Cell Eng Exam 1

Receptor-ligand binding

At time = 0, protein = [P]0, ligand = [L]0, complex = 0. At equilibrium, protein = [P]0-x, ligand = [L]0-x, complex = x = [C].

Gelatin

Denatured collagen. Good for forming hydrogel. Collagen is the primary protein component of the ECM and most collagen is fibrillar. Denatured collagen and type IV collagen are gelatinous

How gene "silencing" enables cell differentiation

Differentiation leads to the distinctive phenotypes of varius cell types. About ~50% of the genome is expressed in any particular cell. The DNA bluprint(s) ~22,000 genes: ~11,000 genes are transcribed in any particular cell type, ~1,000 are "housekeeping" genes expressed in all cell types, the rest (~10,000) of the transcribed genes determine cell type (on a cell by cell basis). The other ~50% of genes are "silent"

Transport

Diffusion: Movement of individual molecules. Convection: Movement of the bulk fluid. Mobility: Movement of charged species by an electric field. Conservation of a species: Choose a region of space then Flow in - flow out + generated - consumed = 0. EX: circulatory system on small scale works by diffusion (diffusionn of O2 into tissue) but large scale is convection.

deltaS = entropy

Disorder, number of states. Max at equilibrium. Macroscale: S=(dH-dG)/T. Microscale: S=Kb ln W. what influences entropy: immobilization of flexible molecules, changes in degrees of freedom, hydrophobic effect, maximum at equilibrium.

Epigenetics

Factors that exert control over the genome that are outside of the primary DNA sequence / genetic code. Common mechanisms for this: gene regulation via histone acetylation, gene regulation via many PTMs (post-translational modifications), gene regulation via DNA methylation, signal pathway activation of genes, gene regulation via glycosylation, control of transcription, various non-coding RNAs, glycosylation.

How leukocytes get out of the bloodstream

First steps are tethering and rolling. First is tethering and rolling via selectins then there is firm adhesion via integrins. Incidentally, the same mechanism is used by metastatic cancer cells to get out of the bloodstream

Fitting

For linear fitting, minimize the sum of the squared error / residuals (linear least squares / linear regression). Phenomena that are nonlinear, pick your own equation and minimize the sum of the squared error / residuals (nonlinear least squares, solution is more complicated)

N value meaning (hill coeff)

If n = 1 - no cooperativity. If n > 1 - positive cooperativity. If n < 1 - negative cooperativity

sLeX on/off switch usefulness

If you step on nail you want the leukocyte to only go to the one foot, but the CD34 is widely expressed on endothelial cells throughout the body, so the leukocyte wouldn't know where to go. Need to turn the switch on to recruit the leukocyte. Switch on in seconds recruitment within minutes.

Transmission Electron Microscopy (TEM)

Imaging with a high energy beam of electrons (like SEM, but it is going THROUGH the image). Runs under high vacuum. Resolution ~ 1 nm. Need sample to be DRY, THIN, and placed on a grid. Image is 2-D

Scanning electron microscopy (SEM)

Imaging with a high energy beam of electrons (not light, electrons). Resolution ~ 1 nm. Need surface of sample to be DRY and conductive (often coated with gold). Image looks 3-D, can see surface morphology.

Importance of non covalent forces

Important for reversible binding (on/off), enzymes (not reversible, substrate binding).

Reynolds Number, Re

Important in fluid flow. Inertial Forces / Viscous Forces. Re < 2,300 = Laminar flow (Most biological systems and devices). Re > 4,000 = Turbulent flow

Effects of substrate adhesiveness

It is a biphasic response. Biding too weakly prevents force generation to move. Binding too tightly prervents detachment. Want somewhere in the middle

Switch-like response

It is an all or none output, an on / off binary output from continuous input. It is important biologically because cells receive a spectrum of continuous input but they make all or none responses. Switch- like responses are enabled by: cooperativity, positive feedback, genetic bistability, opposing enzymes, and cascades.

Which fit is better:

Smaller magnitude of error, and a random pattern of error (if there is a pattern then there's likely some phenomena that's being missed.

Inhibiting selectin-based adhesion

Sometimes want to do this for chronic immune disease, cancer therapy, etc. EX: Glycomimetics (a company) developed "Rivipansel" as a cancer drug

What does deltaG < 0 mean?

Spontaneous, thermodynamically favored

Waddington's epigenetic landscape

Think of an undifferentiated cell rolls down a hill as it differentiates. Originally didn't think you can go back up but now know that we can (reprogram). The landscape is used to explain cell differentiation; new "space" (troughs) opens for gene expression, in tandem, barriers "grow inn" that prevent lateral movement of cells. Today this general type of approach is part of "computational epigenetics"

Guesstimation: science daily says "it's official, there are 42 million protein molecules in a cell"

This estimate is not reasonable ? not sure

Biomedical implications of epigenetics

re/de-activationn of various "blueprints" holds promise for: control of cell fate for use inn tissue engineering and regenerative medicine, and "safe" cancer therapy ( a new experimental drug can make some leukemic cells "mature" into healthy cells).

sLeX bids to each selectin

sLeX is a required binding partner for ALL THREE selectins in vitro. sLeX binds to L, E, and P-selectin in in vitro flow chamber experiments. But, there are additional requirements for sLeX:selectin binding under physiological conditions (i.e., in the body, in vivo).

Fractional saturation of receptor

(# of occupied sites / # of total sites). Y = [C] / [P]0.

Rivipansel

(Unexpectedly) treats Sickle Cell Disease (SCD) by inhibiting selectin-based adhesion. it makes "all blood flow like a river" in sickle cell disease. Pain is reduced in minutes, hospital stays go from ~1 week to 1 day. But what does E-selectin have to do with occlusions formed by misshapen RBCs? Because E-selectin causes occlusion. E-selectin "nucleates" sickled RBC occlusion sites Rivipansel reverses this.

Polysaccharides can be 'free floating'

(e.g. HA) 'free floating' polysaccharide-based structures (such as the proteoglycan, which has hyaluronic acid as its core) are abundant in cartilage (and are also present in several other tissues such as skin). But they can also be covalently attached to the cell surface: alternatively, polysaccharides can be covalently associated with the cell surface (e.g. via CD44 that - in non-variant form - is present on most cell types.

Air force pilot problem: out of the 4,063 pilots, how many were 'average' in size (middle 30%)

0, none of them were actually average in all measurements.

What is the diffusion constant for a protein? (D = kBT / 6pi u r) u is the viscosity

10^-6 cm^2/s. This calculation made using viscosity of water (10^-3 Pa s), kB*T ~ 4x10^-21 J, and assumed protein diameter of ~ 6nm therefore radius of ~ 3nm.

How old is the field of epigenetics?

1930s - chromosomes are linked to inheritance in drosophila. 1953 - the double helix structure of DNA is discovered. Soooo by the 1930s DNA and chromosomes were known to be critical for "inheritance", the chemical structure of DNA was unknown, the genetic code was unknown. 'Epigenetics' were scientifically fuzzy/undefined efforts to explain how one set of chromosomes resulted in the various cell types that constitute multicellular organisms.

What is the approximate size resolution limitation with a fluorescence microscope?

200 nm (imaging resolution limitation is about wavelenght/2)

deltaG0 for protein interactions

5-20 kcal/mol

Beer-lambert law

A = -log(I/I0) = ecl. A=absorbance, I=intensity, e=extinction coefficient (particular to material), c=concentration, l=path length.

Aggrecan

A core protein linker for proteoglycans. Aggrecan links smaller GAGs (KS and CS) to HA. it has a high MW (~250,000) encoded by a single gene. It is highly glycosylated (it is ~90% carbohydrate), mainly with 2 types of GAGs (CS and KS). Each aggrecan contains ~100 CS chains, which are typically ~20kDa each, and the chains are either 4-sulfated, 6-sulfated, or usually both. There are fewer KS chains (up to 60), and they are usually shorter in length (5-15 kDa)

For orientation accuracy what is more important?

A higher concentration GRADIENT of ligand (nnot just a high concentration)

Enzyme linked immunnosorbent assay (ELISA)

A molecular measurement. Can detect the presence of proteins or other antigens in solution. Samples can come from blood, cell lysates, or any other source. Colorimetric (absorbance) signal. Enzyme amplifies the signal (high sensitivity label). With or without secondary antibody (secondary helps amplify signal, sandwich assay).

ECM fun facts

A person has ~15g of HA in their body. A person 'recycles' ⅓ rd of his/her HA each day (each day a person makes ~5 g HA). @ $280 / 1 mg, a person makes $1,400,000 worth of HA a day.

Surface plasmon resonance (SPR)

A plasmon is an optical/electrical phenomenon. First, immobilize ligand to a thin gold surface. Flow the binding partner over the surface. Then record a signal proportional to the mass on the surface. Can get an on / off rate.

Fluorescence resonance energy transfer (FRET)

A quenching mechanism that acts over a 0-10nm distance. One excited state (donor) decays while transmitting energy to a distant molecule (acceptor) that absorbs the same released energy. Emission from donor matches the excitation of the acceptor. Allows the determination of close proximity between two molecules in space and time. Can also measure conformational changes within a molecule (intramolecular).

Personalized medicine

A solution to the idea that no 2 patients are the same. Highly personalized treatments but the biggest pitfall is that it is very expensive. An estimated that the annual cost is ~ $1 trillion so maybe we went toooo personalized. Maybe a simple adjustable chair type solution for this personal medicine problem.

Fluorescence microscopy

A wide array of tags, easy to sue. Can overlay a phase-contrast image to see additional features. Gentle to sample, can image live cells. Resolution limit to ~200 nm. Can see 2D projections of 3D molecules.

Flow Cytometry

AKA: Fluorescence Activated Cell Sorting (FACS). Instead of counting on a microscope individually, thousands of cells per second can move single-file through a laser spot. Excited with 1-4+ lasers to show emission in 10+ colors. Can identify sub-populations of cells. Can select for live cells / measure viability. Can sort your positive cells and keep them for later. Originally slow to run hundreds of samples, plate reader attachments now make it easy. Can phenotype cell with high precision

How many different types of cells in the body?

About 400-500 cell types, nature is able to get this many from one blue print through epigenetics.

What is the size/thickness of an oligosaccharide?

Againn "average". Think about the thickness of the glycocalyx, they should be about the same 10-50 nm. Is an 'average' glycan (at least) 10 nm long. Wellll the average disaccharide is ~ 1 nnm, based on this information, the glycan would be ~ 3nm in length. An average oligosaccharide is <= 3-4 nm but this seems too thin (based on glycocalyx length). This discrepancy can be partially due to the fact that glycans exist on protein scaffolds.

Scatchard Plot

Allows you to visually demonstrate if simple binding model is correct. Gives slop of -1/Kd

Nutrient sensor involved in epigenetics

An emerging candidate is UDP-GlcNAc (after ATP, is the most abundant small molecule metabolite in cells)

Cartilage

An example of how GAGs and proteins work together. Cells comprise only a few perent of volume of cartilage, the major part of the tissue is a highly organized and expanded ECM. contains a network of collagen fibrils (responsible for form and tensile properties), a high concentration of PG (mostly aggrecan) which exerts a swelling pressure on collagen network. It is the retention of aggrecan in compressed form within inextensible collagen network that causes the swelling pressure and makes the tissue ideal for resisting compressive load with minimal deformation making it load bearing.

Genetic Bistability (switch-like)

An example of this is mutual repression. A gene is expressed that represses the promoter of another gene so that only 1 is on at a time, can modulate and switch which gene is expressed via the introduction of an inducer (example of a synthetic biology approach)

Luminescence

Another emission of light. Does not require excitation. Typically requires chemical energy (chemical reaction). Ex: beetle luciferin reacting with firefly luciferase and mg+2 to make oxyluciferin which releases light.

Laminin

Another important multi-adhesive protein. Laminin cross links Type IV collagen to other ECM components and the cell via integrins. Fibronectin has a similar role for type 1, 2, and 3 collagen. Laminin is a main component of basement membrane.

High-thoughput multi-label plate readers

Applicable to absorbance-based assays (Concentration and Cell Viability, want clear bottom plate), Applicable to fluorescence-based assays (GFP expression, Binding Affinity, want black wall plate), Applicable to luminescence-based assays (Luciferase expression and Cell Viability, use white plates). Advantages of plate reader: Fast, Quantitative, Low sample volume, High-throughput, on destructive. Disadvantages: Values are average values per well, Sensitivity may be lower than other methods.

Electrostatics

Attraction between unlike charges. Repulsion between like charges. This force can allow for specificity. Electrostatics can increase reaction rates (can help encounter complexes form more frequently, and can cause the success rate for encounter complexes through orientational steering). Electrostatics can also decrease reaction rate (can prevent encounter complexes from forming (repel), can decrease the success rate for encountering complexes through orientation steering)

Proteoglycans

Basically GAGs with core proteins. Examples include aggrecan. PGs can be decorated with HA, KS, and CS. HA enables PGs to be larger than bacteria

Integrins

Bent (inactive) vs extended (active) conformations. EX: chemoattractants can activate integrin. Integrins have two opposing functions firm adhesion and extravasation. Integrins are complex, versatile, and malleable group of CAMs

Types of proteins per cell

Between 8-11,000 types. Used to think that there are ~5000 but now we know there's about twice as many. Why? Because we have better detection methods they have become more sensitive. From Science Daily: most proteins exist in a narrow range, between 1,000 and 10,000 copies per cell (these were detectable 10 plus years ago) others exist at fewer than 10 copies per cell (these can now be detected)

How does delta deltaG0 of -1 kcal/mol at 298K change Kd?

Binding is 5x stronger (see slides 22-23 for work, 09/07)

sLeX present on CD44 allows E-selectin binding

CD44 can serve as an E-selectin ligand during cancer metatasis (glycolipids have also been implicated as E-selectin binding partners). The role of sulfation (OSO2-) in CD44 : E-selectin binding is not yet fully known. Note that CD44 w/o sialic acid binds to hyaluronan, not to E-selectin.

Verification of selectins

CD62 was confirmed to be involved (selectins are "CD markers"). There are 3 main selectins: E-selectin (on endothelial surface), L-selectin (found on leukocytes), P-selectin (platelet and endothelial). IMPORTANT: selectins can either be on the incoming (i.e. "rolling") cell (L) or on the surface (endothelia, P and E). P selectin is typically on the surface (e.g. the lumen of blood vessels) and i guess E selectin?. The lumen of blood vessels was recapitulated by coating a flow chamber w P-selectin => supported cell tethering and rolling, these experiments verified that selectins were involved in leukocyte homing.

Epigenetics and glycosylation

Can glycosylate histones to alter gene expression

Dynamic Light Scattering (DLS)

Can measure size (0.3 nm - 10 μm) and zeta potential (surface charge). Size is a BULK, intensity-weighted measurement of particles in solution. Measurement is quick and easy. Autocorrelation of scattered light is used to fit for diffusion coefficient (D = kBT / 6pi u r). Hydrodynamic radius is calculated from Stokes-Einstein. This is the standard best practice for analyzing particle size. Can do this in a hydrated environment (proteins, particles, etc.)

Nanoparticle Tracking Analysis (NTA)

Can measure size (10 nm - 1 μm) and absolute particle concentration in solution. Tracks of individual particles are followed over time and each track is fit to Stokes-Einstein. Direct number-weighted average. Single particle fluorescence capability. Microscopes follow how particles diffuse, can see identity info based on fluorescence effects. Limitation: not as big range, doesn't work very well at the top of its range

Calculating hill coefficients

Can obtain the predicted Hill coefficients from EC10 to EC90 data. Equation: n = log(81) / log(EC90/EC10)

Dimensional analysis

Can quickly determine the important aspects of problem. Nondimensional = need all units to cancel out. One strategy is to first arrange parameters of a phenomena to find a characteristic time constant (t). Then compare the ratio of the time constants from different phenomena: Diffusion, Convection, Reaction. Can compare various characteristics of system: Times, Forces. Figure out which variable/parameter dominates

Coulter Counter

Can size 0.4 μm - 1,600 μm. Counts particles as displaced volumes (can estimate size of particle). Particles suspended in a weak electrolyte solution are drawn through a small aperture that separates two electrodes. The voltage across the aperture is the sensing zone

Three components of cell flux

Cell diffusion (μ [=] distance2 / time). Changes in cell speed from concentration (μ/L [=] distance2 / time M). Directional bias of movement (c [=] distance2 / time M). check slide 47 lec 9 for equation.

Explanation for discrepancy in oligosaccharide vs glycocalyx

Certain cell surface glycoportiens (e.g. CD34 extend 30-50 nm (or more) above the cell surface, thereby giving the glycocalyx a greater apparent thickness. Another explanation is because there is a fuzzy line between the glycocalyx and the ECM. the ECM consists of a mixture of covalently and non-covalently associated proteins and polysaccharides. The key point is that polysaccharides form a bridge and exist in a continuum between the glycocalyx and ECM

Salt dependence on binding

Changes to the effective strength of ionic interactions. Nonspecific screening - diminishes magnitude of electrostatic interactions. Specific ion binding - ions can bind to specific sites on a protein, can increase the effective size of the protein.

Binding rates and affinity

Check slide 28 for general scales (09/07)

Common values in cell engineering

Check slide 29 (09/07)

Time scales of receptor mediated response to ligand

Check slide 30 (09/09). Overall responses are pretty quick (within minutes)

Common time scales

Check slide 30 09/07

Equilibrium with ligand depletion

Check slide 34 (09/09) for equations. Use a dimensionless number to calculate the fraction of cell receptors complexed and number of cell receptors complexed.

Biomolecular interaction kinetics

Check slide 40 (09/09) for equations

Debye-Huckel model

Check slide 46 09/07

Thermodynamics

Check slides 09/07 (20) for equations!!

Triglycerides (lipids)

Chemical structure: glycerol with three fatty acid chains. There are also "cis" and "trans" fatty acids; these function as fat storage and help make up adipose tissue

Phospholipids

Chemical structure: glycerol with two fatty acid chains, and a phosphate and amino alcohol; their major function is for biomembranes (cell membrane). Phospholipids spontaneously assemble into micelles (single layer) or liposomes (double layer) and these can be used for drug (and nucleic acid) delivery.

Non-coding RNA

Chromosomal DNA also is the template for many non-coding RNAs that regulate gene expression

Fibronectin

Connects collagen to GAGs. Fibronectins link Types 1, 2, and 3 collagen to the cell by serving as a bridge to integrins.

Glycolipids

Consist of ceramide and a glycan. Chemical structure: long-chain sphingoid base backbone linked to a fatty acid via an amide bond w the 2-amino group and to a polar head group at the C1 position via an ester bond, forming ceramide (sphingoid and fatty acid) then the ceramide is linked to a sugar (e.g. glucose, galactose, or inositol) via a beta-glycosidic bond btw the hemiacetal group of the sugar and the C1 hydroxyl group of the ceramide. The sugar component is usually "elaborated" like there's additional monosaccharides added. Glycans provide lipids with added structures and activities.

DeltaG = Total free energy

Contributions from both enthalpy and entropy. Determines if reaction will occur. Measure by equilibrium constant (Kd); contributions from both enthalpy and entropy

Cooperative vs. noncooperative

Cooperative enzymes give a sigmoidal shape and appear as an all or none response. This response is over a smaller input range (steeper) in comparison to the noncooperative case with the same Kd.

Oxygen binding to hemoglobin

Cooperativity is important to allow binding of O2 to hemoglobin. It can bind 4 molecules of O2 (cooperativity allows binding to get easier every time O2 binds). Structural change helps mediate the all or none binding response. Protein structure allows O2 transport and release function (hemoglobin picks up and drops off O2 quickly)

Many approaches for counting cells

Coulter counter. Microscope and hemocytometer (Count squares and average, Typically count left side and top side lines only) this can take a longer time but it's reliable.

Measuring cell viability/ toxicity

Count cells directly - with hemocytometer and look at Trypan Blue staining of dead cells. Metabolic activity - A colorimetric (absorbance) assay that uses (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or a related kit (CellTiter); More live cells = higher signal. Lactate dehydrogenase (LDH) - is released by dying cells; More dead cells = higher signal. Apoptosis - Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). DNA fragmentation caused by apoptosis. Flow Cytometry - Fluorescent dyes such as propidium iodide can stain dead cells / DNA.

Stokes-Einstein Equation

D = KbT/6piuR. Numerator: Thermal energy (ability for random motion). Denominator: Resistance to movement / drag forces. R: Hydrodynamic radius assuming drag on molecule/particle can be modeled as a sphere. Diffusion is always a function of temperature and viscosity.

DLS vs NTA comparison

DLS is a bulk intensity-weighted measurement. NTA is a direct number-weighted average. In different situation these two methods ca give different measurement outputs (ex: if there is a very large outlier the DLS measurement will go very high but the NTA measurement wouldn't be nearly as effected. DLS is gold standard when particles are all about same size.

How chromatin structure governs gene silencing

DNA can be "open" or tightly-packed configurations. DNA for genes that produce proteins (are expressed) are in the "open" configuration. The other ~50% of genes are tightly packed and "silent" (DNA tightly packed around histone).

Double question: I - if activation energy increases what happens to k? II - if temperature increases what happens to k?

Decreases, increases

Fluorescence

Emission of radiation due to an excited state molecule returning to a ground state. Emission is at a lower energy (longer wavelength) compared to excitation. Quantum yield: efficiency, the fraction of excitations that result in emission; generally increases with solvent polarity. Local environment affects fluorescence. Higher sensitivity than absorption. Can look at a bunch of features at the same time.

deltaH = enthalpy

Energy in molecular bonds. This is an intrinsic value, changes during the reaction. Can measure by isothermal titration calorimetry (ITC). many bonds are important (P-L, P-W, L-W, W-W, P-P, L-L). typically <0

Leukocytes - where do they live/ work?

Even though leukocytes are called "blood cell", they do most of their work in "solid" tissues. Jobs for leukocytes (WBCs) in tissues: some are phagocytic (e.g. macrophages), others produce antibodies, some secrete histamine and heparin, others neutralize histamine. Briefly, they provide a defense against organisms that cause disease and either promote or inhibit inflammatory responses.

Ligand/Receptor targeted drugs

Example: Eylea drug is high-affinity receptor-based drug that soaks up excess VEGF that causes blindness. There is a large market potential for these biologics that bind to their target very well.

Cascade (switch-like)

Example: the MAPK cascade. Signal builds as you move down the cascade (amplifies)

Cell migration

Examples of cell migration: Immune cells defending host, Endothelial cells during angiogenesis, Dermal fibroblasts during wound healing, Tumor cell during metastasis. How do cells move? Random motility, Persistent random walk (sustained movement in a given direction), Taxis (directed movement, i.e. neutrophil attack)

Positive feedback (switch-like)

Examples of this are blood clot formation, anaphase switch in cells (chromosomes separate), breastfeeding, and birth. Positive feedback loops need a clear end/stop otherwise they can spiral out of control.

Hill equation, cooperativity

Fractional saturation: Y = L^n /(Kd + L^n). n is the hill coefficient, which increases with increasing cooperativity. n=1 - no cooperativity, n >1 - positive cooperativity (makes easier), n <1 - negative cooperativity (makes it harder for the next ligand to bind)

GAGs as structural molecules

GAGs provide structure. PGs / GAGs are a minor ECM component by MASS (values range from ~0.2-5% (m/m) for DRY tissue). Though it is a small percentage by mass of dry tissue, remember that GAGs are HIGHLY HYDRATED looking further, polysaccharides can compose up to 70% of the volume of the ECM (in essence, the ECM is a hydrogel). Polysaccharide volume expands 50-100x upon hydration.

GAGs as information carrying entities

GAGs store information that controls stem cell fate... mitosis (cell division) produces identical daughter cells, so how does asymmetric stem cell division occur? Stem cells live in a 'nische' which provides a hospitable environment where a stem cell can survive in stasis for decades. Sulfation patterns on GAGs give binding cues for different growth factors (sulfation results in block copolymer formation, which can lead to sequestration of diff GFs; no sulfation no binding (HA doesn't sequester GFs)). So Stem cells are protected from GF influence bc of a shield of HA and PGs that are non-sulfated (form capsule) but during division one of the daughter cells can be translocated outside of this shield where it is now susceptible to binding of GFs (new microenvironment). This new environment outside of the nich allows for cell differentiation/proliferation.

Brain glycolipids

Gangliosides GM1, GD1a, GD1b, and GT1b are abundant in a healthy brain. GD2 is cancer-associated

Glycocalyx

Glycocalyx completely covers the plasma membrane (surface area equals the surface area of the plasma membrane 2400 um2). The thickness of the glycocalyx is 10-50 nm (plasma membrane 6-10 nm). The glycocalyx is thicker than the plasma membrane. Primarily consists of: oligosaccharides (and occasionally polysaccharides) covalently attached to plasma membrane proteins or lipids

Nonlinear least squares

Goal to minimize the sum of the square error. Analytical solution not easily obtained and so equation must be solved iteratively starting with a good guess for the B parameters. Use computational approaches to solve (i.e. matlab use nlinfit)

HA, maybe buy it, can it treat cancer?

HA is a 'workhorse' scaffold component for biomaterials used in tissue engineering and regenerative medicine. Mole rats are more resistant to cancer because they have extraordinary levels of HA. BUT HA can also promote cancer because it can protect a tumor. The HA capsule can sequester pro-tumorigenic factors, exclude therapeutics. Also, PEGPH20 is a modified form of hyaluronidase, which is now being tested as a cancer drug (it degrades HA). HA is never sulfated

Acetylation as drug target (via HDACi)

HDACi is a histone deacetylase inhibitor which trap chromatin in the 'open' form. They can reactivate cancer suppressing genes (e.g. p21) that had been checked out. Zolinza is the first FDA approved HDACi. Basically this drug inhibits the reveerse reaction.

HA size matters

High molecular weight form of HA which has the protective properties (prevents the transformation of normal cells into cancer cells). Low molecular weight HA can actually be dangerous, it is a potent pro-inflammatory molecule. HA has size-based bioactivity

Fortebio octet

High-throughput approach to measure binding in 96-well plates. Helps to determine kinetic parameters similar to SPR. measures concentration.

The two "classic" epigenetic methods

Histone acetylationn and DNA methylation

Spectroscopy

How electromagnetic radiation interacts with molecules and cells. Types of interactions with light: scattering, absorption, fluorescence emission, photochemistry (chemical rxn with light like x-linking). Types of information you can gather: structural, analytical, and dynamic.

Are WBCs miscounted

How many are there in the blood? # leukocytes in the blood: (8x10^3 leukocytes/ uL)(10^6 uL/L)(6L of blood) = 4.8x10^10 WBCs... but the total number is ~ 1 trillion... why is there a discrepancy... lymphocytes in blood 2-5% of cells (i.e. ~50 billion) lymphocytes on agar (mimic of ECM/tissue) 95-98% of cells (i.e. ~1 trillion)

Types of noncovalent forces

Hydrogen bonding, electrostatics, van der waals forces, hydrophobic interactions

Phospholipid for drug delivery

Hydrophobic drugs can be delivered via micelles and hydrophilic (or crystallized) drugs can be delivered via liposomes. You can engineer and add more bells and whistles to liposomes like a homing peptide or extra protection against an immune response.

Michaelis-menton enzyme kinetics

Hyperbolic stimulus response curve. Requires an 81 fold increase in stimulus to drive the enzyme from 10%-90% maximal response

Two problems: I - given [P]0, [L]0, Kd need to find [C]. II - given [P]0, [L]0, [C] need to find Kd. What do you use to solve problems I and II?

I - simulation, to see how complexes form with each [P] and [L] input. II - model fitting, fitting of all of the binding data to estimate the parameter Kd.

Lipid-based delivery of mRNA (COVID Vaccines)

IONIZABLE LIPID: protonated at certain pH values to facilitate escape. STRUCTURE INTEGRITY: inclusion of cholesterol helps structural integrity, cholesterol also promotes membrane fusion. PEGYLATED LIPID: increases their colloidal stability. CHARGED LIPID: enhances nucleic acid condensation or stabilizes the complexes in serum. These components found in both Pfizer and Moderna vaccines

Depletion effects

If [L] ~ [P] then we start off with [L]0 ~ [P]0 and [L] can be depleted over time. Then what is [L] at equilibrium -> [L] = [L]0 - [C] = [L]0 - Y[P]0. What is the receptor concentration: Rt is the total receptors per cell, cell number per volume is n. Receptors per cell Rt = total P0 as # per volume, divide by Nav to convert to mols of receptor per volume. [P]0 = n*Rt / Nav

Cooperativity

In real examples, there is often the interaction between multivalent binding sites. Ann example of extreme positive cooperatively - once the first ligand blinds, all others bind and give an "all or nothing" type response.

RTK/MAPK signaling cascade

In the extracellular space a hormone (ligand) binds to RTK -> the binding of hormone ligand causes receptor dimerization and autophosphorylation -> next there is recruitment of adaptor protein GRB2 from cytoplasm -> then recruitment of Sos a Guanine nucleotide exchange factor, from the cytoplasm -> then recruitment of Ras, a membrane-associated GTP-binding switch protein -> then activation of Ras (GDP->GTP) -> Ras activates MAPKKK -> MAPKKK binds and phosphorylates MAPKK -> MAPKK binds and phosphorylates MAPK which is then transported to the nucleus and activates transcription factors. This is not a linear signal, it gets amplified with each step.

sLeX binding for L-selectin

In vivo, however, sLeX attachment to CD34 is not enough to ensure binding to L-selectin! sLeX is a binding partner for L-selectin (but NOT E-selectin or P-selectin) when it is presented on O-glycans attached to CD34. Basically, sulfation of sLeX is a rapid "on / off" switch (when sLex is sulfated switch is on an there is high affinity binding for L-selectin)

Ions can screen charges

Increase ion with opposing charges, k decreases. Increase ion with like charges, k increases. Screening charges can reduce attractions or reduce repulsions

As Kd decreases binding affinity

Increases

Reprogramming is epigenetic

Induced pluripotent stem cells are like going up the hill and introduce controlled stimulus to helpt determine cell fate.

Global least squares

Instead of treating the 3 data sets separately (multiple experiments), a better approach is to estimate the unknown parameter globally among all data sets simultaneously. Restrict the parameters to be consistent in all data sets to give better fit. Check slide 48 for equation (09/09)

How do you think cell speed depends on substrate adhesiveness? (adhesiveness = ligand density * affinity)

Intermediate adhesiveness is best because gives a biphasic response!

Engineering approach to selectin research

Introduction of flow chamber set up for studying adhesion interactions under physiological shear stress conditions. The "flow" experiments revealed interesting dynamics. It was found that there were different shear forces.. The flow rate can be converted to a force (shear stress).

Hydrogen bonding

Involves a hydrogen donor (often Nitrogen in proteins) and hydrogen acceptor (often oxygen or lone electron pair). EX: in water, each molecule participates in 4 H-bonds

Chemotaxis coefficient, X

Itself a function of L, RT, Kd, and S. μ, Random Motility Coefficient is a function of n, S, and P.

Kinetic reaction constant

K = (KbT/h)*e^(-Ea/RT). KbT is thermal energy (~4.1x10^-21 J), molecule moving around. H is plank constant ~6.6x10^23 Js. Ea is activation energy [=] j/mol. RT is thermal energy/mol ~2.5x10^3 J/mol. Note: RT = KbT*Na

Receptor-ligand equilibrium

Kd = Koff/Kon. Y = [L] / ([L] + Kd). This is always correct for systems in equilibrium.

Equilibrium constant

Kd = e^(deltaG0 / RT) = koff/kon

Are white blood cells misnamed?

Kinda a trick question, because they don't really live in the blood

Km

Km = (k-1 + kr) / k1 = ( [E] [S] )/ [ES]

Units for Kon

Kon = M^-1s^-1

Cell speed

Lamellipod extension, Contractile force, Relaxation, Displacement of the cell body towards leading lamellipod. Speed and persistence time can vary by orders of magnitude depending on cell function

Histone "tail" determines HIstome

Large number of proteins that interact with histone via the tails. There are at least dozens of proteins (in addition to DNA) that interact with histones.

Typically, set up experiments so that [L]0 >> [P]0. Why?

Ligand costs less than protein (usually protein is more expensive/big). Protein is often on the surface/immobilized and the ligand is free. Chosen so we can assume that [L] ~ [L]0.

Lymphocytes establish first contact by reaching out

Lymphocytes carried in blood or lymph have numerous finger-like structures (called microvilli) studding their surfaces. An idea suggested by SEM images was that homing receptors were on the tips of microvilli. These receptors were identified way later, these receptors were selectins!

Which enzyme's response (MAPKKK MAPKK MAPK) is most switch like?

MAPK! It is a very steep transition from off-> on state. MAPKKK looks like a traditional Michaelis-Menton curve and MAPKK is in between.

Receptor Tyrosine Kinases (RTK)

Major class of cell surface receptors. Ligands for this receptor: soluble or membrane-bound peptide/protein hormones (i.e. growth factors (NGF, PDGF, FGF, EGF), insulin, etc.). The dimerization of the receptor enables activation that propagates the signal in the cell.

Epigenetics by generation

Mice study: there is natural variation that occurs in genetically identical litttermates and this change is due to the mothers diet. In humans: the dutch hunger winter everyone ate very little and scraps to stay alive the long term effects of the famine have be studied and found that the impact on fetuses depended on the stage of pregnancy and even for babies that seemed healthy at birth, something had happened to their development in the womb that affected them - and even their children and grandchildren for decades. Those children grew up to have higher rates of obesity. Another example for humans multi-generational epigenetics is if the mother smokes it affects her, the fetus and the fetus's reproductive cells (3 generations). Men's sperm can also affect the next generatoin (ie smoking).

Assumptions (for fitting?)

Model is correct, experimental noise is normal (Gaussian), all experimental uncertainty is in the dependent variable, no systematic errors, each data point is an independent observation, # of data points > # of parameters to be fitted.

Types of binding

Monovalent: two free enzymes and two free ligands. Multivalent: coupled enzyme and ligand can only bind one at a time. Avidity (improve binding) coupled enzymes and ligands, gives very strong binding interactions

Temperature dependence on binding

Most biological reactions take place at 37deg C. in lab reactions are performed at room temp (25 degC) or in a cold room (4 degC). The cooler the temperature the slower the rate of reaction. Check slide 41 (09/07)

Cell movement in blood

Most of the cells are moving very fast but there are some cells that are moving slowly with a "rolling motion" (along the periphery of vessels). The cells that have slowed down are assessing the surrounding environment trying to find a place to get out.

Multi-Photon microscopy

Multiple, lower energy photons (longer lambda) are used for excitation. Especially useful for imaging live cells and tissues because it reduces photodamage. It penetrates deeper (~1mm). Out of focus emissions are minimized.

Mutant RTK

Mutant RTKs identified in cancers. Signals for proliferation in the absence of growth factors. This is a big research interest in targeting cancer cells.

Does the 11,000 diff genes mean only 11,000 diff types of protein per cell?

NO! There are only about ~11,000 genes transcribed BUT there are more than 11,000 different types of mRNAs, why? This is due to alternative splicing which amplifies genetic diversity into many additional proteins. EX: CD44, a multifunctional adhesion molecule with ~10 mRNA splice variants. ALSO there are post translational modifications that add to genetic diversity. EX: CD44 has ~10 mRNA splice variants PLUS post-translational modifications (e.g. N-glycosylation)

What would be the best method to characterize 50 nm hydrated nanoparticles that can aggregate together?

Nanoparticle tracking! Not fluorescence microscope bc cann't get down with nm. Not DLS because wouldn't be able to see smaller particles, the larger aggregates would take over. Not Coulter counter because wouldbe be able to see smaller particles, only see aggregates. Not SEM because the sample would have to be dry.

Can a model prove a hypothesis?

No, a model can be consistent w/ a hypothesis, but they don't prove (can add strength, show limits)

Hydrophobic interactions

Not an actual bond, structured water around a molecule. When a hydrophobic surface frees itself from solvation there is a gain in entropy. Can be a dominating energy term

Elevator pitch for epigenetics

Nuclear inheritance not based on changes in DNA sequence

Nucleosides

Nucleoside building blocks attached to either a purine (A or G) or a Pyrimidine (C, U, or T) base and with a phosphate backbone can form larger structures. These structures include DNA (double stranded) and RNA ("single stranded", though it folds and couples with itself).

Oligosaccharides vs polysaccharides

Oligosaccharides are small (generally < 15-20 monosaccharides). Polysaccharides can be huge (as big as ten thousand, or more, monosaccharides).

Estimate the number of chemically distinct forms of cD44 that cann be made by human cells

One gene can make 10 different mRNAs. Each mRNA can make a protein that has ~7 different N-glycosylation spots. So each mRNA can produce 100^7 proteins (10^14)

90's controversy over binding partners for tethering /rolling

One method: coated plates with selectins then test the binding of putative ligands. Second method: coat plates with the putative ligands then test selectin binding to the plate. The results were conflicting. The issue? No flow!! While this mechanism actually works in the blood stream where there is lots of flow.

Confocal microscopy

Only a small point is illuminated to eliminate out-of-focus light. Allows clearer images of 3D samples. Imaging is done serially one "slice" at a time (slice with light). Deconvolution techniques can recreate imaged volume (given planes of information and computationally reform image). High-speed techniques for time-lapse, live-cell studies. Colocalization capabilities.

Orientation accuracy

Orientation accuracy as a function of gradient steepness (same concentration): more steep, better accuracy. Orientation accuracy as a function of concentration (same relative gradient steepness): becomes more accurate with increasing concentration but then there is an optimal concentration and past that the concentration is very high that the cell is over stimulated and looses accuracy, biphasic response.

Linear least squares

Output signal y, is modeled as a function of variable x and parameters B. want to minimize the sum of the squared error (to get magnitude of deviation). By solving fo the vector of parameters, B, such that R^2 is at a minimum. Check slide 43 and 45 for equations (09/09)

Super-resolution microscopy (PALM, STORM)

PALM: photoactivation localization microscopy. STORM: stochastic optical reconstruction microscopy. The technique is based on using a point spread function for more precise location and multiple imaging cycles where only a fraction of the fluorophores are on each cycle so that they can be resolved.

PSGL1 is the "host" protein that allows sLeX to bind to P-selectin

PSGL1's full name is P-Selectin Glycoprotein Ligand 1 (there are other binding partners now known for P-selectin, PSGL-1 was the first one discovered). Sulfation is also required for PSGL-1 binding to P-selectin, but this time it is the protein, not the sugar (as was the case for L-selectin) that must be sulfated.

A ligand is diffusing inn a BioMEMS device channel that is flowinng at 1 micron/ s and the channel is 200 microns across

Pe = 2 =~1 neither dominates.

Cell migration paths

Persistence in direction over short time periods. Random direction over longer time periods. As chemoattractant gradient increases, walk is much more directed

Sensitivity analysis: confidence intervals

Pick the most narrow and lowest min interval for best fit. Know that there is a clear true fit for the parameter.

Protein components of ECM

Proteins are the major ECM component by "dry weight". 3 classes of biomacromolecules: 1) GAGs and PGs (they cushion cells and form hydrogels), 2) collagen (in fibril form) (provide mechanical strength), 3) Multiadhesive matrix proteins (ex: aggrecan, fibronectin, laminin basically, 4) inorganic components found in bone.

Opposing enzymes (switch-like)

Reversible covalent modification of an enzyme, catalyzed by two opposing enzymes. The main enzyme's substrate is >>Kd so that it acts with "zero-order" independent of [S].

22nd amino acid

Pyrrolysine, it has a couple other x groups can be considered as a 23rd and 24th amino acids. However this aminno acid is not found in humans (but its founnd in bacteria).

RTK modulation

RTK stimulation modulates many important cell processes including regulation of proliferation, differentiation, promotion of cell survival, changes in metabolism.

Mass action kinetics

Rate is proportional to the frequency with which reacting molecules simultaneously come together

Damkohler Number, Da

Reaction Rate / Convection Rate. Da small = Reaction-limited, bulk flow is fast. Da large = Reaction rate dominates and reaction becomes convection-limited

Thiele Modulus, phi squared

Reaction Rate / Diffusion Rate. thiele small = Reaction-limited, diffusion is fast. thiele large = Reaction rate dominates and reaction becomes diffusion-limited.

What does deltaG = 0 mean?

Reaction is in equilibrium

Enzyme kinetics

Reaction: E + S <- k-1 k1 -> ES -kr-> P + E. There is an assumption of equilibrium (k-1 >> kr), the first steps happens QUICKLY, reaches equilibrium. There is also an assumption of steady-state: d[ES]/dt = 0 when substrate is in excess of the enzyme (enzyme is saturated). The second step is rate limiting: v = d[P]/dt = kr[ES]

Reaction rates

Reaction: aA + bB + ... + zZ -> P. Then the reaction rate is: Rate = k A^a B^b ... Z^z. This is determined by mass action kinetics

GAGs

Repeating disaccharides. There are several types of GAGs all made of repeating monosaccharide dimers. 5 Types included hyaluronan (largest and not sulfated), chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate. Ex: for HA n (repeated units) can be > 20,000 but generally < 100 for other GAGs. GAGs consist of amino sugars and uronic acids (CO2H modified monosaccharides).

Subsensitive enzyme

Requires greater than 81 fold increase in stimulus to drive it from 10%-90% maximal response.

Ultrasensitive enzyme

Requires less than 81 fold increase in stimulus to drive it from 10%-90% maximal response. The upstroke of the stimulus response curve is steeper than that of a hyperbolic Michaelis-Menton enzyme. However, they are relatively less sensitive at small stimuli than MM enzymes. Highly ultrasensitive enzymes behave in an all-or-none, switch-like fashion

Which technique has the best resolution?

SEM, TEM, and AFM have about the same resolution (~ 1nm). So good to consider their other differences (strengths and weaknesses) between these techniques and the types of samples they work on and images they make

Absorbance

Scan the sample across a spectrum (look at different wavelengths, which were absorbed?). Measure light absorbed at each wavelength. For one photon transition: deltaE = hv; electronic transitions: lambda: 200-400 nm (UV), 400-750 nm (visible); chromophores: undergo visible transitions due to double bonds, aromatic groups, nucleic acids, heme, some transition metals. Use beers law to measure absorbance.

21st amino acid

Selenocystein, the "21st amino acid" is co-translationally inserted into polypeptides under the direction of a UGA codon assisted by a specific structural signal in the mRNA. As of 2016, 54 different human "selenoproteins" have been discovered

Leukocyte adhesion after selectin

Several other factors contribute to leukocyte adhesion and extravasation. Cells will suddenly stop, chemoattractant was added that activated integrins cells stop moving (firm adhesion)

Functions of noncovalent forces

Signaling, structure formation, immune recognition, gene expression, metabolism

Histonne acetylation

Simple form: lysines (positively charged) interact strongly with DNA (negatively charged) but histone acetylaitonn "masks" the positive charge, conntributing to the "open" form of chromatin. Acetylation is reversible too. Acetylation covnerts chromatin to the 'open' form because the positive charges of the lysine (K) side chains of histones are masked thus decreasinng their affinity for negatively charged DNA. acetylationn increases gene expression. Acetylation by HATs, deacetylationns by HDs

To overcome homing problem

Strategy #1: biomaterials-based delivery options, have cells embedded in biomaterial and implemented directly to known location. Strategy #2: engineering the cell for homing (if the cell is not encapsulated); advantages of this is simplicity (don't need additional material) and can be used for unknown / inaccessible sites in the body (eg: spinal cord, metastatic cancer), but some challenges include how can a cell be engineered to home to, and stay at, the intended / needed site of action in the body?

Assumptions for receptor kinetics

System is at equilibrium, medium is well mixed, ligand available at uniform concentrations, receptors available uniformly. Number of receptors per cell (Rt = 10^5). Depletion is or is not occurring.

Types of Taxis

Taxis: Directed turning towards gradient - what allows this?. Orthokinesis: Change in movement speed as concentration changes. Klinokinesis: Change in turning frequency as concentration changes. Orthotaxis: Speed is affected by movement direction. Klinotaxis: Random turning affected by movement direction

How is one of many blueprints activated?

The blueprint for one cell type (out of > 400) involves activation of specific signaling pathways: for example, neural differentiation requires Wnt signaling. KEY POINT #1 - by mix and matching various combinations of signaling pathway activities, the > 400 types of human cells are created. KEY POINT #2 - there are factors "outside of", "over", and "beyond" the DNA blueprint (i.e. genetics) that control life.

Implications of the Waddington Epigennetic Landscape

The epigenetic code is the set of epigenetic features that create different phenotypes in different cells. More specifically, ~1000 "housekeeping" genes are expressed in all cells and ~4000 (question this) cell-type-specific genes are also expressed. Most (~70 to 80%) genes are NOT EXPRESSED in a particular cell type

"Expanding the genetic code"

The genetic code usually codes for 20 amino acids. "Redundancy" results in 64 codons that code for only 20 amino acids (usually).

Immune system (briefly)

The immune system works like a single organ but its parts are located in many regions of the body. Integration of the parts occurs through a process called lymphocyte recirculation (or homing). The total circulating pool of lymphocytes is turned over 10-48 times a day.

Non-competitive inhibition (enzyme kinetics)

The inhibitor binds to a different spot on enzyme

DNA methylation

The methyl comes from a SAM molecule and with the help of methyltransferase the methyl is added to a cysteine that "imprints" the DNA so the gene canot be expressed due to tight packing. There are an entire family of MTs with various functions inncludinng maintaining methylation (matches from parent) or de novo methylation (usually during differentiation). All of the MTs work together to determine the "DNA methylome" and imprinting (gene silencing).

What do the PSGL-1 modifications mean?

The modifications a(1,3) fucosylation and a(2,3) sialylation makes the tetrasaccharide a sialyl Lewis X ("sLeX"). Be able to recognize this structure.

Phospholipids assemble into membranes

The phospholipids form a bilayer sheet to form the membrane. Biological membranes have added features such as glycolipids (and embedded glycoproteins). Not counting cholesterol, ~99% of membrane lipids are phospholipids (the other 1% are glycosphingolipids)

Bioengineering - non natural building blocks

The ~20 amino acid limid does hinder protein engineering (artificial proteins). Non-natural amino acids to make proteins can sometimes be useful (example: click reactions)

Histone modification

There are many more histone modifications beyond simple acetylation. Examples are methylation, phosphorylation, isomerization, ubiquitination, and glycosilationn

MAP Kinase pathways

There are multiple MAP Kinase pathways found in eukaryotic cells. Causes different responses like mating, cell-wall remodeling, filamentation, etc. Check slide 21 for more details (09/14).

Enzyme kinetics sensitivity

There are three main categories: michaelis-menton enzyme kinetics, ultrasensitive and subsensitive behavior

Why should cellular engineers be interested in epigenetics

There is a great need for differentiated cells for tissue engineering and regenerative medicine applications. By understanding how "nature" accomplishes this cell differentiation, cell engineers hope to expand their ability to exploit stem cells.

Question everything: one lecture says "typical" cell is 20 um in diameter vs an earlier lecture said average cell is 10 um in diameter

There really isn't any such thing as an average or typical cell because there are so many different cell types. Note that in general, nothing in biology is average. Remember air force pilot cock pit problem (make the seat adjustable don't make the seat for the average pilot)

Atomic Force Microscopy (AFM)

Theres a tip on a cantilever. Tip can scrape a surface or oscillate over a surface. Change in deflection (contact) or frequency (non-contact) of cantilever measures forces. Sample first deposited to a FLAT substrate like mica. Gives a full 3-D scan of the surface topology. Relatively gentle to samples (can have wet, other microscopy is not as gentle). Resolution ~ 1 nm. Biggest disadvantage of this technique is that the Scanning speed is slow (very slow)

Diffusion

Thermal motion of all gases and liquids. Movement from higher concentration to lower concentration. Described by Fick's laws: Flux = J (mol/m^2s) = -D gradientC. D = diffusivity (m^2/s), gradient c = concentration gradient of species (M/m). -gradient J = D gradient^2 c

Enzymes

These proteins are biological catalysts that increase reaction rates by decreasing the activation energy of the reaction. Enzymes do NOT change the thermodynamics but they change reaction kinetics. Enzymes are not consumed by the reaction. Enzymes can be regulated and have high specificity.

Nucleic acids

They code for proteins. They are an information storage and retrieval system. The central dogma of biology is how DNA can be replicated then transcribed to form RNA which is then translated to form proteins. They are also structural and form "materials" (e.g. ribosomal RNA forms ribosomes) and these materials can be natural or made in the lab (DNA origami)

Post translational modificationns

They greatly increase the types of proteins in a cell. So > 11,000 mRNAs then >11,000 cellular proteins and after modifications >>> 11,000 proteome varieties

What do the "active" interfaces of proteins look like compared to the average region of proteins?

They look the same. Other features: enriched aromatic amino acids, avg. 10 bridging H-bonds, avg. 20 water molecules trapped at interface

What are the selectin biding partners

They were suspected to be sugars inn the 90's. P-selectin glycoprotein ligand-1 (PSGL1) is a 240 kDa homodimer consisting of two 120kDa polypeptide chains; it is constitutively expressed on all leukocytes. PSGL-1 is primarily found on the tips of the microvilli. PSGL-1 can bind to P-selectin on the endothelium when decorated with appropriate sugars. The glycans displayed by PSGL-1 must undergo two specific post-translational modifications to function as a counter-receptor for P-selectin: a(1,3) fucosylation and a(2,3) sialylation.

How long will it take a leukocyte to travel from a lymph node to an infected foot based on cells moving through the ECM at ~ 1 cell length per minute?

This would take about 100,000 minutes based on a 1.2 m distance to travel at 12 um / min which is about equal to 70 days (closest guess in poll was 1 JHU length academic term. Luckily leukocytes use blood as a transport medium because it is much much faster. If asked the same question but the WBC were moving through the blood stream then it would be anywhere between 0.5-2 min max (5-6 L/ min blood pumped, at rest, moves all of the blood). The vascurlar is a high speed freeway

Light

Three perpendicular vectors: E - electric field, M - magnetic field, v - velocity of light. Lasers are coherent light of a single wavelength. Light equation: wavelength x frequency = c (speed of light, 3x10^8 m/s). Energy = hv (h is planks constant). Imaging resolution limitation is ~ wavelength / 2

Flux

Tissues are very heterogeneous but We can still typically approximate them as homogeneous with an effective diffusivity, D: Flux = -D d[S]/dx Flux = -D d[S] / dr (one dimension). Flux: Something (molecules, heat, etc.) per area per time. Examples: oxygen, glucose, drug. D for proteins (10-100 kDa) in water: 10^-6 cm2/s. D for proteins (10-100 kDa) in tissues: 10^-7 cm2/s = 10-50 μm2/s

How fast do leukocytes move?

To perform many (most) of their functions, WBCs must move through the body. Leukocytes are "fast" cells with a speed of >= one cell length/minute (which in reality is really slow)

Receptor binding specificity

Total binding of the receptor is often slightly higher than the specific binding to the receptor due to non-specific binding (i.e. due to hydrophobic interactions etc.). Good to add a blocking buffer to separate out non-specific binding.

Lipids (families)

Triglyceride, a fat or oil; Phospholipid; Glycolipid; steroid (e.g. cholesterol)

Common molecule with intrinsic fluorescence?

Tryptophan (amino acid)

Glycans

Typically attached to lipids or proteins. Glycans are sugar structures that are not used for energy. Glycans give structural diversity and change/tune bioactiviy. For proteins: each site of glycosylation typically has many different glycan structures. Example: considering one site of glycosylation there are >100 chemically distinct glycoproteins, with a second glycosylation site >10,000 distinct, adding a third site >1000000 (100^3) distinct proteins.

Experiments with cell migration

Typically take place on a protein coated surface. A gel can be used to release chemoattractant (Under-agarose assay, Transient and steady-state profiles). Time-lapse imaging. Single cell vs. population data. Usually 2-D movement. Mathematical modeling. Important Parameters - Persistent Random Walk: Root-mean square speed, S and Persistence time in a direction, P (t>>P). Check slide 43 lec 9 for equations

Steady state expression of sLeX

Very few cells express sLeX, which is a good thing. For example, hematopoietic stem cells (HSCs) express sialyl-LacNAc but this lacks the fucose to make it be sLeX. "Glycoengineering" sLeX onto HSCs by adding fucose to sialyl-LacNAc dramatically increases transplantation efficiency, decreasing the number of bone marrow donors needed. The numbers involved represent an increase of ~1 to 3 recipients per donor to 100 to 300. Clinically speaking, this is a "Big Thing"

Receptors

Very important for signaling (mechanical, chemical, etc.). They are on the surface of cells and propagate signals in the cell

Water at interface

Water is an active participant. Trade off between the energy to remove water adn the energy of forming new contacts

Van der waals forces (dispersion forces)

Weak interaction at close range (attractive >= 0.4 nm). Induced dipole in non-polar atoms (dipole - induced-dipole, induced-dipole - induced-dipole). EX: used for gecko-inspired bandage

Cell-based therapies - "delivery" of cells

What happens with the reintroduction of cells into a patient? Do they "home" to where they should go? And if they do, do they stay there? -> usually not! (often <1% of cells "home" to the desired location in the body)

Determine the number of receptors complexed

When [L]0 >> [P]0. then [L] = [L]0 - [C] ~= [L]0. Fraction of cell receptors complexed at equilibrium: Y = [L]0 / ([L]0 + Kd). The number of cell receptors complexed per cell at equilibrium: Ceq = Y*Rt, Rt = receptor # per cell

Competitive inhibition (enzyme kinetics)

When the inhibitor binds to the same active site as substrate. Inhibitor, I, binds reversibly and is in rapid equilibrium. At steady state can define both Km and Ki. Ki = [E] [I] / [EI]

When we measure a binding "signal" (y) experimentally, how does this relate to the biological binding event itself, such as fractional biding (x)?

Y = A*x + B. A - constant of proportionality to y. B - nonzero baseline signal

Cooperativity, Hill equation

Y = [L]^n / ([L]^n + Kd). n is the hill coefficient, which increases with increasing cooperativity. The hill equation can be graphically evaluated by a line with slope n

Is DNA methylation reversible?

YES! Methylation can be reversed with TET proteins. This process releases CO2. thinking of waddingtoonns landscape, can think of DNMT activity as creating the barriers preventing lateral movement and TET activity as creating new space and opens gene for expression

When enzyme is saturated

[ES] ~ [E]0, all E is in complex form, this is when there's way more [S] than E. vmax = d[P]/dt = Kr[E]0 = kcat[E]0

~6 different atoms (almost entirely) create the major classes of "small molecules found in humans, these atoms are:

atom - total number Hydrogen - 4.2x10^27 Oxygen - 1.6x10^27 Carbon - 8.0x10^26 Nitrogen - 3.9x10^25 Phosphorous - 9.6x10^24 Sulfur - 2.6x10^24

4 major classes of small molecules function as __________ ______ for synthesis of biomacromolecules

building blocks

Thermodynamics equation

deltaG (free energy) = deltaH (enthalpy) - T*delta S (entropy)

4 building blocks

fatty acids -> lipids amino acids -> proteins nucleotides -> nucleic acids monosaccharides -> complex carbohydrates

Rolling behavior of leukocytes under flow

there seems to be an inverse relationship between flow rate and rolling velocity, low flow rate was high velocity and high flow rate was low velocity. Binding interactions appear to become STRONGER under higher force (like a chinese finger trap) (i.e. leukocyte homing interactions exhibit "catch bond" behavior). There is minimal if any biding without flow, explaining why the 96 well plate assay didn't work (increased flow rate, increased number of cells binding)

Lipids (big picture)

triglycerides,

Total number of atoms in the human body? (guesstimation)

~3.6x10^27 atoms (based on a carbon-based life assumption)

Peclet number, Pe

​​Convection Rate / Diffusion Rate. Pe small = Diffusion dominates (has a smaller time constant). Pe large = Convection (bulk flow) dominates (has a smaller time constant)