This thesis investigated using ellipsometry to analyze ligand binding to G-protein coupled receptors (GPCRs). GPCRs are important cell surface proteins linked to many diseases. Ellipsometry is an optical technique that can quantify ligand-receptor interactions by measuring changes at a surface when polarized light is reflected off. An A549 epithelial cell line expressing the CXCR4 GPCR and its ligand CXCL12 was used. Enzyme-linked immunosorbent assays were performed to determine optimal ligand and antibody concentrations. Baseline characterization of glass slides and binding experiments with ligand and antibody were conducted using ellipsometry. Psi-delta analysis of collected data showed trends in binding.
Journal club presentation on:
Pandya, C., Brown, S., Pieper, U., Šali, A., Dunaway-Mariano, D., Babbitt, P. C., et al. (2013). Consequences of domain insertion on sequence-structure divergence in a superfold. Proceedings of the National Academy of Sciences of the United States of America, 110(36), E3381–7. doi:10.1073/pnas.1305519110
A Systems Biology Approach to Natural Products ResearchHuda Nazeer
Explains the systems biology approach (holistic approach), its advantages and tools used compared to the reductionist approach in natural products research.
Journal club presentation on:
Pandya, C., Brown, S., Pieper, U., Šali, A., Dunaway-Mariano, D., Babbitt, P. C., et al. (2013). Consequences of domain insertion on sequence-structure divergence in a superfold. Proceedings of the National Academy of Sciences of the United States of America, 110(36), E3381–7. doi:10.1073/pnas.1305519110
A Systems Biology Approach to Natural Products ResearchHuda Nazeer
Explains the systems biology approach (holistic approach), its advantages and tools used compared to the reductionist approach in natural products research.
3DSIG 2014 Presentation: Systematic detection of internal symmetry in proteinsSpencer Bliven
These slides are from 3DSIG 2014, presented on July 11.
I describe our investigation of internal symmetry in protein structures. This is quite common (24% of domains), and has many implications for function, folding, and evolution.
I introduce the CE-Symm method, described in
Myers-Turnbull, D., Bliven, S. E., Rose, P. W., Aziz, Z. K., Youkharibache, P., Bourne, P. E., & Prlić, A. (2014). Systematic Detection of Internal Symmetry in Proteins Using CE-Symm. Journal of Molecular Biology, 426(11), 2255–2268. doi:10.1016/j.jmb.2014.03.010
I discuss the results from running CE-Symm across the PDB, as well as some particularly compelling examples.
See also my poster by the same title for more details.
Personalized Medicine and the Omics Revolution by Professor Mike SnyderThe Hive
Personalized medicine is expected to benefit from the combination of genomic information with the global monitoring of molecular components and physiological states. To ascertain whether this can be achieved, we determined the whole genome sequence of an individual at high accuracy and performed an integrated Personal Omics Profiling (iPOP) analysis, combining genomic, transcriptomic, proteomic, metabolomic, and autoantibodyomic information, over a 38-month period that included healthy and two virally infected states. Our iPOP analysis of blood components revealed extensive, dynamic and broad changes in diverse molecular components and biological pathways across healthy and disease conditions. Importantly, genomic information was also used to estimate medical risks, including Type 2 Diabetes, whose onset was observed during the course of our study. Our study demonstrates that longitudinal personal omics profiling can relate genomic information to global functional omics activity for physiological and medical interpretation of healthy and disease states.
Meet the speaker, Professor Michael Snyder (Stanford):
Michael Snyder is the Stanford Ascherman Professor, Chair of Genetics and the Director of the Center of Genomics and Personalized Medicine. He received his Ph.D. from the California Institute of Technology and postdoctoral training at Stanford University. He is a leader in the field of functional genomics and proteomics, and one of the major participants of the ENCODE project. His laboratory study was the first to perform a large-scale functional genomics project in any organism, and has launched many technologies in genomics and proteomics. These including the development of proteome chips, high resolution tiling arrays for the entire human genome, methods for global mapping of transcription factor binding sites (ChIP-chip now replaced by ChIP-seq), paired end sequencing for mapping of structural variation in eukaryotes, de novo genome sequencing of genomes using high throughput technologies and RNA-Seq. These technologies have been used for characterizing genomes, proteomes and regulatory networks. Seminal findings from the Snyder laboratory include; the discovery that much more of the human genome is transcribed and contains regulatory information than was previously appreciated, and a high diversity of transcription factor binding occurs both between and within species. He has also combined different state-of–the-art omics technologies to perform the first longitudinal detailed integrative personal omics profile (iPOP) of person and used this to assess disease risk and monitor disease states for personalized medicine. He is a co-founder of several biotechnology companies including; Protometrix (now part of Life Technologies), Affomix (now part of Illumina), Excelix, and Personalis, and he presently serves on the board of a number of companies.
Detection of misfolded aβ oligomers for sensitive biochemical diagnosis of Al...José Luis Moreno Garvayo
El equipo del Dr. Claudio Soto de la Universidad de Texas ha conseguido demostrar que son capaces de detectar pequeños fragmentos de proteínas mal plegadas, los precursores de las placas seniles, que pueden estar circulando por nuestro cuerpo durante años o décadas antes de que surjan los primeros síntomas de la enfermedad de Alzheimer
Plasma phospholipids identify antecedent memory impairment in older adultsJosé Luis Moreno Garvayo
En este trabajo publicado en la revista Nature medicine el pasado mes de marzo, el equipo del Dr. Federoff plantea un novedoso enfoque que consiste en analizar un grupo de diez fosfolípidos para la detección de la enfermedad de Alzheimer antes de la manifestación clínica de los síntomas.
Slides from my talk describing CE-Symm and my research on internal symmetry. It was given for jLBR, the weekly seminar series for our department at PSI.
PAMAM/5-fluorouracil drug conjugate for targeting E6 and E7 oncoproteins in c...Arun kumar
In the present study, poly(amidoamine)/5-fluorouracil (PAMAM/5-FU) was prepared and used as
a conjugate system for delivering drugs to target E6 and E7 oncoproteins, which are predominant in
cervical cancers. Specifically, molecular docking analysis was used to investigate the interaction
between the PAMAM/5-FU and E6/E7 oncoproteins, which showed that the PAMAM/5-FU conjugate
had a higher affinity for the oncoprotein than for 5-FU. Different generations of PAMAM dendrimers
(0.5G, 1.0G, 1.5G, 2.0G, and 2.5G) were synthesized, characterized and tested as drug carriers for 5-
FU. The PAMAM and PAMAM/5-FU drug conjugate showed less toxicity over COS-7 and HeLa cell
lines. Laser confocal imaging and western blotting for tumor suppressor proteins pRb and p53 were
used to confirm the interaction of PAMAM/5-FU with E6/E7 oncoproteins. Hematological analysis of
PAMAM/5-FU using BALB/c female mice with cervical cancer confirmed the less toxic nature of this
material. Based on these results, the developed PAMAM/5-FU conjugate is a potential candidate for
the treatment of cervical cancer.
26 ASBMB TODAY FEBRUARY 2021Discovering an old DoGs’ neMargaritoWhitt221
26 ASBMB TODAY FEBRUARY 2021
Discovering an old DoGs’
new trick
Heterotrimeric G proteins regulate
a variety of signaling pathways that
control cell development and influ-
ence cell morphology via actin/cyto-
skeleton remodeling. There are four
main families of G proteins: Gi/Go,
Gq, Gs and G12/13. Researchers long
have thought that Gs, unlike its family
members, is coupled specifically and
exclusively to adenylyl cyclases.
In a new study published in the
Journal of Biological Chemistry,
Alejandro Castillo–Kauil of the
Center for Research and Advanced
Studies of the National Polytechnic
Institute and collaborators challenge
this dogmatic view by identifying a
new Gs target. Using biochemical,
molecular biological and chemo-
genetic approaches, the researchers
demonstrated that the Gαs subfamily
of G proteins can regulate the activity
of Rho GTPases such as Rho guanine
nucleotide exchange factor, or Rho-
GEF. The interaction identified by the
group activates the small G protein
Cdc42 by Gs-coupled GPCRs, stimu-
lating a rearrangement of the cyto-
skeleton and inducing formation of
fingerlike protrusions called filopodia.
These results provide new insight
into G protein activity and define a
new role for RhoGEF coupling in G
protein function.
DOI: 10.1074/jbc.AC120.015204
A pathogen’s proteins target
mitochondria
The tick-borne pathogen Coxiella
burnetii causes Q fever, or query fever,
a rare flulike disease that can spread
to humans who inhale dust particles
contaminated by infected farm or
CONTINUED FROM PAGE 25
Noninvasive tool provides oral cancer prognosis
Oral squamous cell carcinoma, which affects about 34,000 people
in the U.S. each year, is found in the cells lining the lips and mouth.
Metastasis to the lymph nodes is a sign of disease progression and may
be accompanied by changes in proteolytic activity. During proteolysis,
enzymes cut up proteins into short fragments called peptides. Recent
work suggests that characterizing the sequence and abundance of these
molecules — a method dubbed peptidomics — might provide new in-
sight on cancer biology and in the clinic. In a recent paper in the journal
Molecular & Cellular Proteomics, Leandro Xavier Neves of the Brazil-
ian Biosciences National Laboratory and a team of Brazilian clinicians
and scientists describe their analysis of oral squamous cell carcinoma
patient saliva using peptidomics.
After extracting peptides from saliva samples, the research team ana-
lyzed and compared the peptide content in samples from patients with
and without metastasis to the lymph nodes. They found more than 1,000
uniquely expressed peptides in each group and an additional 1,628 pep-
tides expressed by both groups. A series of statistical analyses identified
77 peptides of particular interest; all of these peptides are overexpressed
in samples from patients with lymph node metastasis, which supports the
hypothesis that proteolytic activity increases ...
3DSIG 2014 Presentation: Systematic detection of internal symmetry in proteinsSpencer Bliven
These slides are from 3DSIG 2014, presented on July 11.
I describe our investigation of internal symmetry in protein structures. This is quite common (24% of domains), and has many implications for function, folding, and evolution.
I introduce the CE-Symm method, described in
Myers-Turnbull, D., Bliven, S. E., Rose, P. W., Aziz, Z. K., Youkharibache, P., Bourne, P. E., & Prlić, A. (2014). Systematic Detection of Internal Symmetry in Proteins Using CE-Symm. Journal of Molecular Biology, 426(11), 2255–2268. doi:10.1016/j.jmb.2014.03.010
I discuss the results from running CE-Symm across the PDB, as well as some particularly compelling examples.
See also my poster by the same title for more details.
Personalized Medicine and the Omics Revolution by Professor Mike SnyderThe Hive
Personalized medicine is expected to benefit from the combination of genomic information with the global monitoring of molecular components and physiological states. To ascertain whether this can be achieved, we determined the whole genome sequence of an individual at high accuracy and performed an integrated Personal Omics Profiling (iPOP) analysis, combining genomic, transcriptomic, proteomic, metabolomic, and autoantibodyomic information, over a 38-month period that included healthy and two virally infected states. Our iPOP analysis of blood components revealed extensive, dynamic and broad changes in diverse molecular components and biological pathways across healthy and disease conditions. Importantly, genomic information was also used to estimate medical risks, including Type 2 Diabetes, whose onset was observed during the course of our study. Our study demonstrates that longitudinal personal omics profiling can relate genomic information to global functional omics activity for physiological and medical interpretation of healthy and disease states.
Meet the speaker, Professor Michael Snyder (Stanford):
Michael Snyder is the Stanford Ascherman Professor, Chair of Genetics and the Director of the Center of Genomics and Personalized Medicine. He received his Ph.D. from the California Institute of Technology and postdoctoral training at Stanford University. He is a leader in the field of functional genomics and proteomics, and one of the major participants of the ENCODE project. His laboratory study was the first to perform a large-scale functional genomics project in any organism, and has launched many technologies in genomics and proteomics. These including the development of proteome chips, high resolution tiling arrays for the entire human genome, methods for global mapping of transcription factor binding sites (ChIP-chip now replaced by ChIP-seq), paired end sequencing for mapping of structural variation in eukaryotes, de novo genome sequencing of genomes using high throughput technologies and RNA-Seq. These technologies have been used for characterizing genomes, proteomes and regulatory networks. Seminal findings from the Snyder laboratory include; the discovery that much more of the human genome is transcribed and contains regulatory information than was previously appreciated, and a high diversity of transcription factor binding occurs both between and within species. He has also combined different state-of–the-art omics technologies to perform the first longitudinal detailed integrative personal omics profile (iPOP) of person and used this to assess disease risk and monitor disease states for personalized medicine. He is a co-founder of several biotechnology companies including; Protometrix (now part of Life Technologies), Affomix (now part of Illumina), Excelix, and Personalis, and he presently serves on the board of a number of companies.
Detection of misfolded aβ oligomers for sensitive biochemical diagnosis of Al...José Luis Moreno Garvayo
El equipo del Dr. Claudio Soto de la Universidad de Texas ha conseguido demostrar que son capaces de detectar pequeños fragmentos de proteínas mal plegadas, los precursores de las placas seniles, que pueden estar circulando por nuestro cuerpo durante años o décadas antes de que surjan los primeros síntomas de la enfermedad de Alzheimer
Plasma phospholipids identify antecedent memory impairment in older adultsJosé Luis Moreno Garvayo
En este trabajo publicado en la revista Nature medicine el pasado mes de marzo, el equipo del Dr. Federoff plantea un novedoso enfoque que consiste en analizar un grupo de diez fosfolípidos para la detección de la enfermedad de Alzheimer antes de la manifestación clínica de los síntomas.
Slides from my talk describing CE-Symm and my research on internal symmetry. It was given for jLBR, the weekly seminar series for our department at PSI.
PAMAM/5-fluorouracil drug conjugate for targeting E6 and E7 oncoproteins in c...Arun kumar
In the present study, poly(amidoamine)/5-fluorouracil (PAMAM/5-FU) was prepared and used as
a conjugate system for delivering drugs to target E6 and E7 oncoproteins, which are predominant in
cervical cancers. Specifically, molecular docking analysis was used to investigate the interaction
between the PAMAM/5-FU and E6/E7 oncoproteins, which showed that the PAMAM/5-FU conjugate
had a higher affinity for the oncoprotein than for 5-FU. Different generations of PAMAM dendrimers
(0.5G, 1.0G, 1.5G, 2.0G, and 2.5G) were synthesized, characterized and tested as drug carriers for 5-
FU. The PAMAM and PAMAM/5-FU drug conjugate showed less toxicity over COS-7 and HeLa cell
lines. Laser confocal imaging and western blotting for tumor suppressor proteins pRb and p53 were
used to confirm the interaction of PAMAM/5-FU with E6/E7 oncoproteins. Hematological analysis of
PAMAM/5-FU using BALB/c female mice with cervical cancer confirmed the less toxic nature of this
material. Based on these results, the developed PAMAM/5-FU conjugate is a potential candidate for
the treatment of cervical cancer.
26 ASBMB TODAY FEBRUARY 2021Discovering an old DoGs’ neMargaritoWhitt221
26 ASBMB TODAY FEBRUARY 2021
Discovering an old DoGs’
new trick
Heterotrimeric G proteins regulate
a variety of signaling pathways that
control cell development and influ-
ence cell morphology via actin/cyto-
skeleton remodeling. There are four
main families of G proteins: Gi/Go,
Gq, Gs and G12/13. Researchers long
have thought that Gs, unlike its family
members, is coupled specifically and
exclusively to adenylyl cyclases.
In a new study published in the
Journal of Biological Chemistry,
Alejandro Castillo–Kauil of the
Center for Research and Advanced
Studies of the National Polytechnic
Institute and collaborators challenge
this dogmatic view by identifying a
new Gs target. Using biochemical,
molecular biological and chemo-
genetic approaches, the researchers
demonstrated that the Gαs subfamily
of G proteins can regulate the activity
of Rho GTPases such as Rho guanine
nucleotide exchange factor, or Rho-
GEF. The interaction identified by the
group activates the small G protein
Cdc42 by Gs-coupled GPCRs, stimu-
lating a rearrangement of the cyto-
skeleton and inducing formation of
fingerlike protrusions called filopodia.
These results provide new insight
into G protein activity and define a
new role for RhoGEF coupling in G
protein function.
DOI: 10.1074/jbc.AC120.015204
A pathogen’s proteins target
mitochondria
The tick-borne pathogen Coxiella
burnetii causes Q fever, or query fever,
a rare flulike disease that can spread
to humans who inhale dust particles
contaminated by infected farm or
CONTINUED FROM PAGE 25
Noninvasive tool provides oral cancer prognosis
Oral squamous cell carcinoma, which affects about 34,000 people
in the U.S. each year, is found in the cells lining the lips and mouth.
Metastasis to the lymph nodes is a sign of disease progression and may
be accompanied by changes in proteolytic activity. During proteolysis,
enzymes cut up proteins into short fragments called peptides. Recent
work suggests that characterizing the sequence and abundance of these
molecules — a method dubbed peptidomics — might provide new in-
sight on cancer biology and in the clinic. In a recent paper in the journal
Molecular & Cellular Proteomics, Leandro Xavier Neves of the Brazil-
ian Biosciences National Laboratory and a team of Brazilian clinicians
and scientists describe their analysis of oral squamous cell carcinoma
patient saliva using peptidomics.
After extracting peptides from saliva samples, the research team ana-
lyzed and compared the peptide content in samples from patients with
and without metastasis to the lymph nodes. They found more than 1,000
uniquely expressed peptides in each group and an additional 1,628 pep-
tides expressed by both groups. A series of statistical analyses identified
77 peptides of particular interest; all of these peptides are overexpressed
in samples from patients with lymph node metastasis, which supports the
hypothesis that proteolytic activity increases ...
Method for physiologic phenotype characterization at the single-cell level in...Shashaanka Ashili
Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells
under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of
interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system’s capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.
A seminar report on the chemical frontiers of living matter seminar series - ...Glen Carter
This seminar report highlights a select few presentations of cutting-edge research being done in various labs across the Paris Science et Lettre (PSL) network.
Molecular mechanisms of action and potential biomarkers of growth inhibition ...Enrique Moreno Gonzalez
Molecular targeted therapy has emerged as a promising treatment of Hepatocellular carcinoma (HCC). One potential target is the Src family Kinase (SFK). C-Src, a non-receptor tyrosine kinase is a critical link of multiple signal pathways that regulate proliferation, invasion, survival, metastasis, and angiogenesis. In this study, we evaluated the effects of a novel SFK inhibitor, dasatinib (BMS-354825), on SFK/FAK/p130CAS, PI3K/PTEN/Akt/mTOR, Ras/Raf/MAPK and Stats pathways in 9 HCC cell lines.
In vitro experiments of prokaryotic and eukaryotic antimicrobial peptide cyto...AI Publications
These proteinaceous molecules, called antimicrobial peptides (AMPs), are a varied collection of antimicrobial peptides. The ability of AMPs to combat gut infections necessitates further study of the AMP-GI tract interaction. These peptides need to be tested in vitro for cytotoxicity before they may be considered for use in clinical infections. Using the MTT conversion assay, neutral red dye absorption assay, and a comparison to vancomycin, researchers examined the cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin in two gastrointestinal cell types (HT29 and Caco-2). Sheep erythrocyte hemolytic activity was also studied, and the influence of AMPs on paracellular permeability was assessed using transepithelial resistance (TEER) and TEM. Gallidermin, nisin A, magainin I, magainin II, and melittin were the least cytotoxic AMPs. To our knowledge, only Melittin and NIS caused considerable hemolysis. There are two distinct ways that melittin and nisin differ in their ability to kill bacteria. It was the only AMP that had an effect on the permeability of the paracellular space. Intestinal tight junctions and cell–cell adhesion were destroyed by long-term melittin therapy, as were microvilli, cell debris, and cell–cell adhesion. Antimicrobial activity and low cytotoxicity make Gallidermin a promising therapeutic drug. The antibacterial properties of Melittin are limited, but its ability to transport poorly bioavailable medicines may be useful.
The influence of reduced oxygen availability on gene expression in laboratory...Santhi Devasundaram
Virtually all dormant
models against tuberculosis tested in animals used laboratory strain H37Rv or Erdman strain. But major
outbreaks of tuberculosis (TB) occur with the strains that have widely different genotypes and phenotypes
compared to H37Rv. In this study, we used a custom oligonucleotide microarray to determine the overall
transcriptional response of laboratory strain (H37Rv) and most prevalent clinical strains (S7 and S10) of
M. tuberculosis from South India to hypoxia.
The influence of reduced oxygen availability on gene expression in laboratory...
Weber-Thesis
1. Investigation of Ligand Binding to G-protein Coupled Receptors with the
Use of Ellipsometry
A thesis submitted on the 25th of February, 2016
To the Department of Biology and Biomedical Engineering of
Rose-Hulman Institute of Technology
In partial fulfillment of the requirements for the degree of
Bachelor of Science in Biology, May 2016
By
__________________________________________________
Anna Mary Weber
Approved: ____________________________________________
Dr. Jennifer O’Connor, Ph.D., Advisor
3. ABSTRACT
Weber, Anna Mary
B.S. Biology, Rose-Hulman Institute of Technology
May 2016
Investigation of Ligand Binding to G-protein Coupled Receptors with the Use of Ellipsometry
Jennifer O’Connor Ph.D.
G-protein coupled receptors (GPCRs) are integral membrane proteins associated with the
signaling of many biological processes. There has been links identified between GPCRs and
various diseases. The pharmaceutical industry is targeting these proteins, but are unable to track
the efficiency, primarily due to limitations with current technologies. Ellipsometry, an optical
technique, can be used to quantify and explain the binding of ligands to receptors of the cell
surface. A549 cell cultures, which express the GPCR, CXCR4, and its associated ligand,
CXCL12, were used for experimentation. Optimal ligand and antibody concentrations were
determined using the well-establish method, ELISA. Baseline and biological testing was
performed on the ellipsometer. Psi-Delta analysis was performed on the collected data where
trends were visually inspected.
4. ii
TABLE OF CONTENTS
LIST OF FIGURES………….…………………………………………………………………...iii
INTRODUCITON…………….…………………………………………………………..………1
LITERATURE REVIEW….…………………………………………………………..………….3
MATERIALS AND METHODS…………………………………………………………….....…5
Cell Culture…………………………………………………………….....……………….5
Enzyme Linked Immunosorbent Assay…………………………………………………...5
Slide Preparation …………………………………………………………….....…………6
Spectroscopic Ellipsometry……………………………………………………………….6
Ellipsometric Experimentation……………………………………………………………6
Psi-Delta Data Analysis……………………………………………………………...........7
RESULTS ……………………………………………………………………………..…….……8
DISCUSSION……………………………………………………………………………………11
CONCLUSIONS………………………………………………………………………………...12
BIBLIOGRAPHY………………………………………………………………..………………13
5. iii
LIST OF FIGURES
Figure 1: Cell Culture………………………………………………….………………………...14
Figure 2: Schematic of Enzyme Linked Immunosorbent Assay…….…………………………..15
Figure 3: Ellipsometry Apparatus……………………………………………………………….16
Figure 4: Example Psi Delta Output From Ellipsometer….…………………………………….17
Figure 5: Glass Slide Characterization Data…………………………………………………….18
Figure 6: Glass Slide with Ligand Data…...…………………………………………………….19
Figure 7: Glass Slide with Antibody Data…...…………………………………………………..20
Figure 8: Glass Slide with Ligand and Antibody Data…...………………………….…………..21
6. 1
INTRODUCTION
G-protein coupled receptors (GPCRs) are integral cell surface proteins that are comprised of
seven trans-membrane helices, responsible for driving many biological processes through the use
of several signal transduction pathways (Kriechbaumer et al., 2012). When a ligand binds to its
specific receptor, a conformational change occurs resulting in heterotrimeric G proteins to
become activated. These proteins are responsible for the transmission of signal molecules to the
interior of the cell (Oldham et al., 2007). Both cellular signaling and mutation of the proteins
can potentially result in detrimental physiological function.
Various diseases including schizophrenia, cancer, and diabetes have all been directly related to
GPCRs (Kriechbaumer et al., 2012). Because GPCRs are both present in high frequencies in the
human genome, with about 800 locations being recognized to date, and play a crucial role in cell
signaling, they are a prime target for drugs and treatment procedures (Giraldo et al., 2011).
Presently, upwards of 60 percent of pharmaceuticals target GPCRs that have been designed to
treat ailments of many physiological systems (Schoneberg et al., 2004). Well known
pharmaceutical companies like GlaxoSmithKline, Eli Lilly, and Pfizer are all investigating in
treatment options targeting these receptors (Kriechbaumer et al., 2012).
Drug discovery is a lengthy process today taking anywhere from 10-12 years from the initial
research and development phase to the creation of a commercially recognized drug (Zang et al.,
2012). The development phase is time consuming due to the extensive testing procedures and
inefficient screening processes currently on the market. More relevant systems of analysis and
high throughput screening are desired in order to expedite the drug discovery process.
7. 2
Currently, the main method of ligand and receptor binding analysis is done through the use of
cell based screening assays. This method is the most common likely due to the ease of the
process making use of simple components like fluorescence and cell markers (Zang et al., 2012).
Also associated with cell based screening assays, however, are critical assumptions that represent
limitations with this method. It is assumed cell viability is stable throughout and at a
continuously, relatively high level, resulting in potential anomalies during the data analysis
portion (Azouz, 2014). The compromise in cell viability can be attributed to the lengthy
processing time associated with these assays. Due to the limitations in accurately representing
cell receptor and ligand binding, a more efficient method of categorization must be determined.
I tested the use of ellipsometry to analyze the binding of cell and ligands. Ellipsometry is an
optical technique in which the efficacy of ligand bind can be measured through the analysis of a
polarized light spectrum that this refracted off of a specimen. This method has been utilized by
some other researchers and is able to successfully quantify the interaction of membranes and
ligand binding to a GPCR (Kriechbaumer et al., 2012). Ellipsometry is able to track minute
changes in the environment, specifically those at the surface of a membrane making this method
appropriate to investigate interactions.
8. 3
LITERATURE REVIEW
Though GPCRs represent significant physiological importance, both intensive study and
investigation of therapeutic treatment have prevailed as a challenging task. Structural and
functional analysis of some of the proteins are restricted by the inability to create and maintain
situations comparable to that of nature. The membrane lacks stability when introduced into
certain buffers resulting in fluctuations in data analysis (Kriechbaumer et al., 2012).
Because the stability of the membranes alone is questionable, the use of the GPCR in a cellular
system is desirable. For a model system, we used of an epithelial cell line, A549, said to express
a specific GPCR, CXCR4, based upon the findings of previous researchers.
Chemokines are a group of cytokines that act as signaling proteins, functioning primarily as part
of the immune response (Zlotnik et al., 2000). Regulation associated with these proteins is
mainly driven by the interaction of the molecules with GPCRs. In humans, upwards of 40
chemokines have been identified ranging in location from the lymphatic system to the nervous
system (Zlotnik et al., 2000). Chemokine receptors, though representing a small family of
proteins currently, are further subdivided into four subfamilies: CXC, CC, CX3C, and XC. The
nomenclature of the subfamilies is attributed to the arrangement of residues on the side chains of
the proteins (Zlotnik et al., 2000). CXC chemokine receptors (CXCRs) are named for the
separation of the two cysteine residues separated by a one different amino acid.
CXCRs are found on a variety of cells, most notably hematopoietic cells and vascular endothelial
cells. CXCR4 has been characterized and is functionally active said having the ability pair to
9. 4
certain G-proteins. The A549 cell line expresses mRNA for CXCR4 which makes it a good
target for experimentation (Murdoch et al., 1999). CXCR4 binds naturally with its ligand
CXCL12 (Kriechbaumer et al., 2012). The selected cell line, A549 expressing this receptor, was
used in combination with the appropriate chemokine ligand and associated antibody.
10. 5
MATERIALS and METHODS
Cell Culture
A549, human epithelial, cell culture was grown in a Corning cell culture flasks in F-12K
Medium (Kaighn's Modification of Ham's F-12 Medium) supplemented with 10% Fetal Bovine
Serum (FBS). Cultures were incubated at 37°C with media renewal 2 to 3 times per week.
Cultures were grown to confluency and then passaged and either subcultured or frozen down. To
passage cells, growth medium was removed and remaining cell layer was treated with 1 mL 10%
concentrated trypsin and incubated until cells became suspended. For subculture, half of the
trypsin was removed and placed in another culture vessel With F-12K media supplemented with
10% FBS. To freeze cells, confluent cells were treated with trypsin, then centrifuged to
concentrate, and resuspended in F-12K media with 10% DMSO. After acclimating in an ethanol
bath overnight at -80°C, cells were stored in liquid nitrogen.
Enzyme Linked Immunosorbent Assay (ELISA)
A double antibody sandwich ELISA was performed (Current, 2008).. Lyophilized Recombinant
Human/Feline CXCL12/SDF-1 beta and lyophilized Human CXCL12/SDF-1 Antibody were
both reconstituted in molecular grade water. Dilutions were made to 200 ng/mL and 10 μg/mL
of ligand and antibody, respectively. A 96 well ELISA plate was collected. 10 μg/mL of the
antibody was added and 1:2 serially diluted across the wells. The antibody was blocked using a
5% Bovine Serum Albumin solution and incubated for 2 hours. After incubation, the wells were
washed with water and shaken dry. This wash and drying process was repeated two more times.
Blocking buffer was added again and incubated for an additional 10 minutes. The wells were
washed and dried five more times. Any additional liquids were removed with a pipette. 200
11. 6
ng/mL of ligand was added and 1:2 serially diluted across the wells. The plate was incubated for
2 hours. The wash process was repeated at this step 3 times and a blocking buffer was added.
Wells were washed again. Specific antibodies were added and incubated for 2 hours and room
temperature. The wells were washed and horse radish peroxidase conjugated antibodies were
added. Plate was incubated at room temperature for 1 hour (Current, 2008).. Absorbance was
measured using the BioTek ELISA Reader.
Slide Preparation
Standard microscope slides were used for experimentation. Opaque tape pieces were affixed to
entire bottom surface of the slide.
Spectroscopic Ellipsometry
A J.A. Woollam Co., Inc. α-SE spectroscopic ellipsometer apparatus was used for
experimentation in conjunction with Version 4.72 of CompleteEASE Data Acquisition and
Analysis Software for Spectroscopic Ellipsometers.
The measurements were collected at a standard angle of incidence of 70°. Measurement controls
were maintained at a constant collection method through all experiments with standard mode and
sample alignment settings.
Ellipsometric Experimentation
All microscope slides were coated with tape on one side. Aliquots of 100 ng/mL ligand and
5μg/mL antibody were made.
12. 7
For a single component analysis, 10 μL of desired biological sample were added to the non-
coated face of the slide and incubated at 37°C. The slide was washed and shaken dry.
Measurements were taken at above parameters.
For a two component analysis, 10 μL of ligand were added to the non-coated face of the slide
and incubated at 37°C. The slide was washed and shaken dry. 10 μL of antibody were
subsequently added to the same portion of the slide and incubated for 2 hours at 37°C. The slide
was washed and shaken dry again. Measurements were taken at noted parameters.
Psi-Delta Data Analysis
Data from CompleteEASE software was compiled. Numerical data was extracted into Excel and
averages were taken for respective subsets. Psi and Delta values were plotted in Excel and
trends were analyzed visually. Data of each of the biological specimens was compared to that of
the baseline characterization slide data.
13. 8
RESULTS
Cell Culture
During the cell culturing process, we encountered some issues. The incubator was
malfunctioning resulting in the death of some of the cultures. Once repaired, however, cells
were able to be successfully grown and passaged. During passaging, it was determined that 10%
concentrated trypsin must be used suspend the cells. They bind to the culture vessel very well
and were unable to be removed with any lower concentrations.
Six aliquots were frozen down stored in the liquid nitrogen dewer for future use.
ELISA
The ELISA is a well-established method used to characterize binding. The double antibody
sandwich method was utilized to confirm the binding of the antibody to the ligand. A literature
protocol was slightly adapted for the procedure. The absorbance values were measured using an
reader and indicated that the optimal concentrations were somewhere between 100-200 ng/mL
for the ligand and about 10 μg/mL for the antibody.
Slide Preparation
Initial baseline characterization was taken of both plastic petri dishes and glass slides. Petri
dishes were deemed incompatible with the ellipsometer system rather quickly as the sides of the
dish are too tall resulting in unnecessary interactions with the incoming polarized light. The
slides as well, initially resulted in difficulties as the ellipsometer was unable to obtain an accurate
reading as the light was penetrating the glass completely.
14. 9
We researched several different methods to counteract this penetration: the use of either a
frosted slide, a roughened slide, or a coated slide. After further analysis, we were unsure how
the frosted slide would affect the system biologically so decided this method was not
appropriate. With testing both the mechanically roughened and coated slide, data was very
similar. Because mechanical roughing is difficult to control and create consistency, the coated
slides were chose for experimentation. 3M Scotch Matte Finish Magic Tape was used to coat
one side of the microscope slide. In theory, the tape material should be homogenous so each
slide should reflect similar data.
Ellipsometric Experimentation
Due to complications with the ellipsometer, data was unable to be collected for the cell line or
for any of the subsequent measurements involving cells.
For other samples, each set of measurements was taken with 3 replicates and 5 pseudo-replicates.
Data collection for the slides, slides with ligand, and slide with antibody were all quite simple.
Some difficulties arose when taking measurements of the slide and both biologics. This is was
corrected for by changing the orientation of the slide on the table of the ellipsometer.
Optimal concentrations of ligand for ellipsometry was found to be 100ng/mL whereas antibody
concentration was 5μg/mL.
15. 10
Psi-Delta Analysis
Baseline characterization of the slide presented the smoothest data. The psi value represented a
positive correlation with wavelength ranging in values from around 20 – 20.5 (See Figure 5).
The delta value peaked with a value of approximately 2 and then had a fairly steep and steady
decline to values around 0.3.
Values of the biological samples were much more variable compared to those of baseline. Each
of the subsequent measurements were evaluated compared to the baseline to track changes in
both psi and delta values.
Aggregate data of slide and ligand represents a steady upward trend in the psi portion. Values at
lower wavelength are at approximately 24.19 and reach maximum values of 24.55. At the upper
limits of wavelength, it can be seen that there is a slight drop off, however. Delta values are
primarily in the negative region climbing towards 0 at higher wavelengths (See Figure 6). Psi
values were substantially higher compared to that of the baseline, but delta values decreased.
Comparatively, aggregate data of the slide and antibody are very similar. Trends are consistent.
Psi values are marginally higher, only about 0.1 in most cases (See Figure 7). The differentials
from the baseline are on the same scale and the slide and ligand.
When ligand and antibody were both bound introduced, the both psi and delta values decreased
compared to the baseline. The data had a much more linear appearance compared to any of the
other figures (See Figure 8).
16. 11
DISCUSSION
Due to the complications associated with incubator, the cells were unable to be tested as part of
the model system. Implementation of this portion is critical for understanding and analyzing the
full biological system and relating significance. Thus it is necessary to complete further and
more in-depth research to categorize the efficiency of the system for this propose.
However, based upon obtained collected data and preliminary results, the spectroscopic
ellipsometer shows promise for system evaluation. Several criterion support this assertion.
Compared to other systems, ellipsometry utilizes both smaller samples sizes and is able to track
changes in lower concentrations. The most substantial benefit is associated with the acquisition
time. As mentioned, cell based screening assay may take up to several hours which results in a
decrease in cell viability. With ellipsometry, data acquisition is completed on a scale of seconds.
The quick data collection provides the Psi-Delta output which can be further analyzed.
Though psi and delta alone, do not provide much significance other than the potential for visual
inspection of trends, these parameters are useful in the creation of an optical model. Optical
constants and biological factors are implemented to devise a model that will explain changes in
the optical surface properties of the cellular system. Derivation of this model is crucial in
relating the numerical values to the biological significance. Thus explaining what is physically
occurring in the system. This process is necessary in the end goal of tracking efficiency of
ligand binding the GPCR.
17. 12
CONCLUSION
The small sample sizes and quick acquisition time associated with spectroscopic ellipsometry
presents benefits over other analytical techniques presently used in characterization of ligand
binding to receptors. Though this method shows promise, it must be further evaluated with the
implementation of a complete model system. Characterization of the system by utilizing the Psi-
Delta data to develop a model with optical constants and further relating that to biological
properties and significance must be completed in order to fully and accurately describe
interactions.
18. 13
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