Society and Genetics
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"Personalized Medicine: Point-of-Care Diagnostics and
Bio-Nano Devices"
Personalized medicine will be the consequence of the
predictive and preventive features of genomic medicine. Factors driving
personalized medicine include: the desire for individuals to have more autonomy
in their healthcare decision-making; the completion of the human and other
genome projects with the availability of genomic sequences for an increasing
number of organisms; and plans for prolonged human space travel with the need
for low payload technologies. Point-of-care (POC) diagnostics, for which the
POC is the individual, will be the ultimate in personalized care. A POC
diagnostic device on which our group is collaborating will provide rapid
diagnosis of bacterial infections, where the POC could be the physician's
office, hospital emergency department, or the patient's home, for example in a
patient with spinal cord injury who is prone to urinary tract infections. Our
group has participated in the development of single cell cytosensors for
radiation detection in outer space. We are applying genomic microarray bio-nano
devices for improved diagnosis of individual patients and with applications in population-based
programs, such as newborn screening.Cross-disciplinary teams are essential for development of POC and
bio-nano devices, and members of these teams should not be limited to a single
institution, but should draw on the appropriate expertise internationally.
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Andre Nel
Division of NanoMedicine; Nanotoxicology; UCLA Medical Center
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Professor of Medicine
Chief of the Division of NanoMedicine at UCLA
"Predictive Toxicological Paradigms for the Assessment of Nanoparticle Toxicity"
Because of the large number of new nanomaterials that are being produced, it is of increasing importance to develop a platform for safety and risk assessment. It is probably not advisable to follow the example of chemical industry where the production of more than 80,000 industrial chemicals has overwhelmed toxicological screening capabilities. Toxicity testing has only been achieved for a few hundred chemicals and as a result, new examples of chemical toxicity show up every year, often with devastating consequences to humans and the environment. One of the principal stumbling blocks in assessing chemical toxicity has been the cost and the logistics to perform animal and in vivo studies. An intuitively more enlightened approach for nanotechnology would be to develop high throughput screening methods that incorporate a relevant toxicological injury mechanisms that can be related to the physicochemical properties of nanomaterials. I will discuss the emerging paradigms of toxicity that can be linked to the physicochemical properties of engineered nanoparticles with a view to outlining scientific principles that originate at the nano/bio interface and could determine whether interactions fail to occur, are biocompatible or injurious in nature. The major toxicological paradigm that have emerged from nanoparticle toxicity relates to the semiconductor, electronic, UV activation, and redox cycling chemistry of the particles, which allows them to induce tissue damage through the generation of oxygen radicals, electron-hole pairs and oxidant injury. It is possible to follow the oxygen radical generation and oxidant stress injury by abiotic methods as well as a set of hierarchical cellular responses that reflect protective, pro-inflammatory, mitochondrial damaging and pro-apoptotic outcomes. An oxidant injury pathway could translate into adaptive, pro-inflammatory or pro-apoptotic cellular effects in the lung, cardiovascular system, skin and the brain. Another important paradigm relates to the ability of nanoparticles to absorb circulatory or cellular proteins as a function of particle size, surface area, functionalized surface groups, charge, hydrophobicity/hydrophilicity etc. This could induce protein unfolding, protein fibrillation, thiol crosslinking and loss of function, which could lead to neurotoxicity, loss of enzymatic activity, and generation of immunological responses. The thermodynamic properties and free surface energy of nanoparticles as a function of particle size, composition, phase and crystallinity could be responsible for particle dissolution in a biological environment, leading to the generation of cytotoxicity through the release of toxic ions or chemicals. Data are also emerging that indicate that cationic nanoparticles exert toxicity through the so-called proton sponge hypothesis, which postulates that particle uptake via acidifying endosomes leads to cellular toxicity through endosomal rupture, cytosolic deposition and mitochondrial targeting. The particle size, state of aggregation/dispersion, functional surface groups and hydrophobicity also plays an important role in determining the route of cellular uptake, subcellular localization and targeting of subcellular organelles. I will demonstrate that it is possible to devise high throughput screening methods to capture each of these toxicological mechanisms, which can then be used to classify nanoparticles into potentially hazardous and potentially safe. If used as a preliminary screen for newly emerging nanomaterials, these predictive science-based approaches can help to determine which materials should undergo priority testing in animal and in vivo exposure models. The knowledge gained from this approach will also reveal which nanomaterial properties are useful to promote biocompatibility.
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Benny Ng
Student, Chemistry and Biochemistry
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"Encapsulation of Fluorescent Polyelectrolytes in Viral
Capsids and Ribonucleoprotein Vaults"
Benny C. Ng, Jason Lin, Stephanie T. Chan, Marcella Yu,
Ajaykumar Gopal, Harold G. Monbouquette, Leonard H. Rome, Sarah H. Tolbert
My research
investigates incorporation of semiconducting polyelectrolytes, poly(2,5-methoxy-propyloxy
sulfonate phenylene vinylene) [MPS-PPV], into viral capsids and
ribonucleoprotein vaults, both hollow cages.Current studies use an icosahedral plant virus (cowpea
chlorotic mottle virus) and cystein-tagged recombinant vaults to encapsulate
MPS-PPV.The virus has its own
negatively charged polyelectrolyte, RNA, which is attracted to its positively
charged inner surface.Access to
the viruses' interior can be achieved during self-assembly in vitro or by
pH-induced swelling of assembled particles.Capsid proteins of cowpea chlorotic mottle virus can
encapsulate fluorescent MPS-PPV.The morphology of the composite depends on the conformation of the
MPS-PPV in its solution environment.
Vaults are
the largest ribonucleoprotein found in higher eukaryotic cells.Although cellular function of these
protein cages is unknown, its hollow cavity and subcellular localization
suggest that they may be involved in nucleo-cytoplasmic transport.Their hollow capped-barrel shape is
measured to be 40 by 70 nm.
However, a mechanism for access to the vaults' interior has not been
identified.By filling the cage
interior with semiconducting polyelectrolytes, whose photophysics is strongly
dependent on its environment, we can compare polymer conformation in these
confined systems to conformation in solution using fluorescence spectroscopy
and Small-angle X-ray Scattering (SAXS).The combined results from fluorescence measurement, fluorescence
quenching studies, and SAXS measuments indicates that luminescent
semiconducting polymers can be localized in of the vaults interior. The results
indicate that vaults can potentially be used as biologically synthesized
nanocapsules for imaging, delivery and encapsulation applications.
*Poster Presenter
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Jason Poulos
Student, Bioengineering
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"Creating practical freestanding
lipid bilayer technologies"
Reconstitution of pore and channel
proteins in artificial lipid bilayer membranes is of considerable interest
scientifically with sensing and pharmaceutical discovery/screening applications
as well. Traditional methods of forming freestanding lipid bilayers, made a
la minute at the point-of-use, require
expertise and result in fragile and short-lived membranes. These shortcomings
preclude their transportation and render them unsuitable for high throughput
applications. We are developing two lipid bilayer platforms to address these
shortcomings. The first is a technique which freezes a membrane-forming
precursor, allowing for portability and long term storage. When thawed, these
precursors form membranes indistinguishable from those formed with conventional
methods able to support channel protein incorporation. The second is a two
phase system adapted from Funakoshi et al., which utilizes gravity to drive the
formation of high quality membranes. This method is easily automated and
compatible with high-throughput robotic processes.
*Poster Presenter
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Melody
Pupols
Student,
Biological Chemistry
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"Nucleic acid nanocapsules:
Packaging RNA into the vault particle using a two-step targeting strategy"
Vaults are 13MDa naturally occurring
cellular nanoparticles. Named for their multiply arched morphology, vaults are
found in 10,000-100,000 copies per cell and are widely conserved across a vast
diversity of eukaryotes. These cytosolic particles are composed of the 97kDa
major vault protein (MVP), the 193kDa vault poly(ADP)ribose polymerase (VPARP),
the 290kDa telomerase/vault associate protein (TEP1), and one or more
untranslated vault RNAs (vRNA). With an internal cavity of 5 x 1073,
the vault particle is large enough to sequester hundreds of proteins. VPARP is
known to associate with MVP via a 163 amino acid MVP minimum interaction
domain ((m)int). This domain is sufficient when fused to heterologous proteins,
such as the green lantern protein GLP, to target them to the interior of the
vault. Furthermore, exogenous vaults are taken up by mammalian cells. Our goal
is to target ribonucleic acid (RNA) to the interior of the vault particle and
subsequently assess uptake and functionality of these packaged nanoparticles in
vivo. To package RNA into the vault, we have
employed a two-step targeting strategy. We utilized the RNA binding
domain from the bacteriophage coat protein MS2 (MS2RBD), which
binds a specific double stem loop RNA secondary structure. By fusing MS2RBD to
the (m)int domain, we created a fusion protein (MS2RBD-(m)int) which can be
targeted to the interior of the vault as well as bind RNA. We have also created
an RNA transcript encoding the green lantern protein (GLP) into which we
incorporated the double stem loop MS2 binding sites in the 3' untranslated
region. The fusion protein MS2RBD-(m)int should now recognize this GLP
transcript via the untranslated MS2 sites. We have shown that both the
MS2RBD-(m)int protein and the GLP-MS2 RNA copurify with recombinant vaults in a
standard vault purification. We have also performed an important control, and
shown that functional GLP protein is produced in transfected mammalian cells
regardless of the addition of the MS2 sites in the untranslated region of the
RNA. Further studies will be carried out to assess the uptake of these packaged
vaults into mammalian cells and to probe for translation of the packaged
GLP-MS2 transcript via fluorescence microscopy. Though many aspects of vault
uptake into cells and later interaction with the cellular environment are
unknown, it will be interesting to see if RNA can not only be packaged into
vault nanocapsules but translated in vivo
as well.
Funded by NIH
*Poster
Presenter
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Robert Purnell
Student, Bioengineering
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"Single Molecule Measurements of DNA
Immobilized in a Biological Nanopore"
Robert Purnell, Kunal Mehta, and
Jacob J. Schmidt
Dept. of Bioengineering, UCLA
Los Angeles, CA
There is significant interest in the
use of biological and synthetic nanopores to perform sensitive and rapid
sensing of single molecules. Biological nanopores, in particular
alpha-Hemolysin (aHL), offer the capability of sensing a large variety of
single molecules at a rapid rate. This is particularly attractive for DNA
because rapid, single-molecule sequencing would significantly reduce system
costs and enable high-throughput analysis of extremely small samples. Previous
work with ssDNA in aHL has used DNA hairpins, which cannot traverse the aHL
pore, to immobilize single-stranded regions inside the pore and measure the
resulting blockage currents. However, hairpins have been shown to produce
current signals with multiple states, making it difficult to establish a
reference conductance value for each base. In this study, strands are
terminated with a streptavidin-biotin cap on the 5' end of the strand. In
addition to preventing complete translocation, this macromolecule (d = 4 nm)
cannot pass into the interior of the pore (d = 2.5 nm). We have found this
eliminates all unfavorable interactions between the immobilizing agent and the
interior residues of the pore. Using this method, we have observed a significant
decrease in multistate behavior in the current signals and an increase in the
consistency of the blockade currents. Here, we report the blockage currents of
adenine, cytosine and thymine polyhomonucleotide strands immobilized inside the
aHL pore. These results will serve as baseline values for future experiments to
measure differences between the nucleotides dynamically as a heteronucleotide
strand traverses the pore.
*Poster Presenter
|
Leonard Rome
Interim Director, California NanoSystems Institute
Senior Associate Dean of Research, David Geffen School of
Medicine at UCLA
Director for Strategic Planning and Partnerships, Jonsson
Comprehensive Cancer Center
Professor, Biological Chemistry
Member, ACCESS Department - Biological Chem., Brain Research
Institute
|
"Vaults: Engineered Nanoparticles for Delivery of
Therapeutics"
Vaults are novel particles first described in 1986 and found to exist in
thousands of copies in most eukaryotic cells. They have an intricate shape
composed of multiple arches reminiscent of cathedral vaults, hence their name.
Vault size (~74 x 42 x 42 nm), shape and localization suggests that they may
involved in nucleo-cytoplasmic transport. The Rome laboratory at UCLA is
interested in elucidating the function of these unique structures and in
manipulating their structure to give them new functions. Rome and his collaborators
are using the baculovirus expression system to produce recombinant vaults in
order to test the concept that vaults can have a broad nanosystems application
as malleable nanocapsules. Toward this aim they are currently designing
particles with encapsulated fluorescent probes and enzymatically active protein
domains. In addition, a number of strategies are currently being considered to
encapsulate chemically active small molecules, drugs and nucleic acids into the
vault particle. If successful, these vault nanocapsules can be bioengineered to
allow their use in a wide variety of biological applications including drug
delivery, biological sensors, enzyme delivery, controlled release, and
nano-electrical machine (NEMS) applications.
|
Makoto Sato
Student, Urology and Molecular
Pharmacology
|
"Prostate targeted safe and
efficient gene therapy vector; recombinant adenovirus equipped with two-tiered
transcriptional amplification system"
Makoto Sato, Mai Johnson, Russell
Powell and Lily Wu.
Department of Urology and Molecular
Pharmacology, David Geffen School of Medicine at University of California Los
Angeles.
We have developed prostate cancer
gene therapy vehicles with a highly specific and robust expression of a
reporter or therapeutic gene.We
utilize adenovirus as an efficient and manageable vehicle.Adenovirus is a double-stranded DNA
virus.The virion is
non-enveloped, spherical and about seventy to ninety nm in size.Adenovirus has many advantages.1) Its biology is well-understood based
on extensive studies, 2) It is easy to manipulate and produce, 3) It has broad
range of infectivity of dividing and non-dividing cells, 4) It may cause only
mild disease, and 5) There's no integration to host genome.
Prostate cancer is one of the
biggest enemies among men's cancer in the United States.Currently we do not have treatment
options for recurrent hormone refractory prostate cancer (HRPC).Our oncolytic adenovirus can be a
weapon to fight this vicious disease.We employed a prostate-targeted gene expression system to direct a safe
and efficient vector-based gene delivery approach.A combination of the modified PSA promoter/enhancer and the
two-step transcriptional amplification (TSTA) system enabled us to achieve
strong expression of transgenes while maintaining excellent prostate
specificity.An integration of the
efficient vehicle and molecular imaging techniques enabled us to assess in
real-time the in vivo gene expression in
human prostate cancer xenograft models (Zhang L. et al. Cancer Res. 63, 2003,
Sato M. et al. Clin. Cancer Res. 11, 2005).When applied to therapeutic study, the TSTA system exhibited
effective tumor cytotoxicity with negligible liver toxicity compared to the constitutive
active CMV promoter (Johnson M. et al. Mol, Imaging, 4, 2005).Thus we showed that the TSTA adenovirus
vector could be utilized as a diagnostic and/or therapeutic tool for prostate
cancer.
We then explored the application of
the TSTA strategy to an oncolytic approach.In this approach, virus is designed to be able to grow (and
kill) only in tumor but efficiently enough.The TSTA system meets this requirement very well.E1A and E1B, two key regulatory genes
for adenoviral replication, are both expressed by the TSTA system to support
the TSTA oncolytic adenovirus to grow.As expected, the TSTA oncolytic adenovirus exhibited efficient viral
production in prostate cancer cells in in vitro infection study.Non-prostate cell lines showed very low levels of viral production, that
confirms excellent prostate specificity.We believe this efficient and specific TSTA oncolytic approach to be a
new promising gene therapy strategy for the advanced stages of HRPC.
*Poster Presenter
|
Sang Son
Student, Chemistry and Biochemistry
|
"Transport
of Live Yeast and Zebrafish Embryo Using a Digital Microfluidic Platform"
Sang Uk Son
and Robin L Garrell*
Department
of Chemistry and Biochemistry, University of California Los Angeles and
California NanoSystems Institute
In digital
microfluidics, droplets can be manipulated between two plates coated with a dielectric
material, or on single plate, by applying a voltage across electrodes beneath
the dielectric. We show for the first time that live yeast (Saccharomyces
cerevisiae) and a zebrafish (Danio rerio) embryo can be transported in droplets. The transported
yeast were subsequently cultured in an incubator to determine their viability
after transport. This was performed by comparing the number of colonies in
cultures of transported and not-transported yeast; the results confirm the
viability of transported yeast. It was also confirmed that transporting yeast
in droplets did not leave yeast cells behind on the device surface. A zebrafish
embryo, 2 hr post fertilization (0.5 mm diameter) was transported in an
extremely large droplet (150 μL) on a specially made device with 5 mm electrodes. The
transported embryo subsequently developed normally and hatched at room
temperature. Droplet-based dechorination of the zebrafish embryo was also
carried out by mixing a droplet of digestive reagent with a droplet containing
the embryo. These experiments demonstrate the feasibility of using a droplet
microfluidic platform to manipulate live organisms, maintaining their viability
in an automated assay.
|
Adam Stieg
Student, Chemistry and Biochemistry
|
"High-Performance Force Imaging and Spectroscopy on Living
Systems"
Adam Z. Stieg, Haider I. Rasool and
James K. Gimzewski
The biological cell constitutes the
basic unit of life. Morphology, energy metabolism, internal fluid flows,
cytoskeletal development, transport, and signaling pathways all affect the
mechanical properties of living cells. The mechanics involved in many of these
processes are pivotal for understanding the structure-function relationships
and regulatory mechanisms associated with cells. Atomic Force Microscopy (AFM)
has recently become widely recognized as a powerful experimental tool in the
field of cellular biomechanics. Many challenges remain in understanding
structural and nanomechanical influences in biological including cellular
reactions to strain, changes in environment, and shift in disease states. The
interactions of mechanical and biomechanical pathways and their relation to
functional states in cells are all amenable to analysis by AFM. This work
entails the development of a high speed, non-contact AFM system toward the goal
of high resolution imaging and spectroscopic analysis of biological systems in
liquid and ambient conditions through the use of sub-nanometer cantilever
oscillation amplitudes at frequencies in the megahertz regime. Small
oscillation amplitudes serve to localize short-range force interactions,
providing the possibility of true atomic and molecular resolution imaging. Use
of high frequency oscillating probes facilitates stable imaging with minimal
perturbation to the biological system of interest. In addition, high frequency
oscillation allows for higher data acquisition rates during force spectroscopy
and imaging. In combination, these features will surpass the limits of current
technologies and further extend force microscopy into the field of biology
research. Our approach toward the study of biological systems merges
examinations of cellular mechanics with those of well-defined synthetic biological
membranes toward the elucidation of structure-function relationships that
ultimately influence global cellular response. Ranging from molecular organization to cellular
architecture, this approach works synergistically to provide insight into our
understanding of the cell as a whole entity.
*Poster Presenter
|
Mike Teitell
Department of Cellular & Molecular Pathology
Associate Professor, Pediatrics,
Pathology and Laboratory Medicine
Member, California NanoSystems
Institute
|
"Cellular Dynamism During Force
Propagation Revealed by Live Cell Interferometry"
Jason Reed, Joshua Troke, Joanna
Schmit, Sen Han, James K. Gimzewski*, and Michael Teitell*
*Equally contributed by
the Departments of Pathology and Laboratory Medicine and Chemistry and
Biochemistry, UCLA, Los Angeles, CA 90095
Cancer and
many other diseases are characterized by changes in cell morphology, motion and
mechanical rigidity.However, in
live-cell-cytology stimulus-induced morphologic changes typically take 10-30
minutes to detect.Here, we employ
live-cell-interferometry (LCI) to visualize the instantaneous response of a
whole cell to mechanical stimulation, and we detect cytoskeletal remodeling
behavior within 200 seconds. This behavior involved small, rapid changes in
cell content and miniscule changes in shape, which would be difficult to detect
with conventional or phase contrast microscopy alone, and is beyond the dynamic
capability of AFM. We
demonstrate that LCI provides a rapid, quantitative reconstruction of the cell
body with no labeling that is highly complementary to traditional microscopy
and flow cytometry, which require cell surface marker detection and/or
destructive cell fixation for labeling.
|
Shimon Weiss
Department of Chemistry and Biochemistry
Professor, Physiology
Member, California NanoSystems Institute
|
"Single molecule probing of dynamic conformation, molecular interactions and dynamic localizations in-vitro, in live cells and in organisms."
We applied single molecule spectroscopy using alternating laser excitation (ALEX) to the study of transcription initiation by e-coli RNA polymerase. We find that the transcription factor sigma70 is not obligatorily released in the transition from initiation to elongation and that the mechanism for abortive initiation is governed by DNA scrunching.
We also applied ALEX spectroscopy to the study of protein folding. We find that the collapsed state of protein L is not driven by native contacts, and we show that Acyl-CoA binding protein (ACBP) has a residual structure in the denatured state.
Lastly, we demonstrate the use of peptide-coated quantum dots for the study of lipid rafts in live cells' membranes and for molecular imaging in living cells and small organisms.
|
Ben Wu
Co-Director, Weintraub Center for Reconstructive
Biotechnology
Associate Professor, Bioengineering
Member, NanoBiotechnology and Biomaterials, Brain Research
Institute, California NanoSystems Institute, UCLA Cardiovascular Stem Cell
Research Center
|
*This talk will take place during the Executive Meeting on
October 31st, 2007.
|
Jennifer Yang
Student, Bioengineering
|
"Biocompatibility and Versatility of
Amphiphilic Block Copolypeptide Hydrogels in the Central Nervous System"
Chu-Ya Yang,1 Bingbing
Song,2 Andrew P. Nowak,1 Yan Ao,2 Michael V.
Sofroniew,2 Timothy J. Deming1
1Bioengineering Department
and 2Neurobiology Department, University of California, Los Angeles,
CA 90095
Mimics of
natural polymers as well as wholly artificial polypeptide sequences have
potential applications in biotechnology (artificial tissues and drug delivery),
biomineralization (resilient, lightweight, ordered inorganic composites), and
diagnostics (biosensors and medical analysis). However, chemical
polymerizations of -amino acids have historically been plagued by numerous side
reactions that limit the utility of these materials. With the discovery of
transition metal species that mediate the controlled polymerization of -amino
acid N-carboxyanhydrides (NCAs), the ability to produce well-defined synthetic
polypeptides has been greatly improved. These synthetic block copolymers
possess controlled molecular weights, narrow polydispersities, and complex
block architectures. The appropriate choice of amino acid monomers leads to
polypeptides that can assemble into various ordered structures and materials.
We have applied this system to the
synthesis of diblock copolypeptide amphiphiles that contain hydrophilic, poly(L-lysine),
poly(L-glutamic acid), or poly(>L-arginine) combined with
hydrophobic, a-helical
poly(L-leucine). They were found to form stiff, clear hydrogels at
low concentrations (~ 1 wt%) in aqueous media. The strength of these hydrogels
can be adjusted by altering the degree of polymerization, relative segment
lengths, and the secondary structure in the hydrophobic segment. Laser Scanning
Confocal Microscopy and CryoTEM revealed a spontaneously formed microporous
network with large (~ 10 >mm)
water rich voids and percolating cellular networks with ~ 100 nm pores that
appear to be comprised of both membranes and fibers. Rheological characterization
showed these hydrogels also display interesting mechanical properties including
rapid recovery of solid like behavior after being broken down by shear, and
mechanical stability at elevated temperature (~ 90 oC). With proper tuning of the
relative block compositions it was found that hydrogels could be optimized to
possess good solubility and mechanical strength in many useful ionic buffers (~
100- 200 mM) including cell culture media.
A unique combination of properties (e.g. heat stability, microporosity, and injectability) has made
these polypeptide hydrogels attractive as cell scaffold materials since they
are structurally similar to the extracellular matrix (ECM) of many tissues.
Currently, there are no viable methods to achieve axon regeneration after
chronic spinal cord injury (SCI). We have performed in vivo studies using
lysine-block-leucine and arginine-block-leucine hydrogels in mice that showed good biocompatibility.
We are currently conducting experiments with functionalized hydrogels to
support and stimulate axon growth in SCI. Block copolypeptides with different
compositions at different concentrations and in different vehicles were
prepared and tested. The preparation of the hydrogels and the biocompatibility
assays will be presented.
*Poster Presenter
|
Marcella Yu and Lisa Goldsmith
Students, Chemical and Biomolecular
Engineering
|
"Toward Vaults as Drug Delivery
Vehicles: Two Methods for Loading Materials into Vault Interior"
Marcella Yu1, Lisa E.
Goldsmith1, Valerie A. Kickhoefer2, Leonard H.
Rome2,3, Harold G. Monbouquette1,3
1Chemical
and Biomolecular Engineering Department, 2Department of Biological
Chemistry, and 3California NanoSystems Institute, University of
California, Los Angeles, CA
Native
vaults (72.5 × 41 nm) are self-assembled, ribonucleoprotein nanocapsules,
that consist of multiple copies of three proteins (MVP, VPARP, and TEP1) and a
small untranslated RNA.Although
vaults exist in most eukaryotic cells, their function remains unknown.However, this naturally occurring
"organelle" has significant potential as a drug delivery system due to its
biocompatibility, large and accessible lumen, and ability to be taken up by
mammalian cell lines.Our goal is
to develop methods of entrapping drugs or DNA within vaults for targeted
delivery.In this poster, we
investigated the loading of materials into the vault interior using two
different approaches.Both methods
exploit the dynamic structure of vaults in solution, and the ability of a
protein (INT) to bind specifically to the vault interior.
In order to sequester molecules of interest in a stable state
for subsequent delivery, horseradish peroxidase (HRP), which catalyzes the
polymerization of tyramine, was fused to INT. The fusion protein (HRP-INT) was expressed using a
baculovirus expression system in Sf9 insect cells and then packaged inside of
vaults at the crude extract stage.The HRP-INT protein was found to co-purify with vaults and to retain HRP
activity, which suggests that the HRP harbored inside the vault remains
functional.Current studies
include efforts to increase protein expression, and to analyze polymer
synthesis using vaults containing entrapped HRP activity.
(2) INT was utilized as a protein "shuttle" to actively
target an attached 1.8 nm gold probe to the vault interior.Vaults loaded with the gold-INT complex
are separated from unbound probes by immunoprecipitation with antibody-agarose
beads.Successful encapsulation of
the gold-INT complex within vaults has been verified with parallel western blot
and silver development (to detect gold) analyses, by TEM imaging after gold
enhancement, and delayed fluorescence quenching kinetics of encapsulated versus
soluble Au-GFP-INT complex.
*Poster Presenters
|
Hong Zhou
Professor, Microbiology, Immunology
& Molecular Genetics
Member, ACCESS, ACCESS Department -
MIMG, California NanoSystems Institute
|
"Seeing
Biological Nano-machineries by Cryo-Electron Microscopy (cryoEM) and Tomography
(cryoET)"
Recent
advances have made electron imaging an indispensable tool for determining the
three-dimensional (3D) structures and molecular interactions of macromolecular
complexes or biological nano-machineries. The newly established Electron
Imaging Center for Nanomachines at UCLA (http://EICN.CNSI.UCLA.edu) aims to provide this
emerging technology in its finest forms to both nano biology and nano-materials
science researchers. A number of 3D structures of biological assemblies will be
presented as examples to illustrate the potential of this advanced tool at
CNSI. Two slightly different modalities of electron imaging- single particle
cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET)- are
commonly employed to visualize or "see" nano-biological machineries or
particles of different structural property.For nano-particles with a homogenous structural
organization, such as protein/DNA/RNA complexes and viral capsids, cryoEM is
used to record a low-dose image for particles embedded in vitreous ice. Images
of thousands of randomly oriented "single" particles are then averaged to
obtain a 3D structure to near-atomic resolution (0.3-0.6nm). At this
resolution, the majority of amino-acid backbones and some bulky side chains are
resolved in addition to a-helices, b-sheets and loops. Such structural data provide valuable
constraints for building atomic-resolution models through integrative
cryoEM-bioinformatics modeling means. For materials and complexes with
pleomorphic or dynamic structures where averaging is not possible, cryoET is
used to obtain their 3D structures at molecular resolution (2-5 nm) from a tilt
image series of the samples. These structural methods provide exciting
opportunities to biologists, chemists and materials scientists for 3D
structural characterization of a wide variety of nanometer-scale assemblies,
devices and materials.
|
Jeffrey Zink
Professor, Chemistry and Biochemistry, Inorganic Chemistry,
Physical Chemistry
Member, NanoBiotechnology and Biomaterials, NanoElectronics,
Photonics, Architectonics, NanoMechanical and Nanofluidic Systems, California
NanoSystems Institute
|
"Nanovalve and controlled release"
|
|
