Review of Biotechnology and Personalized Medicine PDF

I.​ Introduction stuff (wtv we did for the first 2 days) A.​ NOTE: He said it was not important to take notes but now said that it is 15Qs on the test so I am j reorganizing and rephrasing the slides B.​ mRNA vaccines 1.​ How it works...

I.​ Introduction stuff (wtv we did for the first 2 days) A.​ NOTE: He said it was not important to take notes but now said that it is 15Qs on the test so I am j reorganizing and rephrasing the slides B.​ mRNA vaccines 1.​ How it works a)​ Take mRNA for spike protein of interest b)​ Place it in liposomal vesicles c)​ Your cells will create protein of interest 2.​ Issues with mRNA (not the finished product just with injecting yourself with mRNA) a)​ mRNA can cause immune reaction or be degraded (1)​ Solution was to change a few of the bases (a)​ Modified mRNA b)​ How can you get mRNA into the cell anyways (1)​ Lipid vesicle 3.​ How it compares a)​ Traditional vaccines (1)​ Uses live/weakened or dead pathogen (2)​ Slow to produce antigen of interest (3)​ Immune response stimulated when injected into arm b)​ mRNA vaccines (1)​ Uses mRNA (2)​ Easier to produce mRNA (3)​ Body produces protein of interest and causes immune response C.​ Plants 1.​ GVA a)​ Chemical component found in dill and parsley b)​ Inhibits mitotic division (1)​ Cancer treatment c)​ Not cytotoxic to healthy cells 2.​ Photosynthesis a)​ Photosynthesis was ideal for plants so it did not change much over evolution (1)​ RUBISCO (a)​ Major enzyme in photosynthesis (b)​ Sometimes uses O2 instead of CO2 (i)​ Results in glycolate as byproduct (c)​ Glycolate will spread all around the cell (d)​ Researches changed genetic info to keep glycolate in one part and not all around (i)​ Used tobacco plants for this (e)​ Plant biomass significantly increased b)​ Photosynthesis in mammalian cells  (1)​ NTUs (a)​ Nano Thylakoid units(mini engineered chloroplasts) placed into mice with osteoarthritis (b)​ Condition improved over time (c)​ NTUs cause significant increase in NADH and ATP even in mammalian cells c)​ Plants for drugs (1)​ Biologics (a)​ Drugs made by genetic engineering or plants (b)​ Normally done with mammalian cells (c)​ Elelyso (i)​ Treatment by Pfizer for gaucher disease (ii)​ Uses only plant cells (iii)​ More funding going toward plant biopharmacology D.​ Organ transplants 1.​ Xenotransplantation a)​ Taking the cells or organs from one species and transplanting it to another (1)​ Done with pig kidney (a)​ Pig kidney had genes modified by CRISPR to lower chances of immune rejection (b)​ Receiver of kidney survived for 2 months 2.​ Tissue engineering a)​ Sub discipline of regenerative medicine (1)​ Building tissues in the lab using somatic, embryonic, adult, or STEM cells b)​ Assembles functional constructs that restores, maintains, or improves damaged tissues or whole organs c)​ Tissue engineering has been used to fix internal organs to produce paneth cells (1)​ Paneth cells produce defensins that protect the organ from pathogens (2)​ Missing or damaged paneth cells is a characteristic in intestinal diseases like IBS 3.​ Organoids for research a)​ Multi chamber organoid that resembles the heart has been used for research (1)​ NOT for transplant, used to better understand cardiovascular diseases and heart development 4.​ STEM cells a)​ Mice STEM olfactory cells were placed in rats and restored smell (1)​ Possible to restore degenerated senses in humans via STEM cells  b)​ Regenerating full organs (1)​ Organs can be decellularized and have STEM cells added to regrow the organ E.​ Nanomedicine ​ 1.​ Self boosting vaccines a)​ Vaccines that will give you a booster at specified times b)​ Uses nanoparticles for this c)​ Helpful for places without much medicinal resources so pts do not have to come in constantly for booster shots 2.​ Nanobots used for lung cancer a)​ Tiny magnet controlled robots can go deep into lungs where most treatments can’t b)​ Less invasive than chemo 3.​ CRISPR a)​ Gene editing tool b)​ Was supposed to be used only for lab use but was used on embryo to prevent blood disease c)​ Casgevy (1)​ CRISPR treatment for sickle cell F.​ Synthetic biology 1.​ Creating complex, biologically inspired systems which has functions outside of nature 2.​ Optogenetically responsive mitochondria a)​ Mitochondria that were more light sensitive were developed in vitro (1)​ Mitochondria make more ATP when light shone on it (a)​ Light sensitive photon pump (b)​ In vitro testing showed that organisms with this modified mitochondria live longer G.​ Lab on a chip 1.​ Mimics human organ function on a little computer chip 2.​ Allows researchers to study diseases without harming anyone or anything a)​ Can mimic heart attacks b)​ Replicate BBB H.​ Inherited disease 1.​ DNA tests a)​ Not all are FDA approved b)​ Some offer non diagnostic medical results (1)​ 23 and Me 2.​ Intracellular nuclear injection I.​ Biomarkers ​ 1.​ Biological molecule found in blood or urine that marks normal or abnormal bodily functions a)​ Proteins  b)​ CTC c)​ Cell free DNA (1)​ cfDNA 2.​ Biopsies a)​ Liquid (1)​ Urine or blood (mainly blood) (2)​ Takes a few days for results (3)​ Blood has cfDNA (a)​ Good for diagnosing cancer (4)​ More readily available for patients b)​ Skin (1)​ Takes 6 weeks for results (2)​ Can’t be done at any regular medical office (3)​ Can’t detect cfDNA 3.​ Galleri test a)​ Analyzes cfDNA from 50 types of cancer b)​ Very accurate c)​ Over $1000 d)​ Gets results in 2 weeks 4.​ Theranos a)​ Biomarker company that claimed they can detect blood disorders b)​ Faked their data and was known as a scandal 5.​ CTCs a)​ Circulating tumor cells b)​ Responsible for metastasis c)​ Cynvenio (1)​ Uses Abs to count CTCs (2)​ Vortex (a)​ Improvement of Cynvenio (b)​ Separates CTCs by properties (i)​ WBC and RBC travel down the blood stream quicker, CTCs separated from blood this way and counted (ii)​ Nature inspired (a)​ rivers 6.​ Cancer stem cells a)​ Multiplies indefinitely b)​ Very resistant to chemotherapy c)​ Responsible for relapse d)​ Very low in number 7.​ CTC exosomes a)​ Might be better way of measuring cancer progression than CTCs b)​ Devices for counting CTC exosomes are being made 8.​ iTEARS​  a)​ Collects biomarkers from tears for dry eye diseases b)​ Non invasive c)​ Takes 5 minutes d)​ Not clinically available J.​ Cancer therapies 1.​ Tumor microenvironment a)​ Tumors have their own environment b)​ If we can maintain signaling properly, we can idealize the environment to prevent the tumor from “waking up” and growing (1)​ When CTCs metastasize, they can be dormant for years (2)​ If tumor microenvironment has a lot of collagen type III, it will not reactivate 2.​ Using the immune system a)​ Abs b)​ Modified T cells c)​ Immunomodulators (1)​ Allows cancer cells to signal apoptosis K.​ Biotechnology 1.​ Discipline of developing medicinal stuff with the purpose of benefiting human health and welfare L.​ Personalized medicine 1.​ Treating patients based on their own genetics 2.​ $1000 genome a)​ Maps out your whole genome and determines what treatment is best for you (1)​ Exercise vs diet for weight loss etc. b)​ 23 and Me c)​ Ancestry reports d)​ Trait reports e)​ Etc 3.​ Genesight a)​ Does genetic testing to see how certain medications will interact with you (1)​ Which will work best (2)​ Which will have more side effects for you specifically 4.​ Myriad genetics a)​ Kits predict breast and prostate cancers 5.​ GenomicHealth a)​ Determines how aggressive a patient’s prostate cancer is b)​ Determines best course of treatment 6.​ GINA a)​ Genetic information nondiscrimination act  (1)​ Law passed saying anyone who got personalized medical DNA test done can’t have their results affect their employment or insurance M.​ Orphan/rare diseases 1.​ SMA a)​ Spinal muscle atrophy b)​ Leading genetic cause of infantile death c)​ Can affect whole family d)​ Affects 1/6000 babies worldwide e)​ Caused by faulty genes that affect muscle nerve connections f)​ Treatments for SMA are very expensive 2.​ FOP a)​ Fibrodysplasia ossificans progressive (1)​ Turns cartilage into bone (2)​ Genetic cause known but no treatments available N.​ Bioethics 1.​ hESCs a)​ Human embryonic stem cells b)​ Derived from human blastocysts (1)​ Requires them to be destroyed c)​ Used in STEM cell therapies d)​ Allowed to be used in US clinical trials but can’t receive federal funding if using or destroying human embryos 2.​ Synthetic embryos a)​ In vitro embryos that are used to bypass hESC regulation b)​ Behaves like real human embryos c)​ Can grow a fetus when implanted in monkey (1)​ Is it still considered life? 3.​ Cloning a)​ Dolly the sheep had lots of health issues but none of her clones did b)​ Federally allowed but states can impose laws against cloning O.​ Aging 1.​ Many factors a)​ Mitochondrial health b)​ Epigenetic factors c)​ Telomeres (1)​ Teloyears (a)​ Company that tells you what your cellular age is (i)​ Cellular age is dependent on telomeres d)​ Sirtuin genes (1)​ Longevity genes (2)​ Overexpression of these genes caused 50% increase in fruit fly life span  (3)​ SIRT 6 (a)​ Gene that when overexpressed in mice increased life span by 30% (b)​ Overexpression of this gene results in better usage of energy from fatty acids (c)​ Fewer age dependent cancers and disorders reported in these mice as well (4)​ Resveratrol (a)​ Stimulates human sirtuin genes (b)​ Not for clinical use 2.​ Stem cell theory of aging a)​ Stem cells are better and more plentiful in younger people 3.​ Longeveron a)​ Company that uses mesenchymal stem cells (MSC) for research on potential stem cell therapy for aging 4.​ US military anti aging pill a)​ Works as NAD+ enhancer to get more energy from mitochondria (1)​ Works similarly to sirtuin genes 5.​ Aging can be accelerated or slowed a)​ One factor of aging is epigenetic factors b)​ Chemicals can slow down or speed up aging by acting as an epigenetic factor P.​ Random toxins ​ 1.​ Botulism a)​ Used in botox b)​ Is a muscarinic ACTH inhibitor 2.​ RNAi a)​ Gene silencing b)​ Prevents mRNA coding for certain diseases from getting translated c)​ Used in (1)​ Cancer treatments ​ (2)​ Macular degeneration treatments (3)​ Huntington's disease 3.​ Ricin a)​ Comes from castor bean b)​ Can potentially treat cancer as a conjugate Ab Q.​ Tissue engineering and regenerative medicine 1.​ Tissue engineering a)​ Lab grown tissue that can be used to as a patch for certain areas of the body b)​ Can treat diseases, be used for drug discovery, in vitro toxicology 2.​ Stem cell therapy a)​ Using stem cells to treat certain diseases  3.​ Gene therapy a)​ Using gene transfection to treat certain diseases 4.​ 4 types of stem cells a)​ Adult stem cells (1)​ Most popular type for clinical trials is adMSCs​ (a)​ Fat derived adult stem cells b)​ Fetal stem cells (1)​ Amniotic (2)​ Umbilical cord (3)​ Placental c)​ Embryonic stem cells (1)​ hESCs d)​ Induced pluripotent stem cells 5.​ hESCs vs adult stem cells a)​ hESCs (1)​ Easy to produce (2)​ More pluripotent (a)​ Can differentiate to many dif types of cells b)​ Adult (1)​ More sources (a)​ Hair (b)​ fat (c)​ Teeth (2)​ Analogous (a)​ Can use your own stem cells for yourself (i)​ Can’t do that with hESCs (3)​ Less ethical issues R.​ Conclusion from all this 1.​ Cell biology is a mix of religion, science, government, bioethics, politics, drugs, business, and regulation II.​ Microscopy, Methods, and other stuff A.​ Intro 1.​ Microscope is the foundation of cell bio 2.​ Invented by Hooke in 1650s 3.​ Father of microbiology - Leeuwenhoek (1670s) 4.​ Schleiden, Schwan - Modern cell theory (1830s) 5.​ Choosing a microscope to use depends on methodology a)​ Living cells b)​ Fixed cells c)​ How to process cells etc B.​ Brain 1.​ Reticular theory a)​ Neurons are not cells  b)​ Serve as a vascular system 2.​ Neuronal theory a)​ Neurons are cells C.​ Resolving power 1.​ Light microscope a)​ Plant cells (1)​ 100µm b)​ Animal cells (1)​ 10µm c)​ Mitochondria (1)​ 1µm 2.​ Electron microscope a)​ Viruses, ribosomes (1)​ 100 nm b)​ Proteins (1)​ 10nm c)​ Molecules (1)​ 1nm D.​ Resolution 1.​ Ability to differentiate between 2 different objects 2.​ Light microscope a)​.2µm 3.​ Electron microscope a)​ 2.4 Å 4.​ Abbe’s equation a)​ Equation to determine theoretical resolution (1)​ Distance = (.61 *wavelength) /n sinø (2)​ Generally distance = ½ wavelength b)​ Does not limit super resolution microscopy 5.​ Theoretical resolution a)​ Resolution that is what we get from the equation 6.​ Practical resolution a)​ Resolution we actually get is normally less than theoretical resolution (1)​ Why? (a)​ Cells are mostly water and have little contrast (b)​ Organic compounds absorb light, heat up, and move around E.​ How to get better contrast 1.​ Contrast a)​ Distinguishing between sample and background b)​ More contrast, better details 2.​ Dyes a)​ Colorimetric  (1)​ Absorbs light only at certain wavelengths (2)​ Hematoxylin (a)​ Used to find nucleus (3)​ Eosin (a)​ Used to find cytoplasm b)​ Fluorochromes (1)​ Fluorescent dyes 3.​ Manipulating light a)​ Phase microscope b)​ DIC 4.​ Computer enhancement F.​ Types of microscopes 1.​ Bright field microscopes a)​ Oldest and most often used microscope b)​ Used mainly by pathologists to examine tissue samples c)​ Used to view dead cells d)​ Many steps to using this microscope (1)​ Fixation (a)​ Using chemicals like formaldehyde to cross link proteins (b)​ “Kills” (fixates) the cells (2)​ Dehydration (a)​ Water removed from tissue sample, replaced with ethanol (3)​ Xylene replacement (part of dehydration) (a)​ Replaces ethanol with xylene (4)​ Infiltration (a)​ Removes xylene, sample placed into paraffin (wax) cube (5)​ Microtone (a)​ Cube sliced very thinly (i)​ Sliced kinda in the same way like deli meat (ii)​ Sliced 10-15µm thin (6)​ Stain sample with hematoxylin (a)​ Counter stained with Eosin (7)​ Apply sections to slide e)​ What if you need a sample quicker (1)​ Cryosection (a)​ Quickly freezes sample instead of using paraffin (b)​ Not used normally by pathologists (c)​ Used mainly for Mohs surgery (i)​ Mohs surgery is the surgery to remove skin cancer  (ii)​ Samples are needed quickly to determine exactly where the cancer stops so the surgeons know where to stop cutting out the skin 2.​ Phase microscopy a)​ Looks at living cells (1)​ Non fixated cells b)​ Uses light interference for better contrast c)​ Used mainly by cell culture biologists 3.​ Differential interference contrast (DIC) microscopes a)​ Looks at living cells b)​ Provides 3D image c)​ Used for single cell electrophysiology (1)​ Example: looking at a neuronal response to a drug (a)​ Very slender pipette entered without killing cell and helps measure membrane potential (b)​ Patch clampings (i)​ Monitors inside out and outside out ion flow through a single membrane channel 4.​ Dark Field microscope a)​ Used mainly by microbiologists b)​ Dark background and bright image used for better contrast (1)​ Resolution not changed for better contrast 5.​ Polarizing light microscope a)​ Light passes through light polarizers so only light from 1 plane gets through b)​ Used to detect highly ordered parallel structures (1)​ Neurobiologists use it for detecting microtubeoles (2)​ Muscle cell biologists use it for detecting actin/myosin 6.​ Confocal microscope a)​ Uses several of these microscope aspects into one b)​ Uses computers to take fluoresced cells and make them into a 3D image c)​ Governed by Abbe’s equation but increases the theoretical resolution greatly d)​ Story of confocal microscope (1)​ Patented in 1950s (2)​ Idea matures in 1970s (3)​ First commercial launch in 1988 e)​ Components of the microscope (1)​ Monochromatic lasers (a)​ 1 frequency per laser (2)​ Confocal pinholes (a)​ Narrows the laser to make it more precise and focused (b)​ The image is gathered when the lasers reflect off the structure point by point, computers sum up all the points (3)​ Computer (a)​ Displays the summed image f)​ Advantages (1)​ Less stray image (a)​ Point by point gathering allows for more precise imaging (2)​ Optical sectioning (a)​ Does point by point method in stacked sections which are then stacked to generate 3D image (i)​ The stacks are called Z stacks (3)​ Stereo imaging (a)​ Point by point (4)​ Multiple wavelengths (a)​ Uses several fluorescent dyes at once (i)​ Regular fluorescent microscopy does 1 wavelength at a time (ii)​ Each dye used for each structure wanting to show on image g)​ Problems (1)​ Fluorochromes can photo bleach (a)​ This is just how they are, it is not a problem from the microscope itself h)​ Confocal microscope also has spinning disks (1)​ Easier to use for living cells (2)​ Faster imaging (3)​ Less laser intensity (4)​ Less heat  (5)​ More dynamic 7.​ Vivascope a)​ Not technically a microscope b)​ Uses confocal imaging via a handheld device for skin biopsies c)​ Used by dermatologists d)​ Helps determine point of care e)​ Non-invasive 8.​ Laser capture microdissection (LCM) microscope a)​ Uses a laser to cut off smaller sample from larger one (1)​ Sample size that can get cut off ranges from 7.5-30µm (a)​ Can be individual cell or bunch of cells 9.​ Atomic force microscope a)​ Does not have lenses b)​ Used for surface analysis c)​ Measures and analyzes many forces 10.​Scanning tunneling microscope ​ a)​ Looks at a surface atom by atom b)​ Ultra high resolution c)​ Does not use electron beams or light 11.​Two photon microscope a)​ Designed for deeper tissue imaging b)​ Less phototoxicity compared to other microscopes (1)​ Phototoxicity (a)​ Damage to the sample caused by light c)​ Is preferred over confocal microscopy G.​ Fluorescence applications 1.​ Fluorescent dyes a)​ Excitation wavelength is less than emission wavelength (1)​ This is because wavelength lost due to heat 2.​ Vital fluorescent microscopy a)​ Vital (1)​ Keeps cell alive b)​ Uses fluorochromes to measure and analyze changes in cell behavior c)​ The dye itself is used to fluoresce the cell but would never permeate through the cell (1)​ AM group added to the dye so it can permeate through the cell and fluoresce from within the cell (2)​ Once dye is in the cell, AM group will detach and will cause the dye to fluoresce d)​ Used for (1)​ Mitochondrial activity (a)​ JC-1 dye used  (b)​ Red/orange mitochondria are healthy, green ones are sick (2)​ Live dead assay (a)​ Uses calcein-AM and Propidium Iodide (P.I) (b)​ calcein-Am turns healthy cells green. When cell abt to die, membrane is more permeable and allows calcein-AM to leave and P.I to enter and dyes the nucleus red (3)​ Intracellular calcium levels (a)​ Uses Fluoro 3-AM (b)​ This dye will fluoresce way brighter in response to high calcium levels and can better indicate the concentration of Ca in the cells 3.​ Plate reading spectrofluorometers a)​ Sums and averages the fluorescent signals from all cells in a sample (1)​ Think of the spec in FRI lab (2)​ quantitative (3)​ Important because cells have variability (a)​ Some will produce more proteins than others 4.​ FRAP a)​ Fluorescence recovered after photobleaching b)​ After fluorescing an area of the cell membrane, intense lasers photobleach the fluorochromes, which will be moving around the cell membrane (1)​ Remember FRAP is used on parts of the cell with high membrane fluidity and is used to track how fast the proteins move around the membrane (2)​ Term: lateral fluidity c)​ Over time, the fluorochromes will be recovered but will not be on the same spot they will have moved around the membrane.  d)​ 5.​ TIRF Microscopy -total internal reflection fluorescence - viewing the edges of a cell adjacent to the coverslip a)​ Intracellular injection (1)​ Uses lucifer yellow (2)​ One singular cell will be injected with the dye and the dye will travel throughout the cell via charged ions (a)​ Mainly used by neurologists to see the connectivity of a neuron and exactly where it branches off and which other neurons it is connected to (b)​ Can also be used between 2 non neuronal cells to see if they are connected (i)​ Gap junctions (electrical junctions) serve as an opening for ions between two cells (ii)​ If the dye starts at one cell and travels to the other cell then that means the two are electrically connected (a)​ This can only done vitally (without killing the cell), dye must fluoresce and must diffuse easily without being membrane soluble (otherwise it would leave the cell and enter others via membrane and not junctions) 6.​ Fluorescence immunocytochemistry a)​ Using antibodies to ID proteins in a cell b)​ Limitations to use (1)​ Has to fluoresce (2)​ Has to only bind to one specific target protein  (3)​ Can only bind onto epitope (4)​ Antibodies must be bivalent (a)​ One branch for fluorescent dye the other for binding to antigen (5)​ Has to have high specificity (a)​ Only binds to one protein (6)​ Has to have high affinity (a)​ Binds well to the one specific protein c)​ Direct vs indirect (1)​ Direct (a)​ Antibodies with fluorescent dyes bind to the protein (2)​ Indirect (a)​ Antibodies will detect the antigen and bind to other antibodies with the dye which will bind to the protein (i)​ This method is preferred because it increases affinity and can bind to more proteins (a)​ When attaching the dye to the antibody, it slightly changes the shape and can mess with affinity, which is why indirect method is preferred d)​ Making antibodies (1)​ Polyclonal antibodies (a)​ Made by many different B (spleen) cells in the body (b)​ Typically an animal is injected with the antigen and has several different B cells produce antibodies, blood is collected, and antibodies collected  (i)​ Issues with this is that there is not much specificity as several different antibodies with slightly different structure binds to different epitopes on the antigen, which would mark the wrong protein trying to be tracked, high chance of cross reactivity, supply ends with the life of the animal producing the antibodies (2)​ Monoclonal antibodies (a)​ Mouse is immunized with antigen (b)​ Mouse spleen cells will produce antibodies (c)​ Spleen cells isolated and fused with myeloma and whichever produces most antibodies is replicated Myelomas are cells that are genetically engineered to not be able to make any proteins (such as antibodies) (a)​ This prevents antibodies from being produced by other cells and eliminates the problems that come from polyclonal antibodies (d)​ Cells are placed into HAT medium (i)​ HAT medium will kill any spleen or myeloma cells that have not fused together (a)​ Note: fused cells are called heterokaryon for having 2 nuclei (ii)​ The fused cells will be tested to see which ones produce the most antibodies and work best (e)​ Hybridomas (hybrid cells) are able to be reused as they can be cryopreserved​ (3) Can be used to determine cell polarity (3)​ Cell polarity - intrinsic asymmetry based off shape, structure, or cellular components (a)​ I am not sure the exact mechanism i could not find anything online but this is all he said in class 7.​ ELISA a)​ Enzyme linked immunosorbent assay b)​ Used to quantify protein of interest (1)​ Ex: cells in a culture naturally produce protein X. When we add drug Y, the cells produce more protein X than when the drug Y was not there. But how much of drug Y is used to make protein X? (a)​ If you have done an MIC assay, it is similar but instead of measuring cell inhibition you measure protein production c)​ How it works (1)​ The protein being tested is isolated (2)​ Primary antibodies attach to secondary antibodies (3)​ Secondary antibodies have enzymes that react in the presence of a substrate (a)​ Sometimes the enzymes will turn a different color (i)​ This is the colorimetric approach (b)​ Sometimes the enzyme will fluoresce (i)​ Fluorescent approach (4)​ Sample is placed in the spectrometer and OD adjusted to a certain value  (a)​ The brighter or more vibrant the coloring, the higher concentration of the protein H.​ Cell death 1.​ How does a cell die a)​ Necrosis (1)​ Pathological cell death (2)​ External cause (a)​ Poison (3)​ Causes cell to explode b)​ Apoptosis (1)​ Genetic cell death (2)​ Internal cause (a)​ Chemotherapy (b)​ Radiation (c)​ T-cells (3)​ Cell implodes (4)​ Annexin V (five) (a)​ Tags apoptotic cells (i)​ Bonds to phosphatidylserine which is normally on the inner membrane of the cell (ii)​ When cell starts to undergo apoptosis, it goes to the outer membrane where Annexin V binds (a)​ When Annexin V binds we can see the membrane fluoresce I.​ Fluorescent proteins 1.​ Intro a)​ Fluorescent proteins sort of act like dyes but because they are proteins they are easily synthesized in vitro b)​ GFP (introduced/developed by Roger Tsien) (1)​ Green fluorescent protein (2)​ Found in jellyfish (3)​ Serves as a reporter molecule (a)​ Will fluoresce when something happening in the cell (b)​ 2 types of reporter molecules (i)​ Continuous (a)​ Will always fluoresce (i)​ Ex: putting human cell with GFP into pig blastocyte to grow human organs in the pig (ii)​ All the human organs will fluoresce green because GFP here serves as a continuous reporter (ii)​ Regulated reporter (a)​ Will turn on and off depending on what is happening in the cell (b)​ Ex: gene of interest has GFP coding gene added to it, if cell fluoresces that means gene of interest was coded as well (i)​ Can be used to tell if certain protein is being expressed (4)​ Can be used alongside other FPs with other colors 2.​ FRET a)​ Forster resonance energy transfer  b)​ Tells how far apart 2 proteins are from each other using FPs (1)​ If protein A has FP that fluoresces blue and it is close to Protein b with a yellow FP, the fluorescence emitted would be yellow. If they were not close together it would be blue c)​ Can also be used to measure enzyme activity (1)​ Some enzymes work by tinkering around proteins (a)​ Can connect or distance proteins (2)​ FRET can determine how far apart two proteins are from each other and thus enzymatic activity (a)​ If enzyme is supposed to bring proteins closer tog but FRET is not working then the enzyme is not working like it should (3)​ FRET for kinase (a)​ Kinase is a protein that phosphorylates proteins (b)​ Phosphatase dephosphorylates proteins (c)​ 2 proteins might only come together if phosphorylated (i)​ These two proteins are marked w dif FPs (ii)​ FRET used to see how far apart they are (a)​ If there is a high conc. Of the two proteins together that means kinase is very active and vice versa for phosphatase 3.​ Biosensors a)​ Protein that reveals change in cell behavior (1)​ Calmodulin (a)​ Coils up in the presence of calcium (i)​ Calcium is essential for neurotransmission (b)​ FPs placed at either end of the calmodulin (c)​ FRET measures distance between them (i)​ Higher distance, not as much calcium J.​ Miscellaneous techniques for non electron microscopy 1.​ Autoradiography a)​ Uses radioactive probe to track cell processes (1)​ During Synthesis phase in mitosis, DNA is replicated (2)​ To see which cells are undergoing S phase, radioactive thiamine placed into cell culture and binds to cells in S phase (3)​ Film placed on the culture and radioactive thiamine will release beta particles that will show on the film as a black dot (4)​ This can also be used to detect certain types of cancers b)​ Other uses of autoradiography (1)​ Radioactive leucine and methionine (amino acids) used to track protein synthesis and protein travel around the cell 2.​ FISH a)​ Fluorescence in situ hybridization ​ (1)​ Uses fluorochromes to (a)​ ID mRNA ​ (i)​ Probe is complementary RNA strand with fluorochrome attached to it (ii)​ RNA binds to mRNA (iii)​ mRNA can be tracked (b)​ ID DNA (i)​ DNA is degraded (ii)​ Same steps as mRNA (iii)​ Used to track telomeres of chromosomes 3.​ In situ hybridization in detecting HPV a)​ HeLa cells have HPV (1)​ Serves as positive control (a)​ Group that will show up positive for whatever is being tested b)​ HPV negative cells serve as negative control c)​ Use fluoresced genetic sequencing probe to see if HPV genetic material is present (1)​ If sample fluoresces and so does positive control, HPV positive 4.​ Single cell intracellular injection a)​ Single cell micropipette intracellular injection (1)​ Same idea as lucifer yellow (2)​ Advantages (a)​ Somatic cellular nuclear transfer requires this method (i)​ Somatic cellular nuclear transfer is a cloning technique where nucleus of somatic cell is  placed into egg cell that does not have a nucleus (3)​ Disadvantages (a)​ One cell at a time (very slow if needing to do more than 1) b)​ Electroporation (1)​ Sample is charged with electromagnetic field (2)​ Electromagnetic pulse causes pores to open up in cell membrane and anything from outside the cell can enter briefly (3)​ Can only be used for tough cells (a)​ Weaker more fluid cells could fully rupture c)​ Liposomes (1)​ Uses lipid vesicles to transfect genetic material or other molecules into cell (2)​ Vesicle is positively charged, phosphate heads of membrane are negatively charged (3)​ Liposomes used in mRNA vaccines (4)​ Cons to this method (a)​ Lysosomes in the cell can destroy the liposome d)​ Viral transfection (1)​ Genes added to viral capsid (2)​ Virus infects cells (3)​ Transfects genes and inserts genes into cell genome (a)​ Retrovirus is used for this method (4)​ Issues (a)​ Not FDA approved (i)​ Deliberately injecting yourself with a virus (b)​ Immune system might kill the virus before it reaches the cell K.​ Electron microscope  1.​ 2 main types a)​ Electron transmission microscope (1)​ TEM b)​ Scanning electron microscope (1)​ SEM 2.​ Resolution a)​ TEM (1)​ Follows new equation (2)​ wavelength of electrons = 12.3/sqrt voltage (3)​ d=2.4å (4)​ The shorter the wavelength the better the resolution (5)​ TEM works by transmitting electron s through specimen (6)​ SEM works by “scanning” the sample and giving surface imaging 3.​ Properties of TEM and Light microscope a)​ For easy notes, 1=TEM, 2=light microscope b)​ Illumination (1)​ Electron (2)​ Light c)​ Sections (1)​ 50-90nm thin (2)​ 10-25µm thin d)​ Lenses (1)​ Electromagnetic (2)​ Glass e)​ Imaging (1)​ Screen (2)​ Eyes L.​ TEM techniques 1.​ Plastic thin sectioning a)​ Tissue fixated (1)​ Glutaraldehyde cross links proteins (2)​ OsO4 cross links phospholipids b)​ Dehydration c)​ Infiltration (1)​ Uses epoxy plastic d)​ Ultra microtone (generally a diamond knife) used to cut samples ultra thin e)​ Staining (1)​ Electron microscopes can only detect staining if the stains are from heavy metals (2)​ Lead used for staining membrane (3)​ Uranium used for counter staining (a)​ Staining everything else  (4)​ Note (a)​ Negative staining can also be used (b)​ Negative staining is when everything but the sample is stained 2.​ Freeze fracture a)​ Cell is frozen b)​ Cracked open with a knife (1)​ This will remove outer membrane c)​ Platinum and carbon cover the cell to make a mold of the interior 3.​ Ultrastructural immunocytochemistry a)​ Same idea as immunocytochemistry except instead of dyes, gold particles are used on antibodies (1)​ Gold is relatively cheaper and comes in many sizes (2)​ Will show up on TEM as little black dots (3)​ Double label protocol (a)​ Use gold particles of two different sizes to see it better when it shows as a black dot 4.​ Ultrastructural autoradiography a)​ Ultra thin sample placed in silver halide solution b)​ Radioisotope placed c)​ Slide is fixed and developed (1)​ Includes washing off silver that is not bound to radioisotope d)​ Silver detected on TEM III.​ Cell separation and purification (falls under methods but wanted to space out a bit) A.​ Note 1.​ We will be looking at several different techniques, we are going to say what is the preferred method for insulin receptor separation and purification 2.​ This section will go step by step (each big letter is a different step unless otherwise stated) B.​ Cell dissociation 1.​ Separating clump of cells into several singular cells ​ a)​ This step is not required for homogenous mixture samples 2.​ EGTA​ a)​ Serves as a calcium chelator (1)​ Compromises Ca2+ (a)​ Ca2+ is used by cells to adhere them together (b)​ Compromising Ca2+ will inhibit cells from sticking together 3.​ Protease a)​ Cuts up proteins (1)​ Protease examples (a)​ Liberase (i)​ Used in edmonton procedure (a)​ Separates pancreatic islet cells to treat T1 diabetes (ii)​ Used in cell therapy (b)​ Trypsin (i)​ Used in research only C.​ Cell sorting 1.​ Discarding cells that do not have insulin receptors 2.​ 5 way to do this a)​ FACS (a type of flow cytometry) (1)​ Fluorescence activated cell sorting (2)​ mAbs used with fluorochromes to test for a protein (a)​ Multiple dif mAbs w a dif fluorochrome for each type is used (3)​ (a)​ This is what happens once samples are tagged w fluoresced mAbs (4)​ This method is not used much in general because making mAbs for each specific protein takes a long time b)​ GUAVA (1)​ Analyzes and counts fluoresced cells w out sorting them (a)​ Fluorescent dyes added to culture (b)​ Can count and differentiate up to 12 different fluoresced wavelengths at once c)​ Velocity sedimentation (1)​ Separates cells based on size (2)​ Cells placed in ficoll media (a)​ Media has a distinguishable gradient ​ (i)​ Ex: top has noticeably lower density than bottom part of the media (3)​ Big cells fall through quicker and small ones take more time (4)​ Bottom of tube has collection funnel to let larger cells drop through to separate tube d)​ Panning (1)​ Not really used anymore  (2)​ Protein of interest coats a plate (3)​ Cells will be added to the culture (4)​ Plate is gently shaken and cells with binding proteins will stick to the plate (a)​ Ex: since we are isolating insulin receptors, the plate would be covered in insulin and cells with insulin receptors would stick on to the plate (5)​ Plate is rinsed so non bound cells will fall off (a)​ Issues with this method (i)​ Proteins with high affinity can only be used (a)​ If not the cells might not bind so well and cells of interest can be washed off (6)​ Other way to do panning (a)​ Beads with iron core has antibodies on its surface (b)​ Beads placed in medium with the cells (c)​ Beads with antibodies will bind to proteins of interest on the cells (d)​ Magnets used to pull in beads away from rest of solution with cells attached to beads e)​ Veridex (1)​ FDA approved technique of counting CTC (2)​ Uses anti-EpCAM mAb coated iron cored beads (a)​ Binds to EpCAM which is a protein found almost exclusively on CTCs (3)​ Beads bind to CTC (4)​ Can be used to determine stage of cancer and monitor if the cancer is resolving or worsening for a specific type of cancer being tested (a)​ Note: this is not really used for diagnosing cancer (Galleri test better for that) this is used for monitoring cancer progression D.​ Cell fractioning 1.​ Breaks up cells of interest into different parts 2.​ Cell lysis (many ways to do this) a)​ Sonication (1)​ Disrupts cell membrane (2)​ Would not be used in our case because we want membrane to be in tact b)​ Detergents ​ (1)​ Solubilizes cell membrane (2)​ More details later c)​ Trituration (1)​ Very small syringe pulls cells up and down  (2)​ Shear stress causes cells to rupture (a)​ Membrane would be intact so we would use this in our example (3)​ This method removes organelles 3.​ Differential centrifugation a)​ Sample placed in buffered isotonic solution (1)​ This separates nucleus from membrane (a)​ Remember we are at the point where the sample is a jumbled mess of cell parts (2)​ Supernatant removed (a)​ Top part of the solution (3)​ Supernatant centrifuged and separated by density (a)​ Bottom layer (microsomal fraction) of centrifuged tube has what we are looking for (b)​ 2 parts to microsomal fraction (i)​ Rough (a)​ Has pieces of rough ER (i)​ Contains ribosomes (ii)​ Smooth (a)​ Has pieces of smooth ER (i)​ Plasma membrane and other stuff (4)​ Microsomal fraction placed in ficoll solution with well defined gradient (a)​ Centrifuged to a point of equilibrium density (i)​ This is known as density gradient centrifugation (ii)​ Equilibrium density - a point where everything in the solution is at matching density with solution (a)​ Organelle with density X is located at part of solution with density X (iii)​ Smooth microsome has lower density as rough, will show up on top of solution 4.​ Separating proteins from rest of smooth microsomal fraction a)​ Detergent separates proteins from membrane and form protein micelle (labeled in diagram below as protein detergent complex)  b)​ Sample undergoes dialysis to remove ions that might have been in the protein micelles and at equilibrium will contain only proteins E.​ Protein separation and enrichment 1.​ Things to consider when choosing a method a)​ Do we want functional or non functional proteins b)​ How much do we know about the tested protein (1)​ Size (2)​ Polarity (3)​ Etc 2.​ Liquid chromatography (3 dif ways to do this) a)​ Ion exchange chromatography (1)​ Method of choice when we do not know much about the protein (2)​ Separates protein via charge (very broad approach) (a)​ Most proteins are negatively charged (b)​ Positively charged DEAE bead used to attract negatively charged proteins (c)​ NaCl gradient used to separate (elute) proteins after beads are isolated (i)​ Beads with proteins will pass through low to higher concentrations of NaCl to detach  proteins from beads and proteins can slip through b)​ Gel filtration (1)​ This method is used if the protein of interest is known to be bigger than other proteins in the sample (2)​ Protein sample is placed in porous gel beads (a)​ Small proteins and ions will slip through the pores and cause them to move down slower (b)​ Big proteins do not fit through the holes so they go around and get to the bottom quicker c)​ Affinity chromatography (1)​ Separates protein of interest by using proteins it would interact with (a)​ Ex: insulin for insulin receptors (2)​ Non porous, non charged beads have protein that would interact with protein of interest (3)​ NaCl used to elute beads so they unbind to the protein of interest (a)​ By this point all non protein of interest has been flushed from the sample (4)​ Proteins unsalted to remove NaCl IV.​ Protein purification (still under methods) A.​ Gel electrophoresis 1.​ Uses acrylamide sieve as gel 2.​ Native gel electrophoresis a)​ Proteins remain in their native (unbroken) state b)​ Separates proteins via molecular waves 3.​ SDS gel electrophoresis a)​ Gives better resolution than native (1)​ Note: resolution refers to how well we can see the bands on the gel (a)​ Darker bands mean more protein b)​ Proteins denatured by boiling (1)​ Urea added to break H bonds (2)​ Beta mercaptoethanol added to break disulfide bonds (3)​ Protein now looks like this (a)​ C–––––––N c)​ SDS is added (1)​ SDS is a detergent w a - charge (2)​ Gives linear protein - charge d)​ Because protein is linear and not scrunched up it is easier to see on the gel 4.​ Isoelectric focusing (IEF)  a)​ Gel electrophoresis that uses a pH gradient b)​ Proteins will fall until they reach their isoelectric point (1)​ Point where POI has no net charge c)​ Blood serum can be separated into 40 bands like this 5.​ 2D gel electrophoresis a)​ Gives best resolution b)​ Bands from IEF placed in gel (1)​ SDS is conducted c)​ Can distinguish between phosphorylated proteins and non phosphorylated proteins B.​ Western blots 1.​ Uses gel electrophoresis and blotting paper 2.​ Proteins are placed in gel for gel electrophoresis a)​ Proteins are separated by molecular weight by molecular waves b)​ Proteins taken out of gel and placed onto blotting paper c)​ mAbs added to bind solely to protein of interest (1)​ Technique very similar to ELISA conducted (a)​ Difference is ELISA is done in wells and this is done on blotting paper 3.​ If results needed quicker, use protein simple western blot  4.​ Photoreactive AAs and western blots (I could not understand him so well in class and nothing is showing up on google so fix this up if you can with a comment) a)​ Context: (1)​ When a protein binds with its receptor (insulin to insulin receptor), it can be reversible (2)​ If a covalent bond were to be formed between them, it won’t be reversible (3)​ Photoreactive AAs will form a covalent bond when light shone on them (4)​ We have protein X and it can bind to protein Y, but we do not know what protein Y is, we can use western blotting to see what it is b)​ Protein X placed on western blot without UV light, multiple bands will show up (1)​ Bands for X, Y, and other proteins c)​ UV light will be shone on other western blot (1)​ All the bands should be the same but there will be a new one (a)​ X-Y band C.​ Mass spectrometry 1.​ SELDI-TOF/MALDI-TOF a)​ Characterizes proteins b)​ Has resolution of 1 AA c)​ How it works (1)​ Protein sample ionized and placed on chip (a)​ Chip has chromatographic light surfaces (b)​ Chip can either be charged or have mAbs to keep protein in place (2)​ Laser shines on the sample (3)​ Ions will fly out and hit the detector  (a)​ Smaller ions will hit faster (i)​ Detector measures TOF (a)​ Time of flight (b)​ Molecular weight can be determined using this method d)​ Clinical application (1)​ Beta amyloid detection (2)​ Context (a)​ Beta amyloid 42 causes dementia(in high amts), beta amyloid 40 is normal (b)​ Difference between them is just 2 AA (3)​ Chip surface has mAbs that are used to hold ALL beta amyloids in place (a)​ Epitope is specific to AA chain in all amyloids (4)​ SELDI-TOF used to see if sample has more beta amyloid 40 or 42 (a)​ 40 is lighter because it is only 40AA so it would hit the detector before 42 2.​ Protein A/G immunoprecipitation a)​ Protein A is produced by S. aureus b)​ Protein A can bind to ALL mAbs c)​ How it works (1)​ Add protein A to bead surface (2)​ Place beads into solution containing POI and mAbs (3)​ POI binds to mAbs (4)​ mAbs bind to protein A (5)​ Solution is centrifuged and beads precipitate at the bottom (6)​ Protein eluted and can use gel electrophoresis to see it as bands 3.​ Autoradiography (for protein purification) a)​ Used in gel electrophoresis b)​ Probes we use for this (1)​ S35 methionine (a)​ Tracks newly synthesized proteins (i)​ Disulfide bonds (2)​ P32 (a)​ Tracks phosphorylated proteins c)​ Steps (1)​ Probe added (2)​ Gel electrophoresis done (a)​ SDS is preferred but any method works (3)​ Analyze radioactive bands on gel d)​ This method will only show bands for phosphorylated and newly synthesized proteins e)​ Example (1)​ We want to see what proteins human cells produce when introduced to insulin (a)​ Use autoradiography using S35 as probe (b)​ Control well (well with human cells and no insulin) will show X amount of bands (i)​ The cells will always be producing new proteins so S35 will show even in the control well (c)​ Insulin added to testing well (i)​ Will show same bands as control well AND a few more bands (a)​ These new bands are proteins synthesized in response to the insulin 4.​ Densitometry a)​ Gives quantitative density value from gel electrophoresis instead of qualitative  (1)​ Instead of saying “this band is more down” we are told “this band has density X” V.​ Cell cultures (still part of methods) A.​ Intro 1.​ Terms a)​ Cell culture (1)​ Ability to grow any kind of cell in artificial environment b)​ In vitro (1)​ In cell culture c)​ In vivo (1)​ In living animal d)​ In situ (1)​ In place (a)​ In the cell itself 2.​ History a)​ 1900s (1)​ Brain tested to see if reticular or neuronal theory was true (2)​ Tested in cell culture b)​ 1950s (1)​ George Gey established HeLa cells in culture c)​ 1961 (1)​ Discovered Hayflick limit (a)​ Non cancerous cells will reproduce 25-50x in vitro before apoptosis 3.​ The power of cell cultures a)​ Can work with one single cell type (1)​ Before cultures we used tissue samples that had many dif types of cells b)​ Can generate infinite number of cancer cells in vitro c)​ Can have full control of environment d)​ Can use recombinant DNA technology (1)​ Biologics (a)​ mAbs (2)​ FRET (3)​ GFPs (4)​ Etc e)​ Novel cells (1)​ Cells that can knock in/knock out certain genes in the culture f)​ STEM cell therapies 4.​ Limitations of cell cultures a)​ Cell culture conditions can affect cell phenotype b)​ Cell can change phenotype over time due to genetic drift (1)​ This happened with HeLa cells B.​ MDCK cells 1.​ Kidney cells that are very similar to fibroblasts a)​ Fibroblasts are cells that are associated with creation of conjunction tissue b)​ These cells can rearrange themselves to a different 3D shape and can even revert to what they originally looked like C.​ Hepatocytes 1.​ Liver cells 2.​ Primary hepatocytes a)​ Liver cells that are the first of their line (1)​ Freshly isolated from the liver b)​ Can’t divide in vitro c)​ Used in toxicology studies (1)​ Liver is used to detoxify the blood (2)​ Primary hepatocytes will lose their function over time (a)​ These cells will have 100% viability for first 2 weeks of being in culture (b)​ If we add cytochrome P450 (a toxin) the culture will detoxify it for 1 week but after that will start to die off D.​ Types of mammalian cell cultures 1.​ Cell strains a)​ Cell freshly isolated from the original place (1)​ Primary b)​ Can only divide up to hayflick’s limit c)​ How to get cell strains (1)​ Take tissue sample and add EGTA and protease (2)​ Add cells to culture and repeat step 1 to make a subculture (a)​ Each time we do this it is called a serial pass (3)​ Keep serial passing until hayflick’s limit has been reached d)​ Advantages (1)​ Because they are primary, they are practically identical to the sample it was taken from 2.​ Cell line a)​ Immortal cells that divide forever (1)​ CHO cells (2)​ HeLa cells b)​ Types of cell lines (1)​ Embryonic (a)​ 3T3 (mouse fibroblast line) (2)​ Cancer cells (a)​ 3 ways to get this (i)​ Tumor (ii)​ Exposing cell culture to mutagen  (iii)​ Spontaneous (a)​ Cells in a culture spontaneously become cancerous (3)​ Advantages (a)​ Unlimited number of cells (b)​ Cancer research (4)​ Disadvantages (a)​ Cells are atypical of their original sample (b)​ Cells have defects (c)​ Can’t be used for in vivo studies 3.​ Telomerase immortalized cell lines a)​ Cell strain or line immortalized by transfecting with telomerase to prevent them from shortening during mitosis b)​ Shorter telomeres in WBC associated with higher dementia rates E.​ Growth media 1.​ Serum supplemented media a)​ FCS/FBS (1)​ Used as a growth medium because the serum has growth factors (2)​ Can’t be used if we want to grow cells for cell therapy (a)​ The serum can contain prions and other stuff that can harm us so we do not want to put those cells in us if they grew in that medium (b)​ Cancer cells do not need growth factors (3)​ Serum is also expensive 2.​ Serum free media (defined medium) a)​ Less expensive b)​ Cells grown in this media can be used for clinical applications F.​ Parabiotic mice to see how growth factors work 1.​ Context a)​ Parabiotic means that they share a circulatory system b)​ 3 sets of mice were used (1)​ 2 young (2)​ 1 young 1 old (3)​ 2 old c)​ Each set of mice shared a circulatory system d)​ Set 1 (1)​ One mouse had its leg muscle injured via liquid nitrogen (2)​ The muscle healed because of growth factor e)​ Set 2 (1)​ The old mouse had its leg injured in the same way (2)​ It healed for the same reason  (a)​ Even though the old mouse (which does not produce growth factors) had its leg injured, it healed f)​ Set 3 (1)​ One old mouse had its leg injured in the same way (2)​ No growth factors in either of them caused the mouse to have his leg not fixed up G.​ Growth substates 1.​ Cell culture dishes a)​ Well plates 2.​ Microcarrier beads a)​ Tiny beads that promote cell attachment b)​ Batch settlements (1)​ Is a closed culture system (2)​ used to grow a large number of cells (3)​ biologics can be collected and media will filter back through 3.​ Roller bottles a)​ Bottles that has cells growing in them that are lightly rotated (1)​ Think shaking incubation 4.​ Tubes a)​ Mimics in vivo vascular system b)​ Think BTR 5.​ transcytosis a)​ Movement of molecules from one cell to another (1)​ Lactating mother will have her Abs go from blood stream to breast milk (2)​ Breastfeeding baby will have Abs go from milk to blood stream (a)​ This happens even with vaccine (i)​ For COVID vaccine it happened 6 weeks after mother got vaccinated 6.​ Neurospheres a)​ Free floating cluster of neural stem cells that can differentiate into any kind of neural cell (1)​ Used to treat CNS diseases VI.​ Tissue engineering (still a method) A.​ Intro B.​ Tissue engineering is the usage of cells to generate lab grown constructs or organoids C.​ first tissue engineering in the 1980’s, a terrible start 1.​ the vacanti mouse - a mouse with a bioengineered ear on its back D.​ 3 main sections 1.​ Engineering cells  a)​ CRISPR 2.​ Engineering materials a)​ Growth factors b)​ Scaffolds 3.​ Tissue architecture engineering a)​ Organoids E.​ Scaffolds 1.​ Cells are isolated and then are placed in a porous 3D scaffold a)​ The cells will grow around the pores to be shaped into a certain way to resemble a scaffold (1)​ The scaffold is used for holding surrounding tissue and mimic in vivo functions 2.​ 2 main types of scaffold a)​ Biodegradable (1)​ Temporary (2)​ Surrounding cells will incorporate the scaffold cells as part of tissue and will not need permanent scaffold b)​ Non biodegradable scaffold F.​ Tissue engineered constructs 1.​ Human bladder a)​ A kid had spina bifida and needed a new bladder b)​ Bladder was grown in lab and successfully transplanted 2.​ Engineered liver a)​ ELAD (1)​ Extracorporeal assisted liver device (2)​ Uses cartridges with C3A cells to give your liver a break (a)​ If your liver is suffering from a disease, it would need some time to recover, so the C3A will “take over” the liver’s job so it can recover properly (b)​ We do not use primary hepatocytes for the cartridges because they are very difficult to produce (i)​ They do not replicate in vitro  (ii)​ Would need cells from many many donors just for the cartridges 3.​ Carticel a)​ Autologous knee cartilage regeneration (1)​ Will take your cartilage cells (autologous) and grow them in vitro, will be transplanted back into the area once the cartilage grew in the culture G.​ Human epidermis construct 1.​ Tissue engineered human skin 2.​ Steps to do this a)​ Seed cells in culture with microporous membrane (serves as a scaffold) (1)​ Cells being used should be NHEK (a)​ Normal human epidermal keratinocytes b)​ Increase concentration of extracellular calcium (1)​ Calcium is used for cell adhesion c)​ Lower media level for air liquid interference (1)​ This forms stratum corneum (a)​ Outermost layer of the epidermis d)​ Test the tissue engineered skin (1)​ Structural analysis (a)​ TEM (b)​ Determines if epidermis is structurally correct (2)​ Biochemical analysis (a)​ Uses Keratin for western blot analysis (i)​ Epidermis is made of keratin (3)​ Functional analyses (a)​ Tests if the epidermis functions like skin (b)​ One way to do this is too see how leaky the skin is (i)​ Can some stuff permeate through? Too much? Too little? H.​ Pros to tissue engineering 1.​ We can see how a virus enters your body a)​ Which receptors of which cells allows for your body to get infected (1)​ For COVID, we found it was nasal cells and for the lungs they entered through ACE2 protein I.​ Cons to tissue engineering 1.​ We do not know how to make viable blood vessels and organoids VII.​ Textbook stuff A.​ Chapter 1 1.​ Proteins a)​ Made from amino acids b)​ Enzymes c)​ Structure (1)​ Cytoskeleton d)​ Process (1)​ DNA transcripted into RNA (2)​ RNA nuclease enzyme that does this (a)​ A -T,C-G (3)​ RNA goes from nucleus to cytoplasm (4)​ Ribosome does translation (a)​ RNA read in codons (b)​ Each codon codes for certain amino acid (c)​ Amino acids form peptide bonds (5)​ Transcription factors (a)​ Turns genes “on and off” (6)​ Mutations (a)​ Can sometimes cause wrong proteins to be made (i)​ Codon redundancy prevents this 2.​ Phospholipids a)​ Cell membrane (1)​ Every cell has phospholipid bilayer (2)​ Important for regulating what goes in and out (a)​ Permeable to small polar molecules 3.​ Prokaryotes a)​ Made up of archaea and eubacteria (bacteria) b)​ Very simple cell structure c)​ No nucleus (1)​ Uses plasmids instead (2)​ mRNA just floats around so translation occurs very quickly 4.​ Eukaryotes a)​ Plant and animal cells b)​ Has a nucleus c)​ More complicated cell structure (1)​ Except for human rbc 5.​ Cytoskeleton a)​ Cytoplasm containing fiber proteins to keep organelles in place (1)​ Microtubules (2)​ Microfilaments (3)​ Intermediate filaments b)​ Also helps cell retain its shape 6.​ Nucleus a)​ Largest organelle in animal cells  b)​ Stores DNA c)​ Surrounded by double membrane 7.​ Endoplasmic reticulum a)​ Smooth ER (1)​ Synthesizes lipids (2)​ Smooth because no ribosomes b)​ Rough ER (1)​ Rough because lots of ribosomes (2)​ Protein synthesis 8.​ Golgi Apparatus a)​ Modifies proteins to work properly b)​ Packages proteins and sends them off 9.​ Endosomes a)​ Brings in particles and proteins from outside the cell into the cell (1)​ Cell bulges inward to scoop the proteins (2)​ Endosomes sort through the proteins (3)​ This process is called endocytosis 10.​Lysosomes a)​ Contains enzymes that breakdown unnecessary or dangerous materials (1)​ Lysosomal enzymes work best at low pH (2)​ Lysosome maintains low pH (a)​ If lysosomal enzymes were released into cytosol where pH is higher, enzymes would not work as well 11.​Plant vacuoles a)​ Stores ions water and nutrients in plant cells b)​ Acidic pH 12.​Peroxisomes a)​ Found in almost all eukaryotic cells b)​ Used to degrade fatty acid chains and CO2 13.​Mitochondria a)​ Produces ATP b)​ Cellular respiration (1)​ 28 ATP molecules from one glucose molecule c)​ Endosymbiosis (1)​ Explanation as to why we have mitochondria (2)​ Mitochondria has its own DNA 14.​Chloroplast a)​ Photosynthesis b)​ Has its own DNA 15.​Mitosis a)​ Interphase (1)​ G1  (a)​ Everything but chromosomes are duplicated (b)​ G0 (i)​ If something is wrong, cell goes into arrest (a)​ Will stop mitosis until problem is fixed (i)​ If problem is not fixed cell will undergo apoptosis (ii)​ If cell has problems but does not arrest, it can cause a tumor (2)​ S (a)​ Synthesis phase (b)​ Chromosomes replicated (3)​ G2 (a)​ RNA, lipid, protein synthesis (b)​ Fixes any mutations during DNA replication (4)​ M (a)​ Mitotic phase (i)​ PMAT (a)​ Not discussed in the book as of now 16.​Model organisms a)​ Single celled eukaryotes that did not change much over time b)​ Best for research due to broad applicability to other cells c)​ Yeast (1)​ Used to study fundamental aspects of eukaryotic structure and function (2)​ Can be haploid or diploid (3)​ Mutations in yeast led to finding key cell cycle proteins d)​ Algae (1)​ Used to study brain function (a)​ How algae responds to Ca2+ 17.​Miscellaneous a)​ multicellular organisms need cell-cell and cell-matrix adhesion b)​ Cells make up tissue which makes up organs c)​ More DNA ≠ more proteins to code for (1)​ 90% of human DNA does not code for proteins (2)​ Humans have 30x more DNA than roundworms but make same amt of proteins d)​ Genomics (1)​ Study of DNA sequencing and DNA mapping (2)​ Phylogenetic trees (3)​ Helps find common ancestors based off common DNA sequencing e)​ Metazoan organisms  (1)​ Early group of eukaryotes that underwent cell differentiation and organized cells into tissue and organs (2)​ All multicellular eukaryotes with mitochondria are metazoan (3)​ Used heavily in research (a)​ Metazoans are somewhat closely related to each other (b)​ Some metazoans hardly changed over evolution and can serve as a “broadscope view” of adaptations over time and applicability of findings to other metazoans B.​ Chapter 3 (skipped a lot for now) 1.​ Intro a)​ Amino acid chains will either fold into one unique shape or a few similar shapes (1)​ Similar shapes are called conformations (2)​ Happens when other molecules or ions interfere with non covalent or covalent bonds b)​ Protein function dictated by protein shape 2.​ Hierarchy of protein structures a)​ Primary structure (1)​ Linear amino acid chain (2)​ Peptide bonds (a)​ N from aa on right side bonds with C on left aa (3)​ Peptides (a)​ Chain consisting of less than 30aa (4)​ Polypeptides (a)​ Chain consisting of generally 200+ aa (i)​ Longest protein is 34k aa long (ii)​ Protein mass measured in dalton (a)​ 1 dalton = 1amu b)​ Secondary structure (1)​ The alpha helix (a)​ Aa forms a spiral structure (b)​ H and O form H bonds to keep the structure (2)​ Beta sheet (a)​ Short aa chains that are perpendicular to the helix (b)​ Held by H bonds (3)​ Beta turn (a)​ 4 aa forms a U shape at the surface of the protein (b)​ Helps fold proteins into compact structures (c)​ Held by H bonds at the ends of the U structure c)​ Structural motif (supersecondary structure)  (1)​ Combination of 2+ secondary structures that serve as a functional protein d)​ Tertiary structure (1)​ Hydrophobic interactions along protein side chains (a)​ Van der waal forces, disulfide bonds (sometimes) and H bonds (b)​ Weak inner connections cause the protein to fluctuate shape slightly (2)​ 3D restructuring of the protein from secondary structure (3)​ Domains (a)​ Regions of a protein structure (b)​ Functional region (i)​ Part of protein that exhibits certain activity (c)​ Structural domain (i)​ Area with 40+aa (ii)​ Stable structure with at least one secondary structure or structural motif (iii)​ Structural domain can also be a functional domain (d)​ Topological domains C.​ Chapter 4 1.​ Cultures a)​ A place for cells to grow in vitro b)​ Must have solid surface and media for nutrients for animal cells (1)​ Bacterial and yeast cultures can grow in liquid media c)​ Cell cultures and strains have finite life span (1)​ Oncongenically transformed (mutated) cells can grow indefinitely (a)​ HeLa d)​ Stem cells in cultures can differentiate and form organoids 2.​ Deconvolution a)​ An image processing technique that improves contrast and resolution in computer imaging for microscopy 3.​ Optogenetics a)​ Using light to activate or deactivate membrane channels in the cell membrane (1)​ Can “turn on or off” certain cell processes (2)​ Idea for this came from phototaxis (plant attraction to light) 4.​ Point source fluorescence ​ ​ a)​ finding a single fluorescently tagged object via super high resolution camera (1)​ Images multiple photons to create a Gaussian curve of fluorescence intensity  (a)​ Better outlines the object at higher resolution than what is normally possible 5.​ Cryo Electron microscopy a)​ Used for electron microscopes b)​ Aqueous solution of sample is frozen in liquid nitrogen (1)​ Being frozen lets us see with the electron microscope without staining or fixing or dehydrating c)​ Microscope takes millions of point by point images and makes 3D model of the sample (1)​ Like confocal microscopy 6.​ Isolating cell organelles a)​ Disrupting cell (1)​ Salt water will cause it to shrivel up and flood out its contents b)​ Centrifugation (1)​ Separates whatever is in the solution by density c)​ Monoclonal antibodies can bind to proteins found solely on organelle specific proteins d)​ Proteomics used to find out protein composition of organelle (1)​ Proteomics discussed in chapter 3