Experimental Methods in Systems Biology Coursera Quiz Answers

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Week 01: Experimental Methods in Systems Biology Coursera Quiz Answers

Quiz 1

Q1. Choose all that apply. Which of the following are ways in which systems biology is often distinctive from traditional biological research? Note that these answers depict generalities rather than unique cases.

Systems biology focuses on how individual molecular components affect a single cellular or physiological function
Experiments in systems biology are often proposed based on insight from quantitative computational models
Systems biology-based hypotheses often involve quantitative and dynamic features of biological systems
Systems biology relies on formal statistical modeling to generate experimentally testable hypotheses
Systems biology concerns only experiments done on an “omics” scale
Systems biology is concerned with how interactions between sets of biological molecules give rise to cellular behaviors and phenotypes
Systems biology may seek to explain how tissue level observations arise from biomolecular phenomena in cells

Q2. Emergent and relevant properties of biomolecular networks can be difficult to predict without knowing __ of network interactions;

Magnitudes
Localizations
Dynamics
All of the above

Q3. Choose all that apply. I am a scientist interested in mechanisms of cancer metastasis, and specifically, how cell invasion through tissue (or dense tissue –like substances) is facilitated by common genetic aberrations. Experiments with which of the following organisms may prove useful for my research?

Drosophila melanogaster
Saccharomyces cerevisiae
Escherichia coli
Mus musculus
Cell lines

Q4. Choose all that apply. It is possible to control _ with light in cells

Cell protrusion
Enzymatic activity
Protein localization
Cell differentiation
Cell proliferation
Protein-protein interactions;

Q5. Which of the following is an advantage of current genome engineering methods over plasmid-based selection for stable expression of an exogenous protein?

Plasmid-based selection doesn’t allow visualization of the exogenous protein
Genome engineering allows precise control over the location of the exogenous DNA
Genome engineering has no off target integration
Plasmid-based selection precludes additional control of transcription rate or protein half-life

Q6. True or false. The Tet repressor blocks expression of genes of interest in tet-off systems

true
false

Q7. True or false. PCR enables exponential amplification of transcripts for accurate quantification

true
false

Q8. Choose all that apply. Which of the following are true regarding cDNA microarray technologies?

One must define beforehand what one is looking for in a microarray
They have limited ability to analyze alternative splicing
PCR steps increase the ability of the microarray to be quantitative
Binding of labeled oligos to spots follows saturable kinetics and therefore limits the dynamic range of the technology

Q9. Choose all that apply. High throughput sequencing can

Quantify protein-DNA interactions
Quantify the transcriptome
Identify SNPs
Probe epigenetic regulation via histone modifications
Probe epigenetic regulation via DNA methylation
Map protein-DNA binding sites;

Q10. True or false. Bisulfite sequencing allows identification of methylated adenine DNA residues by converting them to uracil

true
false

Q11. Choose all that apply Fluorescence in situ hybridization

Can quantify single cell gene expression
Can identify loss or gain of gene copy number
Is compatible with single cells
Can identify single nucleic acid molecules

Q12. Choose all that apply. Which are key features of Unique Molecular Identifiers to provide a potential way to quantify gene expression despite PCR steps?

The ability of each barcode to attach to each transcript is unbiased
The PCR steps are designed to amplify only the unique molecular identifiers
They label every transcript present in a sample
The number of unique barcodes is much greater than the number of transcripts in the sample

Q13. True or false. Western blotting is generally more quantitative than reverse phase protein arrays.

true
false

Q14. Choose all that apply. Western blotting

Can detect changes in phosphorylation over time
Is highly specific because it uses two different antibodies to detect the same target
Relies on separation of proteins in a mixture by their charge
Can be used to quantify protein levels;

Q15. True or false. Antibodies that work well for western blotting will work well for RPPA

true
false

Q16. Mass spectrometry for proteomics has two mass analyzers because:

They can be run in parallel to increase throughput which allows for greater coverage of the proteome
It allow analysis of larger and fragmented peptides to infer sequences
The first mass analyzer provides ionization so that the second mass analyzer can also analyze charge
Two analyzers in series multiply the ability for precise mass determination and thus better identification

Q17. True or false. Flow cytometry allows analysis of protein expression at the single cell level

true
false

Q18. True or false. Flow cytometry allows repeated time course measurements in a single cell

true
false

Q19. True or false. Flow cytometry has higher throughput than fluorescence microscopy, but fluorescence microscopy allows for denser time courses

true
false

Q20. True or false. Fluorescence microscopy can measure analytes in both live cells and fixed cells. It can measure both nucleic acids and proteins.

true
false;

Week 02: Experimental Methods in Systems Biology Coursera Quiz Answers

Quiz 2

Q1. Sanger sequencing is not amenable to massively parallel analysis because

The chain termination chemistry would cause sequence convolution between neighboring sequencing reactions
Massively parallel analysis requires very short reads, but Sanger sequencing only gives relatively long reads
Fluorescence-based observation of nucleotides is not sensitive enough to allow chain termination chemistry in small molecule numbers
The chain termination chemistry dictates that identification of each base pair requires separating sequencing reaction products by size

Q2. True or false. Cluster overlap is desired to maximize sequencing depth by deconvoluting spatially overlapping fluorescence emission.

true
false

Q3. Choose all that apply. One deep mRNA sequencing experiment allows

Inference of translational control by specific miRNA
Analysis of transcript levels across the entire transcriptome
Quantification of ratios of splice isoform abundance
Prediction of mRNA half life
Identification of novel splice isoforms
Observation of long non coding RNAs

Q4. True or false. It is important that the fragment size distribution be matched to read length, so that reads are longer than the average fragment to guarantee full readability and maximum information content.

true
false

Q5. True or false. RPKM and FPKM units are identical when single end read experiments are done.

true
false

Q6. True or false. Pacific Biosciences 3rd generation sequencing technology can detect base modifications such as methylation, but has a high error rate

true
false

Q7. True or false. The concentration of the library run over the flow cell is critical because too high of a concentration will cause difficulty in base calling.

true
false

Q8. True or false. Sequencing a DNA strand by sequential addition of nucleotides rather than by termination chemistry is the innovation that allowed 2nd generation sequencing technologies

true
false

Q9. True or false. The ideal method for mRNA seq data quantification is counting reads

true
false

Q10. True or false. Paired end sequencing experiments take approximately twice as long as single end sequencing experiments

true
false;

Week 03: Experimental Methods in Systems Biology Coursera Quiz Answers

Quiz 3

Q1. Why is it important that protein and peptide mixtures be subject to separation steps prior to tandem mass spec analysis?

During electrospray ionization, bound peptides can recombine forming unexpected fusion artifacts
Unseparated proteins tend to bind to each other causing aggregation artifacts in the mass analysis
The first mass analyzer can only select a subset of peptides for further sequencing analysis at a given moment in time

Q2. True or false. Time of flight is a mass analyzer that depends on the fact that heavier ions travel quicker than lighter ions when a voltage is applied

true
false

Q3. Choose all that apply. The iBAQ score;

Takes into account that proteins have different trypsin digest profiles and therefore may show up more or less frequently during mass spec analysis
Only needs a single identified peptide, but improves as more peptides are identified
Allows estimation of absolute protein levels
Relies on expected patterns endogenous isotope labels that occur with natural frequencies

Q4. True or false. It is important that any mass labeling method for quantification give peptides a unique chromatography elution profile

true
false

Q5. Choose all that apply. The Q Exactive instrument

Uses a liquid chromatography column prior to the electrospray source to further separate peptides with nanoliter flow rates
Requires separation of proteins by molecular weight prior to input
Is a tandem MALDI/Electrospray/Orbitrap device
Uses an electrospray ionization source as the input to the orbitrap
Couples a quadrupole mass analyzer with an orbitrap mass analyzer

Q6. Choose all that apply. An orbitrap

Detects different m/z by their different rates of linear oscillation radially
Uses Fourier transforms of dynamic radial oscillations to infer the spectral composition of the m/z mixtures
Works in a batch mode to analyze groups of ions simultaneously
Keeps injected ions in a helical orbit
Relies on alternating AC and DC currents to accelerate ions along its length after the oscillatory burn-in phase is complete

Q7. A mass spectrometer

Independently measures mass and charge of molecules
Fundamentally can filter by the mass to charge ratio of any molecule that is ionized
Requires both positive and negative charges on each molecule to be analyzed
Utilizes ionization of solvent to push molecules into the mass analyzer

Q8. True or false. Electrospray ionization is the interface between a liquid chromotography column and mass analyzer and it uses a high voltage coupled with solvent evaporation from sprayed droplets to send ionized peptides to the mass spec

true
false

Q9. True or false. A key feature of the iTRAQ reagents is that they have a mass balancing group to ensure they equally pass through the MS1 scan such that their unique mass tags are then observable in the MS2 scan after fragmentation

true
false

Q10. True or false. Collision-induced dissociation involves rapidly accelerating ions into a highly charged plate then reversing the voltage to induce peptide bond shearing

true
false;

Week 04: Experimental Methods in Systems Biology Coursera Quiz Answers

Quiz 1: Midterm Exam

Q1. Essay 1.

Dr. Bark Mirtwistle wants to explore interactions between two genetic alterations—loss of p53, and EGFR copy number amplification—in the context of glioblastoma multiforme. He is particularly interested in the proliferation and apoptosis response of cell lines to molecularly targeted therapies (e.g. EGFR inhibitors), and especially in the responses of single cells and how they can be variable across a cell population. Choose the sentence that best continues the essay

The assay of choice for this study will be live-cell imaging combined with mRNAseq
Immunocompromised mice with EGFR-amplified, p53 null human glioma cell xenografts injected into the brain will be the model system to begin with.
The first biological system will be S. cerevisiae, engineered to express human EGFR at high levels, to identify interacting proteins.
E. coli will be the biological system of interest, to highly express in large numbers of cells these two proteins and see if they interact;
The biological system he will start with is a glioma cell line that is positive for EGFR amplification but wild type for p53

Q2. Choose the sentence that best continues the essay

Top proteins interacting with EGFR and p53 will be stably expressed in a glioma cell lines
Using CRISPR genome engineering, he will tag the amplified EGFR genes with a DD domain to control their half-life and expressio
Using CRISPR genome engineering, he will take these EGFR amplified cells and create p53 knockouts
The xenografts will be subjected to blue light stimulation to induce overexpression of EGFR with light-induced transcriptional systems
The interaction between p53 and EGFR proteins will be engineered to be controllable by red light by tagging them with Phy and PIF

Q3. Choose the sentence that best continues the essay

He will subject the cells to treatment with a targeted EGFR inhibitor (erlotinib), a targeted p53 drug (Nutlin), or their combination (erlotinib + nutlin), a vehicle control, and a positive control for anti-proliferative effects (paclitaxel) and apoptosis (TRAIL) for 48 hours
Upon treatment of the cells with the appropriate wavelength of light, he will monitor proliferation and apoptosis responses with live-cell imaging to understand how p53 and EGFR expression or their interactions give rise to proliferation or death;
Mice will be subjected to treatment with a targeted EGFR inhibitor (erlotinib), a targeted p53 drug (Nutlin), or their combination (erlotinib + nutlin), a vehicle control, and a positive control for anti-proliferative effects (paclitaxel) and apoptosis (TRAIL) for 48 hours
The top interacting proteins with both EGFR and P53 are 14-3-3 and Actin. These proteins will be knocked down via siRNA or overexpressed with an exogenous plasmid and subjected to treatment or EGFR inhibitor (erlotinib), a targeted p53 drug (Nutlin), or their combination (erlotinib + nutlin), and a vehicle control.
Graded levels of Shield1 will be applied to the cells in the presence of a targeted EGFR inhibitor (erlotinib), a targeted p53 drug (Nutlin), or their combination (erlotinib + nutlin), a vehicle control, and a positive control for anti-proliferative effects (paclitaxel) and apoptosis (TRAIL) for 48 hours.

Q4. Choose the sentence that best continues the essay

He will then assay all the conditions with western blotting to understand what signaling pathway post translational modifications are affected by the treatment conditions as a function of EGFR and p53 status.
Live-cell probes for p53 and EGFR will be followed in terms of localization and abundance to understand how they respond to the given perturbations
Proliferation and apoptosis will be monitored over time on a single cell level with flow cytometry, to evaluate the heterogeneity of the population response dynamically in terms of the phenotype, and how that is related to the genetic status
To understand where to focus the single cell studies, he will first perform a mass-spectrometry based proteomics screen to look for the proteins that are differentially altered among the above conditions, as well as assess proliferation and apoptosis via flow cytometry assays;

Q5. Choose the sentence that best continues the essay

After the top hits are identified, two-photon imaging will be applied to observe single cell signaling, proliferation, and apoptosis responses in the xenografts as a function of p53 or EGFR genomic status and drug treatment
Single cell mRNA-seq on at least 100 cells per tumor will be employed from dissociated xenograft tumor cells to evaluate the extent of transcriptional variability as a function of P53 and EGFR status and drug treatment. Unique molecular identifiers will be used to ensure the resulting data are quantitative.
Once a sharper biochemical focus is defined, he will attempt to look at 2-3 selected activities in space and time using live-cell imaging in response to the same conditions as above, to evaluate cell-to-cell differences in relevant biochemical activities and correlation with proliferation or apoptosis
Xenograft tumors will be harvested at several time points post-treatment, then subjected to flow cytometry analysis to observe single cell responses of proliferation and apoptosis, western blotting to assay key signaling pathways identified in the prior studies, as well as qPCR for transcriptional responses of the same

Q6. Essay 2.

Dr. Blarbelly McHuffle, a postdoc in Prof. Major Bigshot’s lab, is performing mRNA sequencing experiments to understand how two compounds alone and their combination affect gene expression in cultured cardiomyocytes differentiated from human induced pluripotent stem cells.

Choose the sentence that best continues the essay;

The biological system, in addition to a single human induced pluripotent stem cell line, will include, C. elegans and D. melanogaster cardiomyocytes.
The biological system, in addition to a panel of human induced pluripotent stem cells from different people, will include, C. elegans and D. melanogaster cardiomyocytes.
The biological system will be a single human induced pluripotent stem cell line, to avoid variation associated with multiple genomic backgrounds.
The biological system will be a panel of human induced pluripotent stem cells from different people, because the response in a cell derived from a single individual may be problematic and dependent on genomic nuances only present in that individual.

Q7. Choose the sentence that best continues the essay

Cells will be subjected to treatment with the combination of the two compounds, as well as vehicle control.
Cells will be treated with a vehicle control, the first compound alone, the second compound alone, and the combination of compounds, in singlet.
Cells will be treated in triplicate with both compounds alone, as well as the combination.
Cells will be treated with a vehicle control, the first compound alone, the second compound alone, and the combination of compounds, in triplicate

Q8. Choose the sentence that best continues the essay;

After treatment, total RNA will be extracted from the cells, and the quality of the RNA in terms of length distribution and concentration will be verified prior to proceeding.
Isolated mRNA from the cell lystates obtained after treatment will be subjected to library preparation.
The next step will be to perform an RNA isolation, so that ribosomal RNA can be removed from the mRNA
After treatment, mRNA will be extracted from the cells, and subjected to adapter ligation

Q9. Choose the sentence that best continues the essay

If samples are of sufficient quality, library preparation will commence which includes conversion of mRNA to cDNA, ligation of adapters, and finally, fragmentation
mRNA isolates will be subjected to purification by size followed by digestion to generate fragments suitable for library preparation, where adapters are ligated and then limited PCR amplification is performed
mRNA will be isolated from total RNA, which is then ready for input to the single molecule real time sequencing machine after extensive sample cleanup
Libraries will be generated for those samples passing quality control, which includes mRNA enrichment, fragmentation of the mRNAs, first and second strand synthesis, ligation of adapters, and limited PCR amplification
To facilitate detection of eventual base pair incorporation during the mRNA sequencing reactions, the phosphodiester bonds in the RNA will be converted to sulfonated reducible forms. These chemically modified mRNAs are then subjected to library preparation which includes fragmentation, ligation of adapters, and PCR amplification;

Q10. Choose the sentence that best continues the essay

. Libraries will be added to a flow cell, amplified to generate clusters at as high a density as possible where clusters are overlapping (overlapping clusters can be separated computationally via advanced deconvolution processing), and then subjected to Illumina single end sequencing, since he is not primarily interested in novel splice variants.
After the mRNAs bind the immobilized DNA polymerases in the sequencing wells, sequencing can commence by monitoring fluctuations in fluorescence corresponding to binding/incorporation events of base pairs to the growing DNA strand.
. Libraries will be added to a flow cell, amplified to generate clusters at an appropriate density, and then subjected to Illumina paired end sequencing, since he is not primarily interested in novel splice variants.
Libraries will be added to a flow cell, amplified to generate clusters at an appropriate density, and then subjected to Illumina single end sequencing, since he is not primarily interested in novel splice variants
Libraries will be added to a flow cell, amplified to generate clusters at an appropriate density, and then subjected to Illumina paired end sequencing, since he is primarily interested in novel splice variants, rather than expression patterns

Q11. Choose the sentence that best continues the essay

Poisson count based statistics will be used to identify differences between the conditions in terms of differentially expressed genes measured by read count aligning to each gene. This will give rise to lists of differential expression between the treatment conditions and give insight into the compound effects on cardiomyocytes
By comparing how many sequencing errors and mutations occur in a compound-specific manner from the resulting sequence data, we can gain insight into the compounds’ effect on cardiomyocytes. We must be careful to align to the same reference genome in each case of model organism.;
The resulting sequencing data will be analyzed for differential expression amongst the treatment conditions via the Tophat/Cufflinks/Cuffdiff pipeline based on the FPKM metric. This will give rise to lists of differential expression between the treatment conditions and give insight into the compound effects on cardiomyocytes
The resulting sequencing data will be analyzed for differential expression amongst the treatment conditions via counts of reads aligning to transcripts and genes. The counts control are subtracted from counts at treatment as a quantitative measure of differential expression. This is valid because the same sequencing depth will be used for each sample.

Q12. Essay 3. Lostly Q. Givenup, a PhD student in Prof. Dontcare’s lab, is trying to do proteomics while his advisor is away traveling months on end. Lostly’s project is focused on finding interactions with a novel protein called Exciting, a globular small molecular weight protein, which has been suggested to play a role in Debilitating Disease through genome wide association studies. Choose the sentence that best continues the essay.

Lostly should quit graduate school because his name is horrible, and his mentor does not care.
By finding interaction partners, it is hoped that these will map onto some prior knowledge pathways to give insight into potential mechanisms of Exciting in Debilitating Disease. These potential mechanisms would give leads on further studies and potential therapeutics.
Protein-protein interactions are often indicative of how proteins are mediating their effects. Proteomics methods such as these can identify such interactions by simultaneous observation of peptides from both proteins in the mass spec at the same time, indicated by fused sequences.
Proteins usually interact with DNA to exert their biological effects, and such proteomics methods should in principal be able to identify unique interactions of Exciting that are of relevance to Debilitating Disease

Q13. Choose the sentence that best continues the essay

By looking at protein abundance in total cell lysates between the two conditions, this will indicate which proteins are more likely to interact with Exciting
The first step will be to express Exciting in E. coli and purify it, so that it can be used as bait to pull out interacting proteins from cell lysates
Protein-protein interactions will be indicated by first cross-linking all proteins with formaldehyde, then purifying Exciting via immunoprecipitation to bring with it all those that were bound to it and therefore cross-linked.
Unfortunately Exciting is a new protein and doesn’t have any available antibodies. Therefore Lostly will find interactions by isolating Exciting bound to DNA rather than to antibodies

Q14. Choose the sentence that best continues the essay

Proteins interacting with Exciting will be pulled out from cell lysates from a cell model of Debilitating Disease , as well as those from S. cerevisae as a negative control. A positive control will be mammalian cells over-expressing Exciting
Proteins interacting with Exciting will be pulled out from cell lysates from a cell model of Deblitating Disease, as well as cells that are not associated with Debilitating Disease. It is hoped that this will reveal Debilitating Disease specific as well as general roles for Exciting. A negative control will be the pull down using the protein purification tag of Exciting but without Exciting itself.
Mice with Exciting knocked-out will have their embryonic fibroblasts purified (Exciting knock-out is embryonic lethal), and the proteins from these cell lysates will be compared to proteins from Exciting-containing mouse embryonic fibroblasts.
Using an antibody against Exciting, Exciting will be immunoprecipitated in a weak buffer that retains most protein-protein interactions, so that proteins interacting with Exciting will be immunoprecipitated as well.

Q15. Choose the sentence that best continues the essay

Liquid chromatography with a strong cation exchange resin bound to Exciting will help to resolve peptides that are interacting with Exciting versus those that do not, as well as create simpler fractions coming off the column allowing deeper mass spec analysis
Protein mixtures will be subjected to proteolytic digestion, so that peptides not interacting with Exciting can be purified by liquid chromatography and SDS-PAGE and the mixtures simplified by fractionating.
The Exciting interacting protein pull down will be subjected to fractionation by molecular weight via SDS-PAGE
Native PAGE will be run on the Exciting interacting protein pull downs so that complexes running at higher molecular weight can be further purified to increase the likelihood that true interactions are being enriched. The resulting gel will be cut into slices to further simplify the protein mixtures

Q16. Choose the sentence that best continues the essay

Prof. Bigshot tells Prof. Dontcare he should really be checking on Lostly. Prof. Dontcare realizes the experiment is totally messed up, and sends an email from Aruba to assign Lostly to making buffers for 2 months for the rest of the super postdocs in Prof. Bigshot’s lab.
The fractionated proteins will be proteolytically digested, tagged using iTRAQ reagents, the various samples (from equivalent fractions) mixed in equimolar ratios, and then purified for injection into the LC/MS/MS
The fractionated proteins will be proteolytically digested, mixed with super SILAC standards in equimolar amounts, then purified for injection into the LC/MS/MS.
The fractionated proteins will be proteolytically digested, then subjected to peptide mass fingerprinting through MALDI-TOF to identify which proteins are in each fraction. This will be followed by LC/MS/MS to focus the analysis on only those identified proteins.

Q17. Choose the sentence that best continues the essay

. Each fraction will be input into the LC/MS/MS, and the resulting data combined to analyze differential expression amongst the conditions via comparison of the peak intensities of the shifted isotope masses from SILAC standards in the different samples
Each fraction will be input into the LC/MS/MS, and the resulting data combined to analyze differential expression amongst the conditions via comparison of the different spectral counts of the number of peptides that align to each protein
Each fraction will be input into the LC/MS/MS, and the resulting data combined to analyze differential expression amongst the conditions via comparison of the different iTRAQ peak quantities
Each fraction will be input into the LC/MS/MS, and the resulting data combined to analyze differential expression amongst the conditions via comparison of the different iBAQ scores from the different samples, normalized by the SILAC standards;

Week 05: Experimental Methods in Systems Biology Coursera Quiz Answers

Quiz 4

Q1. Flow cytometry differs from FACS because in FACS cells are sorted prior to analysis.

true
false

Q2. Choose all that apply. Essential components of a flow cytometer include:

Laser
Camera
LED
Flow cell
autosampler
Fluorescence filter

Q3. Choose all that apply. Light scattering properties of cells

Include forward scattering, which is indicative of cell granularity
Give information for gating cells vs. debris
Can be used to distinguish multiple cell types in blood without the need for fluorescence
Include hyper angle scattering, which compositely informs sorting voltages as well as cell morphology
Include forward scattering, which is indicative of cell size

Q4. Two fluorophores that exhibit emission spectra overlap cannot be co-analyzed in a flow cytometer

true
false

Q5. Choose all that apply.The flow cell

Can accommodate a range of flow rates
Is the best nightclub around
Uses hydrodynamic focusing to align the cell sample flow within sheath fluid
Uses integrated rectangular grids to facilitate the elimination of cell doublets or higher order clumps
Performs best at flow rates toward the higher end of the range

Q6. Choose all that apply. Fluorescence filters on the flow cytometer

Allow multicolor imaging even with a single laser
Work with dichroic mirrors that define the light path
Sit in front of a detector to ensure that only a defined range of light wavelengths enters that particular detector
Predominantly consist of bandwidth filters, which not only ensure laser excitation gets removed from the signal, but also reduce fluorescence emission intensity to within the working range of the photomultiplier tube
Consist of both longpass and bandwidth filters

Q7. Choose all that apply. Little researcher Johnny wants to do cell cycle analysis and also look for active DNA replication. The following would be advisable and/or recommended:

Select spectrally overlapping dyes for DNA and anti-BrdU so that only a single laser is needed
Collect events at a slow flow rate
Ensure that fluorescence is collected on a log scale to maximize dynamic range and sensitivity
Gate events using the DNA stain channel area and width measurements

Q8. Flow cytometry is capable of quantifying markers both on the exterior surface and within the interior of the cell simultaneously

true
false

Q9. Choose all that apply. Events on the flow cytometer

Correspond to cells
Consist of a time dependent signal
Can be characterized by a height, area and width

Q10. Accurate quantitation of DNA levels requires collection of the DNA stain fluorescence on a log scale to maximize dynamic range

false
true

Q11. One can discriminate which events are debris from which events are cells by gating on those that have both large FSC and SSC

false
true

Q12. Choose all that apply. Doublet discrimination

Can be achieved by gating on a fluorescent signal available in all cells, or by gating by side scatter (
Is particularly important in cell cycle analysis to determine whether an event is a 4N cell or two 2N cells
Involves creating a gate that distinguishes single cell events from events that are likely to be two cells together.
Allows FRET analysis by resolving interacting fluorophores into their singlet and doublet states
Becomes more important as flow rate is decreased
Consists of gating event area vs. width, with the rationale that singlets will exhibit large width with respect to the same area

Q13. Choose all that apply. Fluorescence barcoding

Is limited to a maximum of 27 samples
Allows mixing of different samples to facilitate data acquisition
Typically improves data quality because all samples are being stained and run simultaneously
Uses multiple dyes to uniquely label different populations of cells that could, for example, have been treated in different ways

Q14. Choose all that apply. Mass cytometry

Is capable of detecting abundances of heavy atoms (e.g. metals) on the single cell level.
Couples flow cytometry with mass spectrometry
Requires cell destruction for analysis due to the ionization method
Facilitates complex and advanced cell sorting strategies based on its ability to make highly multiplexed measurements
Can multiplex on the order of 32 parameters simultaneously in single cells;

Q15. A mass cytometer cannot filter ions arising from heavy metal tags from the ions arising from the normal cellular biological molecules (e.g. metabolites, proteins, lipids, etc.), so therefore relies on time of flight detection to look for heavy metals.

false
true

Q16. Cell cycle analysis is a straightforward technique applicable to both flow and mass cytometry

false
true

Q17. Because a mass cytometer is essentially a flow cytometer coupled to a mass spectrometer, the flow cytometer piece can gate cells from debris and then input single cell material to the mass spectrometer, while the mass spectrometer enables highly multiplexed analysis of these single cells

true
false

Q18. Choose all that apply. Mass cytometry may be a better choice than flow cytometry when

When quantifying lowly expressed targets, since the mass spec is highly sensitive as compared to fluorescence detection
One is interested in many parameters in single cells, but there is only limited sample available
When signal to noise is a critical factor
When one has many samples to run, because the mass cytometer can measure so many things per sample

Q19. Compensation is the process by which the fluorescence emission intensity due to one fluorophore, as measured by one channel, is subtracted out of the detected intensity in a second channel, so that the signal in a second channel can be attributed to the presence of a second fluorophore’s emission intensity

false
true

Q20. Choose all that apply. Obtaining good quality quantitative data in flow cytometry consists of

Carefully titrating antibodies to ensure that signal fluctuations are not due to staining variability
Operating at the lowest flow rate possible (true)
Validating antibodies to ensure minimal non-specific binding and low signal to noise
Using log scale detection to maximize signal to noise
Including compensation controls in each experiment

Week 06: Experimental Methods in Systems Biology Coursera Quiz Answers

Quiz 5

Q1. The function of a dichroic mirror is to both reflect excitation light towards the specimen and to deflect residual excitation light away from the detector

false
true

Q2. Choose all that apply. In fluorescence microscopy, typically

Residual excitation light reflected from the specimen reaches the dichroic mirror and subsequently gets recycled
The objective is the unit that provides magnification of the image
Detectors can be one of many things that sense light, from an eye to a camera to a photomultiplier tube
The dichroic mirror reflects excitation light to the specimen but only lets fluorescence emission light back through
The excitation filter is the first step of selecting wavelengths of light to send to the specimen

Q3. The difference between bandpass filters and longpass filters is in that the former (bandpass) first filters the shorter wavelengths of light, then allows longer wavelengths through, whereas the latter (longpass) first filters the longer wavelengths of light, then allows the the shorter wavelengths through.

true
false

Q4. Choose all that apply. Fluorescence microscopy excitation sources can be

LEDs, which usually have a narrowly defined wavelength, but one that is broader than lasers
Xenon or mercury lamps, which emit a broad range of wavelengths but typically must be replaced more often than LEDs
Electron beams, which emit a very narrow range of energy that allows visualization beyond the diffraction limit of visible light
Lasers, which have usually have a very narrowly defined wavelength

Q5. Choose all that apply. Live-cell imaging very often requires

Maintaining a proper atmosphere, which can include humidity, CO2, and O2 levels
Stable temperature control
Using high excitation power to reduce camera acquisition time
Maintaining a proper atmosphere, which usually dictates proper media conditions such as osmolarity and pH

Q6. Total internal reflection fluorescence (TIRF) uses excitation light at a critical angle which results in illumination of a very thin section at the bottom of the specimen

true
false

Q7. Choose all that apply. Laser scanning confocal microscopy

Can reconstruct 3D images of a specimen by sequentially acquiring images at different focal planes in a so-called z-stack
Is typically not recommended for live cell imaging due to the large loss of emission light in the sectioning process which requires higher excitation power
Usually provides a better z direction resolution than a spinning disc confocal, but at the cost of slower acquisition
Requires rastering the excitation in both x and y directions to image an entire plane
Requires two laser beams to define the current rastering point

Q8. Choose all that apply. Widefield fluorescence

Is often preferred in live-cell imaging because it allows for collection of a maximum amount of emission light, as compared to confocal techniques
Consists of very wide confocal sectioning
Collects light from in-focus and out-of-focus planes
Is often suitable for cell monolayers which rarely exceed 20 microns Correct option 3

Q9. Choose all that apply. Spinning disc confocal microscopy

Uses a rotating disc to pass the specimen across the excitation light in a defined pattern for rapid acquisition of confocal images
Is typically quicker than laser scanning confocals because it collects light from many points in parallel, as opposed to one point at a time.
For live-cell imaging is optimized by including a layer of microlenses that focus excitation light through the pinholes in the spinning disc
Is preferred for live-cell imaging over laser scanning confocals because it typically requires lower excitation power and can acquire confocal sections faster, albeit at the cost of reduced axial resolution

Q10. Transmitted light microscopy can be combined with fluorescence microscopy to reveal potentially important morphological features of cells along with behavior of fluorescence probes, but often results is loss of light which hinders fluorescence image acquisition

true
false

Q11. Small molecule dyes that bind DNA are used for live-cell imaging because they have a high probability of binding strongly to any cell as well as having high signal due to the large amount of DNA in most cells.

false
true

Q12. Choose all that apply. Small molecule dyes

Can “light-up” the nucleus, mitochondria, endoplasmic reticulum, golgi apparatus, plasma membrane, and other cellular compartments with reasonable specificity.
Easily enter all cells
Can inform cell generation tracking by monitoring their intensity over long term time lapse, based on the principal of symmetric parsing of the dye at each cell division.
Provide straightforward means of labeling specific cellular components
Often have fluorescent protein equivalents in terms of spectral excitation/emission properties as well as cellular localization.

Q13. Biosensor design is almost unlimited, they typically work based on one of two fundamental principles: changes in fluorescence localization or energy transfer between fluorophores.

false
true

Q14. Choose all that apply. Fluorescence resonance energy transfer

Can occur when the excitation spectra of the donor overlaps with the emission spectra of the acceptor
Functions by the donor fluorescence emission causing excitation of the acceptor before the donor fluorescence emission is captured by the camera (or detector)
Typically requires two fluorescent proteins to be in physical proximity, since usual Forster radii are in the range of nanometers
Like many aspects of microscopy, has a four-letter word acronym (in this case FRET)
Is highly localization dependent

Q15. Choose all that apply. FRET quantification

In the case of intramolecular FRET, can be accomplished with only 2 channels, although one should still have positive and negative controls, as well as conditions where the two fluorophores of interest are imaged singly.
Is markedly simpler for intramolecular FRET, when the fluorophore stoichiometry is fixed and known, as opposed to intermolecular FRET, when the fluorophore stoichiometry is not known a priori.
By ratiometry can result in non-linear relationships between the measured ratio and fraction of probes undergoing intramolecular FRET, depending on the form of the ratio used
Typically requires acquisition of 4 fluorescence channels, combinatorially iterating among donor excitation, acceptor excitation, donor emission, and acceptor emission.
Can be easily converted to absolute concentration measurements, based on a calibration curve

Q16. Choose all that apply. Fluorescent probes currently exist that permit measurement of which of the following in live cells?

Small G-protein activities
Kinase activities
Levels of particular metabolites
Protein trafficking;

Week 07: Experimental Methods in Systems Biology Coursera Quiz Answers

Quiz 6

Q1. Choose all that apply. Volcano plots

Can facilitate determining the single genes that change the most, but not necessarily the ones with highest statistical significance.
Plot fold change vs. p-value (statistical significance) to clearly depict those single genes which have large and statistically significant changes.
Allow for simple visualization of the “most important” high fold change, high statistical significance single gene results of an omics-scale experiment.
Combine prior knowledge with single gene results to get a more comprehensive view of cellular changes.

Q2. Heatmaps to visualize omic-scale data sets are enhanced by combination with clustering algorithms that allow simultaneous observation of how genes or conditions group together on a dendrogram.

true
false

Q3. Single gene conclusions from omic experiments based differential expression alone are often very limited because they do not look at the data in the context of prior knowledge.

false
true

Q4. Choose all that apply. Gene set enrichment analysis

Is limited because determination of statistical significance of the overlap is unclear .
Considers enrichment of your list of genes with previously defined gene sets by (most often) requiring a gene ranking system and looking at overlap between your list and previous gene sets, in order of the ranking .
Compares how a list of differentially expressed genes obtained from your experiment overlaps with a host of previously known gene sets defined by prior knowledge .
Compares what is happening to multiple genes simulataneously as opposed to considering results “gene-at-a-time” .

Q5. Gene ontology is a classification system for placing genes into a hierarchical context that is applicable to most cells and organisms.

false
true

Q6. Choose all that apply. Inference of transcription factors from omics data

Provides hypotheses as to what transcriptional regulatory events might have contributed to the observed coordinate changes in gene expression, but requires further validation experiments for conformation.
Considers relationships between proteins and mRNAs to be regulated primarily by micro RNAs when correlation is not observed .
Relies on a wealth of prior knowledge that has defined specificity of transcription factor/promoter interactions.
Is based on the assumption that many coordinate changes in gene expression might be described by changes in activity of a smaller group of transcription factors.

Q7. Choose all that apply. Protein-protein interaction analysis

Uses prior knowledge of protein-protein interactions to connect a list of genes to each other and gene products not on the list.
Biases in protein-protein interaction analysis include those that are well-studied tend to have more interactions (rich get richer) and experimental techniques tend to favor stable, long-lasting interactions as opposed to transient ones.
The links between previously known protein-protein interactions and those in your gene list may be described by a network model.;

Q8. It is possible to analyze lists of genes and generate plausible hypotheses that are consistent with a wide range of prior knowledge about kinase activities that play a role in measured expression changes.

false
true

Q9. Choose all that apply. While network models generated from prior knowledge and lists of differentially expressed genes can give rise to novel hypotheses , it is difficult to use such models to

Analyze the temporal behavior of the biological network .
Assess the role of protein translocation between cellular compartments in regulating responses.
Predict responses to new perturbations.
Quantify the contributions of various network entities in the role of the considered biological process.

Q10. HIPK2 was identified as an important regulator of HIV-associated nephropathy by combining omic-scale transcript measurements with a wealth of prior knowledge.

false
true

Q11. Choose all that apply. When analyzing dynamic responses of cells to stimuli, single cell analysis can be quite important because

Cellular heterogeneity diversifies cell responses, which can mask the true system behavior.
Tracking the same cell over time by flow cytometry can reveal oscillatory behavior.
Many cellular systems are non-linear in terms of input-output behavior, so that heterogeneous cells in a population may be in completely different states when assayed in response to the same stimulus.
When only a population average is observed, fully synchronized dynamic responses cannot be inferred .

Q12. Bistability in a biochemical system can cause different cells of a clonal population to be in completely different states.

true
false

Q13. P53 responds to DNA damage with damped oscillations that dictate whether a cell lives or dies.

true
false

Q14. The magnitude of ERK activation following EGF treatment dictates whether a cell will enter S-phase or not .

true
false;

Q15. Ultrasensitivity is defined as a sigmoid with a hill coefficient less than 1, which makes the response extremely steep with respect to increasing input magnitude.

true
false

Q16. Bimodality of cell population responses implies bistability or ultrasensitivity of the underlying biochemical mechanisms .

true
false

Q17. Choose all that apply. Protein expression variability

Can dictate phenotypic variability and cell fate .
Arises mainly due to stochastic transitions of genes between active and inactive states corresponding to transcriptional competence.
Is affected by mRNA and protein degradation.

Q18. Model simulations can suggest interesting hypotheses but can not conclusively prove something is true. They can however rule hypotheses out as not true given the assumed model.

true
false;

Week 08: Experimental Methods in Systems Biology Coursera Quiz Answers

Final Exam

Q1. Dr. Bark Mirtwistle is back and wants to explore interactions between two genetic alterations—loss of PTEN and EGFR copy number amplification—in the context of glioblastoma multiforme drug responses. He is interested in the proliferation and apoptosis response of cell lines to molecularly targeted therapies (e.g. EGFR inhibitors), and especially in the responses of single cells and how their variation across a cell population can contribute to drug resistance.

The biological system he will start with are 2 strains of immunocompromised mice, one with inducible, brain glial cell-specific EGFR amplification, and the other with inducible, brain glial cell-specific PTEN ablation. Upon induction brain tumors form.
The biological systems he will start with are primary neurons as normal controls, EGFR amplified glioma cells, PTEN null glioma cells, and mouse embryonic fibroblasts that are EGFR amplified and PTEN null. These cell lines will be seeded into cell culture dishes for further analysis.
The biological system he will start with are a panel of four glioma cell lines that are +/- EGFR copy number amplification and +/- loss of PTEN. These four cell lines will be implanted intracranially into the brains of immunocompromised mice.
The biological system he will start with are a panel of four glioma cell lines that are +/- EGFR copy number amplification and +/- loss of PTEN. These four cell lines will be seeded into cell culture dishes for further analysis.

Q2. Choose the answer that best continues the essay.

Mice will be treated with the standard of care regimen, which includes radiation and chemotherapy with temozolomide.
Two drugs that are associated with these mutations are an EGFR inhibitor gefitinib and a PI-3K inhibitor BKM120 (PI-3K is the enzyme that opposes PTEN). Seeded cells will be treated with control, gefitinib alone, BKM120 alone, or the combination.
Two drugs that are associated with these mutations are an EGFR inhibitor gefitinib and a PI-3K inhibitor BKM120 (PI-3K is the enzyme that opposes PTEN). Mice will be given daily regimens of vehicle control, gefitinib alone, BKM120 alone, or the combination, and tumor growth monitored over a 3 month time course for at least 3 mice per condition and time point.
The cells will be treated with temozolomide, a standard of care for glioblastoma, as well as radiation.

Q3. Choose the answer that best continues the essay.

At weekly time points following the beginning of drug treatment, as approved by the Coursera thought experiment animal safety committee for ensuring the protection of most life forms that have backbones and maybe some of those that do not (CTEASCEPMLFHBMSTDN), animals will be sacrificed and tumors surgically removed and dissociated into single cell suspensions. Half of these single cell suspensions will be stained with metal conjugated antibodies against epitopes specific for glioma cells vs. non-tumor cells (e.g. endothelial cells) and then sorted to enrich for glioma cells with mass cytometry. The glioma cells will be seeded in culture dishes and allowed to expand for further experiments. The other half of the sample will be fixed and permeabilized for subsequent analysis.
At weekly time points following the beginning of drug treatment, as approved by the Coursera thought experiment animal safety committee for ensuring the protection of most life forms that have backbones and maybe some of those that do not (CTEASCEPMLFHBMSTDN), animals will be sacrificed and tumors surgically removed. The levels of proliferation and apoptosis in the tumors will be assayed by western blot against relevant protein markers of these phenotypes, which will inform the efficacy of the various treatments on the different mutation backgrounds.
At weekly time points following the beginning of drug treatment, as approved by the Coursera thought experiment animal safety committee for ensuring the protection of most life forms that have backbones and maybe some of those that do not (CTEASCEPMLFHBMSTDN), animals will be sacrificed and tumors surgically removed and dissociated into single cell suspensions. Half of these single cell suspensions will be stained with fluorescent antibodies against epitopes specific for glioma cells vs. non-tumor cells (e.g. endothelial cells) and then subjected to FACS to enrich for glioma cells. The glioma cells will be seeded in culture dishes and allowed to expand for further experiments. The other half of the sample will be fixed and permeabilized for subsequent analysis.
The treated cells will be assayed for apoptosis via flow cytometry with annexin-V-FITC staining at several time points following treatment (up to 72 hours), to understand the fraction of cells responding to the various treatments given the different mutation backgrounds. The apoptotic cells will further be sorted via FACS and seeded in culture dishes and allowed to expand for further experiments.

Q4. Choose the answer that best continues the essay.

Once the seeded glioma cells have expanded to sufficient numbers, they will be treated with vehicle control, gefitinib alone, BKM120 alone, or the combination for 48 hours. Each sample of cells will be lifted from the culture dish and prepared into two equal size single cell suspensions for analysis of apoptosis and proliferation using flow cytometry. Proliferation will be assessed by a dual staining with propidium iodide and anti-BrdU FITC antibodies (a BrdU pulse will be applied prior to lifting the cells), with propidium iodide (PI) fluorescence collected on a linear scale and anti-BrdU fluorescence on a log scale. Apoptosis will be assessed by staining with Annexin-V-FITC and PI, without permeablization, with fluorescence of both collected on a linear scale. In each case, excitation with a 488 nm laser will be applied, cells will be gated by PI-positivity, doublets will be gated out by Annexin-V-FITC area vs. Annexin-V-FITC width, and then proliferative and apoptotic responses quantified via fraction of singlet cells in different gates as follows. Those cells that are BrdU positive will be considered S-phase, those with high DNA content as G2/M, and those with low DNA content as G0/G1. Those cells that are Annexin-V positive will be considered apoptotic, and those that are Annexin-V positive and PI positive as dead.
Once the seeded glioma cells have expanded to sufficient numbers, they will be treated with vehicle control, gefitinib alone, BKM120 alone, or the combination for 48 hours. Each sample of cells will be lifted from the culture dish and prepared into two equal size single cell suspensions for analysis of apoptosis and proliferation using flow cytometry. Proliferation will be assessed by a dual staining with propidium iodide and anti-BrdU FITC antibodies (a BrdU pulse will be applied prior to lifting the cells), with propidium iodide (PI) fluorescence collected on a linear scale and anti-BrdU fluorescence on a log scale. Apoptosis will be assessed by staining with Annexin-V-FITC and PI, without permeablization, with fluorescence of both collected on a log scale. In each case, excitation with a 488 nm laser will be applied, cells will be gated by FSC vs. SSC, doublets will be gated out by SSC area vs. SSC width, and then proliferative and apoptotic responses quantified via fraction of singlet cells in different gates as follows. Those cells that are BrdU positive will be considered S-phase, those with high DNA content as G2/M, and those with low DNA content as G0/G1. Those cells that are Annexin-V positive will be considered apoptotic, and those that are Annexin-V positive and PI positive as dead.
Once the seeded glioma cells have expanded to sufficient numbers, they will be treated with vehicle control, gefitinib alone, BKM120 alone, or the combination for 48 hours. Each sample of cells will be lifted from the culture dish and prepared single cell suspensions for analysis of apoptosis and proliferation using flow cytometry. Proliferation will be assessed by a dual staining with propidium iodide and anti-BrdU FITC antibodies (a BrdU pulse will be applied prior to lifting the cells), with propidium iodide (PI) fluorescence collected on a linear scale and anti-BrdU fluorescence on a log scale. Apoptosis will be assessed by staining with Annexin-V-FITC and PI, with fluorescence of both collected on a log scale. In each case, excitation with a 488 nm laser will be applied, cells will be gated by FSC vs. SSC, doublets will be gated out by SSC area vs. SSC width, and then proliferative and apoptotic responses quantified via fraction of singlet cells in different gates as follows. Those cells that are BrdU positive will be considered S-phase, those with high DNA content as G2/M, and those with low DNA content as G0/G1. Those cells that are Annexin-V positive will be considered apoptotic, and those that are Annexin-V positive and PI positive as dead.
Once the seeded glioma cells have expanded to sufficient numbers, they will be treated with vehicle control, gefitinib alone, BKM120 alone, or the combination for 48 hours. Each sample of cells will be lifted from the culture dish and prepared into two equal size single cell suspensions for analysis of apoptosis and proliferation using flow cytometry. Proliferation will be assessed by a dual staining with propidium iodide and anti-BrdU FITC antibodies (a BrdU pulse will be applied prior to lifting the cells), with propidium iodide (PI) fluorescence both collected on a log scale. Apoptosis will be assessed by staining with Annexin-V-FITC and PI, without permeablization, with fluorescence of both collected on a linear scale. In each case, excitation with a 633 nm laser will be applied, cells will be gated by FSC vs. SSC, doublets will be gated out by SSC area vs. SSC width, and then proliferative and apoptotic responses quantified via fraction of singlet cells in different gates as follows. Those cells that are BrdU positive and Annexin-V positive will be considered S-phase, those with high DNA content as G2/M, and those with low DNA content as G0/G1. Those cells that are Annexin-V positive and BrdU positive will be considered apoptotic, and those that are Annexin-V positive and PI negative as dead.;

Q5. Choose the answer that best continues the esssay.

Mass cytometry will be applied to deeply measure pharmacodynamic responses across the treated cells that have been lifted into a single cell suspension (primary target of drugs—phospho-EGFR for gefitinib and phospho-Akt for BKM120), simultaneously with cell-to-cell variability in the expression of EGFR, PTEN and PI-3K, as well as delineate cell type by accepted lineage markers for glioma stem cells. A panel of 15 such antibodies will be incubated with the cells, each labeled with a different heavy metal element. Additionally, cells will be labeled with a metal-conjugated DNA intercalator to enable discrimination between cells and debris.
Live-cell imaging will be applied to deeply measure pharmacodynamic responses across the harvested cells from the tumor (pathway target of drugs—phospho-EGFR for gefitinib and phospho-Akt for BKM120), simultaneously with cell-to-cell variability in the expression of EGFR, PTEN and PI-3K, as well as delineate cell type by accepted lineage markers for glioma stem cells. A panel of 15 such antibodies will be incubated with the cells, each labeled with a different heavy metal element. Additionally, cells will be labeled with a metal-conjugated DNA intercalator to enable discrimination between cells and debris.
Mass cytometry will be applied to deeply measure pharmacodynamic responses across the fixed and permeabilized cells from the tumor (pathway target of drugs—phospho-EGFR for gefitinib and phospho-Akt for BKM120), simultaneously with cell-to-cell variability in the expression of EGFR, PTEN and PI-3K, as well as delineate cell type by accepted lineage markers for glioma stem cells. A panel of 15 such antibodies will be incubated with the cells, each labeled with a different heavy metal element. Additionally, cells will be labeled with a metal-conjugated DNA intercalator to enable discrimination between cells and debris.
Mass cytometry will be applied to deeply measure pharmacodynamic responses across the fixed and permeabilized cells from the tumor (pathway target of drugs—phospho-EGFR for gefitinib and phospho-Akt for BKM120), simultaneously with cell-to-cell variability in the expression of EGFR, PTEN and PI-3K, as well as delineate cell type by accepted lineage markers for glioma stem cells. A panel of 150 such antibodies will be incubated with the cells, each labeled with a different heavy metal element.

Q6. A recently published study demonstrated that in a brain tumor cell line with EGFR amplification and PTEN loss, the responsiveness of particular cells within the population to dual treatment with gefitinib and BKM120 was dictated by whether the ERK signaling pathway was activated or inhibited within 30 min. of drug treatment. This ERK activation happens in about 50% of the cells. However, the factors leading to this ERK pathway activation or inhibition are unclear. We are interested in understanding the signaling mechanisms that may be operating in different cells of the population to activate or inhibit ERK signaling in this way.

The biological system of interest will be the above noted cell line, seeded on a substrate suitable for live-cell imaging. Cells will be engineered to express a live-cell intramolecular CFP-YFP FRET-based reporter of ERK activity.
The biological system of interest will be a mouse brain tumor model with EGFR amplification and PTEN loss. The cells will be engineered to express a live-cell intramolecular CFP-YFP FRET-based reporter of ERK activity.
The biological system of interest will be the above noted cell line, implanted intracranially into mice. Prior to implantation, cells will be engineered to express a live-cell intramolecular CFP-YFP FRET-based reporter of ERK activity.
The biological system of interest will be D. melanogaster with EGFR amplification and PTEN loss. Cells will be engineered to express a live-cell intramolecular CFP-YFP FRET-based reporter of ERK activity.

Q7. Choose the answer that best continues the essay.

Dual treatment with gefitinib and BKM120 for 30 minutes will be done, and then cells will be sorted based on CFP fluorescence (those that have ERK turned on) and subjected to mRNA sequencing. The top differentially expressed genes are K1, K2 and K3 kinases, which are selected for further analysis.
Dual treatment with gefitinib and BKM120 for 24 hours will be done, and responses measured with mRNA sequencing. The top differentially expressed genes will be candidates for subsequent experiments, which turn out to be the kinases K1, K2 and K3.
To develop hypotheses for what proteins may be involved with such crosstalk, lists of genes associated with gefitinib, BKM120, and the ERK signaling pathways (all available in public databases) will be connected via previously known protein-protein interactions followed by kinase enrichment analysis using bioinformatic software tools. The top three kinase hits that have selective inhibitors will be selected for testing in subsequent experiments. These kinase inhibitors are K1, K2 and K3.
Dual treatment with gefitinib and BKM120 for 24 hours will be done, and responses measured with mass spectrometry-based proteomics after enrichment for phosphopeptides. The top differentially expressed proteins will be candidates for subsequent experiments, which turn out to be the kinases K1, K2 and K3.

Q8. Choose the answer that best continues the essay.

Cells will be seeded and placed in the microscope environment with proper temperature, atmosphere and pH control, and several fields of view will be selected with cells that have appropriate healthy morphology and are expressing the FRET reporter at adequate levels (intermediate fluorescence). An imaging protocol will be set up which includes image acquisition at every field of view at 3 minute intervals, and each acquisition will include CFP excitation-CFP emission, CFP excitation-YFP emission, YFP excitation-CFP emission, and YFP excitation-YFP emission. Each channel may not be needed for analysis but are included for contingency.
Mice will be placed in the microscope environment with proper temperature, atmosphere and pH control, several fields of view will be selected with cells that have appropriate healthy morphology and are expressing the FRET reporter at adequate levels (intermediate fluorescence). An imaging protocol will be set up which includes image acquisition at every field of view at 3 minute intervals, and each acquisition will include CFP excitation-CFP emission, CFP excitation-YFP emission, YFP excitation-CFP emission, and YFP excitation-YFP emission. Each channel may not be needed for analysis but are included for contingency.
Cells will be seeded and placed in the microscope environment with proper temperature, atmosphere and pH control, and several fields of view will be selected with cells that have appropriate healthy morphology and are expressing the FRET reporter high levels to ensure the best signal to noise. An imaging protocol will be set up which includes image acquisition at every field of view at 3 minute intervals, and each acquisition will include CFP excitation-CFP emission and CFP excitation-YFP emission.

Q9. Choose the answer that best continues the essay.

After a 15 min baseline of images are acquired, cells will be treated with BKM120 (B) and gefitinib (G) and then images acquired for another 30 minutes. In subsequent experiments, cells will be imaged similarly but instead treated with either control (DMSO), K1 inhibitor (i) alone, K2i alone, K3i alone, B+G+K1i, B+G+K2i, and B+G+K3i.
After a 15 min baseline of images are acquired, cells will be treated with BKM120 (B) and gefitinib (G) and then images acquired for another 24 hours. In subsequent experiments, cells will be imaged similarly but instead treated with either control (DMSO), K1 inhibitor (i) alone, K2i alone, K3i alone, B+G+K1i, B+G+K2i, and B+G+K3i.
Cells will be treated with BKM120 (B) and gefitinib (G) and then images acquired for another 30 minutes. In subsequent experiments, cells will be imaged similarly but instead treated with either control (DMSO), K1 inhibitor (i) alone, K2i alone, K3i alone, B+G+K1i, B+G+K2i, and B+G+K3i.
After a 15 min baseline of images are acquired, cells will be treated with BKM120 (B) and gefitinib (G) and then images acquired for another 30 minutes. In subsequent experiments, cells will be imaged similarly but instead treated with either control (DMSO), B alone, or G alone.;

Q10. Choose the answer that best continues the essay.

To address the question of whether any of the kinase inhibitors K1, K2 or K3 had an effect on ERK pathway activation or inhibition in response to dual gefitinib and BKM120 treatment, quantitative image analysis will be performed. Background will be subtracted from every field of view, cells identified with regions of interest, and then total CFP excitation-CFP emission intensity in each cell will be divided by total YFP excitation-YFP emission intensity in that same cell. This ratio will be plotted over the time course for every cell.
To address the question of whether any of the kinase inhibitors K1, K2 or K3 had an effect on ERK pathway activation or inhibition in response to dual gefitinib and BKM120 treatment, quantitative image analysis will be performed. The background from the first field of view will be subtracted from every field of view, and then total CFP excitation-CFP emission intensity in each field of view will be divided by total YFP excitation-YFP emission intensity in that same cell. This ratio will be plotted over the time course for every field of view.
To address the question of whether any of the kinase inhibitors K1, K2 or K3 had an effect on ERK pathway activation or inhibition in response to dual gefitinib and BKM120 treatment, quantitative image analysis will be performed. Background will be subtracted from every field of view, cells identified with regions of interest, and then total CFP excitation-YFP emission intensity (FRET channel) in each cell will be plotted over the time course for every cell.
The imaging data will be given to the NSA for facial recognition analysis and they will conclude that they need to monitor glioma cell phone calls with paracrine ligand capture assays.

Q11. Eight years into his PhD career, poor Lostly Q. Givenup still has no meaningful findings or publications, although he has now, thanks to the help of the postdocs in Prof. Bigshot’s lab, finally managed to obtain quality proteomics data for proteins that interact with the novel protein Exciting. When he emailed Prof. Dontcare about the results, a month later Prof. Dontcare responded with “looks good”.

Lostly decides to stop considering Prof. Dontcare’s opinion, or lack thereof, and takes matters into his own hands. He has attended some interesting seminars on systems biology analysis and decides that such analysis might help him generate meaningful findings from this proteomics data set.
Lostly goes to Mallorca to find Prof. Dontcare, and discuss the data in person. However Prof. Dontcare will not get out of the pool or stop drinking mojitos.
Lostly finally decides this project is going nowhere and gets a real job doing construction, only to find he quite liked having nobody caring what he was doing every day. However, Lostly does find it nice that at the end of the day he can look at and touch what he has accomplished.
Lostly decides to switch labs, because he wants to spend another five years or more getting another project going.

Q12. Choose the answer that best continues the essay.

His first step is to take this list of interacting proteins and determine their corresponding genes. He ranks this gene list by the measured strength of the interaction, and then performs gene set enrichment analysis to find statistically significant overlap with other previously defined gene sets. He also performs gene ontology analysis (on the unranked list) to provide further clues as to cell types that Exciting may be associated with.
His first step is to take this list of interacting proteins and determine their transcription factors. He then performs gene ontology analysis on the transcription factors to provide further clues as to biological processes, molecular functions, and cellular compartments that Exciting may be associated with.
His first step is to take this list of interacting proteins and determine their corresponding genes. He ranks this gene list by the measured strength of the interaction, and then performs gene set enrichment analysis to find statistically significant overlap with other previously defined gene sets. He also performs gene ontology analysis (on the unranked list) to provide further clues as to biological processes, molecular functions, and cellular compartments that Exciting may be associated with.
His first step is to take this list of interacting proteins and rank them by the measured strength of the interaction. He then performs gene set enrichment analysis to find statistically significant overlap with other previously defined gene sets. He also performs gene ontology analysis (on the unranked list) to provide further clues as to biological processes, molecular functions, and cellular compartments that Exciting may be associated with.

Q13. Choose the answer that best continues the essay.

The previous analysis suggests that Exciting is involved in transcriptional responses as well as kinase signal transduction. Lostly is fortunate to have a very good genomics core facility at his institution and submits two samples (in triplicate) for mRNAseq, +/- siRNA against Exciting from a cell culture model of debilitating disease. He performs differential expression analysis via the cufflinks/cuffdiff pipeline to derive a list of genes who’s transcript levels are potentially regulated by Exciting. He inputs this list into ChEA, which informs him that TF1 and TF2 are likely involved with the observed transcriptional response. He further inputs this list, along with the interacting protein list, into Expression2Kinases, which informs him that Kin1 is very likely involved with regulating Exciting.
The previous analysis suggests that Exciting is involved in transcriptional responses as well as kinase signal transduction. Lostly is fortunate to have a very good genomics core facility at his institution and submits two samples (in triplicate) for mRNAseq from a cell culture model of debilitating disease. He performs differential expression analysis via the cufflinks/cuffdiff pipeline to derive a list of genes who’s transcript levels are potentially regulated by Exciting. He inputs this list, along with the interacting protein list, into Expression2Kinases, which informs him that TF1, TF2, (transcription factors) and Kin1 (a kinase) are very likely involved with regulating Exciting.
The previous analysis suggests that Exciting is involved in transcriptional responses as well as kinase signal transduction. Therefore he performs transcription factor analysis based on the Exciting promoter, to output a list of transcription factors. He further inputs this list, along with the interacting protein list, into Expression2Kinases, which informs him that TF1, TF2, (transcription factors) and Kin1 (a kinase) are very likely involved with regulating Exciting.
The previous analysis suggests that Exciting is involved in transcriptional responses as well as kinase signal transduction. Lostly is fortunate to have a very good genomics core facility at his institution and submits two samples (in triplicate) for mRNAseq. He performs differential expression analysis based on difference in counts to derive a list of genes who’s transcript levels are potentially regulated by Exciting. He inputs this list into ChEA, which informs him that TF1 and TF2 are likely involved with the observed transcriptional response. He further inputs this list, along with the interacting protein list, into Expression2Kinases, which informs him that Kin1 is very likely involved with regulating Exciting.

Q14. Choose the answer that best continues the essay.

Many prior studies have shown that oscillatory activation patterns (over several hours) of Kin1 activity in response to oxidative stress gives rise to upregulation of gene expression mediated by TF1 and TF2, which promotes a protective antixoxidant response. Lostly therefore hypothesizes that Exciting may play a key role in facilitating Kin1-mediated crosstalk between TF1 and TF2. It is already known that the levels of Exciting do not change in response to oxidative stress. Luckily, there is a live-cell reporter for Kin1 activity that Lostly’s lab bench neighbor has access to and has validated. This reporter is based on localization of GFP fluorescence to the cytoplasm vs. the plasma membrane. Lostly transfects the live-cell reporter of Kin1 activity into the cell line model of Debilitating Disease, treats cells +/- siRNA against Exciting, +/- cycloheximide and +/- oxidative stress, and observes Kin1 activity in several single cells under each condition using flow cytometry, every hour for an eight hour time course.
Many prior studies have shown that oscillatory activation patterns (over several hours) of Kin1 activity in response to oxidative stress gives rise to upregulation of gene expression mediated by TF1 and TF2, which promotes a protective antixoxidant response. Lostly therefore hypothesizes that Exciting may play a key role in transmitting the Kin1 signal to TF1 and TF2, or perhaps vice versa, from TF1 and TF2-regulated genes to Kin1. It is already known that the levels of Exciting do not change in response to oxidative stress. Luckily, there is a live-cell reporter for Kin1 activity that Lostly’s lab bench neighbor has access to and has validated. This reporter is based on localization of GFP fluorescence to the cytoplasm vs. the plasma membrane. Lostly transfects the live-cell reporter of Kin1 activity into the cell line model of Debilitating Disease, treats cells +/- siRNA against Exciting, +/- cycloheximide and +/- oxidative stress, and observes Kin1 activity in several single cells under each condition using flow cytometry, every hour for an eight hour time course.
Many prior studies have shown that oscillatory activation patterns (over several hours) of Kin1 activity in response to oxidative stress gives rise to upregulation of gene expression mediated by TF1 and TF2, which promotes a protective antixoxidant response. Lostly therefore hypothesizes that Exciting may play a key role in transmitting the Kin1 signal to TF1 and TF2, or perhaps vice versa, from TF1 and TF2-regulated genes to Kin1. It is already known that the levels of Exciting do not change in response to oxidative stress. Luckily, there is a live-cell reporter for Kin1 activity that Lostly’s lab bench neighbor has access to and has validated. This reporter is based on localization of GFP fluorescence to the cytoplasm vs. the plasma membrane. Lostly, using the cell line model of Debilitating Disease, creates a line stably expressing the live-cell reporter of Kin1 activity. He performs live-cell experiments with these cells +/- siRNA against Exciting, +/- cycloheximide and +/- oxidative stress, and observes Kin1 activity in several single cells under each condition, over an 8 hour time lapse.
Many prior studies have shown that oscillatory activation patterns (over several hours) of Kin1 activity in response to oxidative stress gives rise to upregulation of gene expression mediated by TF1 and TF2, which promotes a protective antixoxidant response. Lostly therefore hypothesizes that Exciting may play a key role in transmitting the Kin1 signal to TF1 and TF2, or perhaps vice versa, from TF1 and TF2-regulated genes to Kin1. It is already known that the levels of Exciting do not change in response to oxidative stress. Luckily, there is a live-cell reporter for Kin1 activity that Lostly’s lab bench neighbor has access to and has validated. This reporter is based on localization of GFP fluorescence to the cytoplasm vs. the plasma membrane. Lostly transfects the live-cell reporter of Kin1 activity into the cell line model of Debilitating Disease, treats +/- oxidative stress, and observes Kin1 activity in several single cells under each condition using flow cytometry, every hour for an eight hour time course.;

Q15. Choose the answer that best continues the essay.

Lostly carefully analyzes the time course data to quantify the activity dynamics of Kin1 in response to oxidative stress. He takes the ratio of fluorescence intensity of FSC vs. GFP fluorescence as the measure of Kin1 activity (FSC is related to the cell size, and therefore reflective of the edges of the membrane). He finds very complex patterns of oxidative stress-induced oscillatory activity that are heterogeneous and unsynchronized across cells when Exciting is present, but more homogeneous and non-oscillatory responses when Exciting is absent. When cycloheximide is used, the oscillations also stop. He decides to interpret these observations with a differential equation-based model to understand whether these data suggest Exciting is upstream or downstream of TF1 and TF2, and how that regulates Kin1 activity. He performs simulations under several different assumptions, where Exciting (i) mediates negative feedback to Kin1 after TF1 and TF2 induce gene expression, (ii) mediates positive feedback to Kin1 after TF1 and TF21 induce gene expression, or (iii) is necessary for TF1 and TF2 to induce gene expression. The modeling analysis suggests that scenario (i) or (ii) is compatible with the observations, so more experiments are needed to distinguish between the different possibilities.
Lostly carefully analyzes the time course data to quantify the activity dynamics of Kin1 in response to oxidative stress. He subtracts background, and then defines regions of interest in each cell for each time point that correspond to the cytoplasm or plasma membrane. He takes the ratio of fluorescence intensity of one to the other as the measure of Kin1 activity. He finds very complex patterns of oxidative stress-induced oscillatory activity that are heterogeneous and unsynchronized across cells when Exciting is present, but more homogeneous and non-oscillatory responses when Exciting is absent. When cycloheximide is used, the oscillations also stop. He decides to interpret these observations with a differential equation-based model to understand whether these data suggest Exciting is upstream or downstream of TF1 and TF2, and how that regulates Kin1 activity. He performs simulations under several different assumptions, where Exciting (i) mediates negative feedback to Kin1 after TF1 and TF2 induce gene expression, (ii) mediates positive feedback to Kin1 after TF1 and TF21 induce gene expression, or (iii) is necessary for TF1 and TF2 to induce gene expression. The modeling analysis suggests that both scenarios (i) and (ii) are compatible with the observations, so more experiments are needed to distinguish between the different possibilities.
Lostly carefully analyzes the time course data to quantify the activity dynamics of Kin1 in response to oxidative stress. He subtracts background, and then defines regions of interest in each cell for each time point that correspond to the cytoplasm or plasma membrane. He takes the ratio of fluorescence intensity of one to the other as the measure of Kin1 activity. He finds very complex patterns of oxidative stress-induced oscillatory activity that are heterogeneous and unsynchronized across cells when Exciting is present, but more homogeneous and non-oscillatory responses when Exciting is absent. When cycloheximide is used, the oscillations also stop. He decides to interpret these observations with a differential equation-based model to understand whether these data suggest Exciting is upstream or downstream of TF1 and TF2, and how that regulates Kin1 activity. He performs simulations under several different assumptions, where Exciting (i) mediates negative feedback to Kin1 after TF1 and TF2 induce gene expression, (ii) mediates positive feedback to Kin1 after TF1 and TF21 induce gene expression, or (iii) is necessary for TF1 and TF2 to induce gene expression. The modeling analysis suggests that both scenarios (i) and (iii) are compatible with the observations, so more experiments are needed to distinguish between the different possibilities.
Lostly carefully analyzes the time course data to quantify the activity dynamics of Kin1 in response to oxidative stress. He takes the ratio of fluorescence intensity of FSC vs. GFP fluorescence as the measure of Kin1 activity (FSC is related to the cell size, and therefore reflective of the edges of the membrane). He finds very complex patterns of oxidative stress-induced oscillatory activity that are heterogeneous and unsynchronized across cells when Exciting is present, but more homogeneous and non-oscillatory responses when Exciting is absent. When cycloheximide is used, the oscillations also stop. He decides to interpret these observations with a differential equation-based model to understand whether these data suggest Exciting is upstream or downstream of TF1 and TF2, and how that regulates Kin1 activity. He performs simulations under several different assumptions, where Exciting (i) mediates negative feedback to Kin1 after TF1 and TF2 induce gene expression, (ii) mediates positive feedback to Kin1 after TF1 and TF21 induce gene expression, or (iii) is necessary for TF1 and TF2 to induce gene expression. The modeling analysis suggests that both scenarios (i) and (iii) are compatible with the observations, so more experiments are needed to distinguish between the different possibilities.;

Review:

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