Principles of Cell Bio Exam 1

MALDI-TOF

(Matrix Associated Laser Desorption Ionization - Time of Flight) Mass Spectrometry

SELDI-TOF

(Surface Enhanced Laser Desorption Ionization - Time of Flight)

1674

- AVL - protozoa, bacteria

Conjugated antibodies

- Can bind to only cancer cells and then selectively kill them

TIRF Microscopy

-addresses another issue with confocal microscopy -intracellular injection of fluorochromes to trace neurons or analyze cell-cell junctions example: Lucifer yellow can be used to trace neurons neuron non neurons i.e. epithelial cells gap junctions: pass small molecules like Ca2+, AMP, etc

Growth Media Requirements

-serum supplemented medium e.g. 10% fetal bouine serum (FBS) contains growth factors most cell cultures: not good for stem cell therapy -defined medium all is known, no FBS critical for stem cell therapy -Standard Cell Culture Dishes ranges from one well to 384 well dishes

plastin thin selecting technique for TEM

-tissue -fix with glutaraldehyde to cross link proteins, OsO4 to cross link lipids -dehydration -infiltration epoxy plastic -Ultramicrotome, sections 50-90 nm thin -sections - suspended in a copper grid -stain with lead to stain the membrane, and uranium which is radioactive and acts as a counterstain

LM - resolution is about x microns

0.2 microns

using standard western blots with two lanes only:

1 is control and 2 is experimental. use monoclonal antibody against X a band on X that is not on Y will be the antibody with X which u can take out to analyze the antibody

$2 million therapy

10,000 cases annually in US qualifies SMA (Spinal Muscle Atrophy) for being an "orphan disease" (Orphan Act of 1983 grants special patent privileges). <200,000 cases in US to qualify as orphan disease. SMA - Leading genetic cause of death in infants and can affect an entire family. SMA affects 1 in 6,000 babies born worldwide each year Caused by deleted and defective genes whose normal protein products are required for proper muscle-nerve connections....but there is hope.. Biogen's "Spinraza" costs $750,000/yr and is an antisense oligonucleotide that blocks the function of the defective gene through blocking protein synthesis Novartis developed "Zolgensma" to treat SMA which is a $2M gene therapy product that introduces the good gene into the body via gene therapy. 2021 - $1.35b in net sales of Zolgensma. But.......... Dateline: 8/15/2022 - Two patients died of liver failure due to Zolgensma orphan/rare disease: legally defined differently in each nation: in america, less than 200 cases in the US important to define because it helps with money and resource allocation SMA: normally, neurons interact with muscles. with SMA, the neuron degenerates and the muscle must be innervated, otherwise it'll degenerate too very rare orphan disease biogen: boston based nation, Spinraza is a drug that's antisense (type of RNA that binds to mRNA not allowing it to transform into a protein), blocks function of defective genes. we want to have a good gene, not just block the bad ones

what measurement do you have to use a light microscope

10-15 microns

Herr Abbe

1873 released a scientific paper describing the mathematics leading to the perfection of this wonderful invention. -aberration, diffraction and coma were described and understood

confocal microscopy

1957- first patent, an "idea" 1970s - Idea developed further 1988- Fist confocal microscopy commercially appeared

fluorescence microscopy

1960s- fluorescence microscopy depends on fluorochromes fluorescent excitation 𝝺: 485 nm —-> heat emission 𝝺: 530 nm —> heat 1970s - popular

vital fluorescence microscopy and spectrofluorimetry

1980s variety of fluorochromes that can based on changes in emission intensity of color reflects change in cell physiology problem dye wouldn't enter cell solution: dye - am can enter cell! because of the esterases (enzymes), the dye is cleaved off

Engineered human skin construct

1980s: can we build the first human skin tissue engineered construct human epidermal lectrotirocytes

2D gel electrophoresis

2-D gel electrophoresis - best resolution, combines IEF and SDS gel electrophoresis IEF lots of bands on tubular gel, tight bands with highest resolution then run SDS, run proteins based on two different approaches distinguish phosphorylated proteins from non phosphorylated protein

Dateline: 2021/2022 - Antibodies from COVID positive or mRNA vaccinated mothers appear x and x week in breast milk

4 to 6 weeks

SARS-CoV-2 Infections Under the Nose of New Organoid Model"

A human nose organoid has been developed to study how the first events of a viral infection take place and the complex interactions between the host and virus. Using the tissue engineered model, researchers showed differences between infections caused by SARS-CoV-2 and RSV (Respiratory Syncytial Virus). The study provides an advance in the development of a novel tissue engineered nose organoid model and in the understanding of the host cellular response to respiratory infections

Nanotechnology for Isolating Disease Biomarkers from Exosomes in tears

A nanomembrane system that harvests exosomes from tears named iTEARS has been developed Collecting exosomes from tears is less invasive than from blood and may predict diseases, etc. and only takes 5 minutes. Can detect different types of dry eye syndrome, and others. Not in the clinic yet.

Liquid Biopsies and Alzheimer's Disease - Amyloid β42 can be distinguished from amyloid β40 with SELDI-TOF

A new study reveals that the brain's ability to clear the main ingredient of Alzheimer's plaques (red - amyloid beta 42) slows with age. The findings could help explain why risk of the disease increases with age. The 1-40 fragment is normal and shorter than the 42 by two amino acids.

Carticel

A product of Genzyme Biosurgery cartilage

Human Brain Organoids Implanted into Mice Connect to Animals' Cortex and Respond to External Sensory Stimuli"

A research team says it has shown for the first time that human brain organoids implanted in mice have established functional connectivity to the animals' cortex and responded to external sensory stimuli. The implanted organoids reacted to visual stimuli in the same way as surrounding tissues, an observation that researchers were able to make in real time over several months thanks to an experimental setup that combines transparent graphene microelectrode arrays and two-photon imaging.

Limitations of Cell Cultures: Dateline: 2/9/2022 - Technology Networks Cell Science - "Cell Cultures Are Not As Stable as You'd Think"

A team of researchers led by marine ecologist Carlos Duarte and bioscientist Mo Li monitored the environment of cells grown in flasks placed in a controlled incubator — a standard cell culture method called batch culture — over a three-day period. Three different cell types were used: human pluripotent stem cells, a cancer cell line and a type of white blood cell. Optical sensors were attached to some of the flasks belonging to each cell type in order to monitor changes in dissolved oxygen and carbon dioxide levels as the cells grew. Other flasks were removed every eight hours and then discarded after measuring cell growth rates and culture acidity.

Fetal Stem Cells

Amniotic, umbilical cord, placental But as of June 2019 fed funds can't be used

Mechanisms in Aging Process Illuminated by Light Powered Mitochondria"

Another example of synthetic biology is the recent report of developing "optogenetically responsive mitochondria" to generate more ATP from mitochondria. Cells were generated in C.elegans (translucent roundworm - image below) that have a light sensitive proton pump in the mitochondria that when illuminated is similar to recharging rechargeable batteries. When the light is on more protons are pumped by the optogenetically designed proton pump and more ATP is generated So what happens to these C.elegans? They are healthier and their lifespan is extended. The researches compare this to a "solar panel system" for mitochondria.

A key Requirement for Tissue Engineered Constructs

Biodegradable or Permanent Biological Scaffolds -biodegradable -non-biodegradable

Liquid Biopsy versus Traditional Tissue Biopsy

Blood biopsy analysis takes a few days, not up to 6 weeks - much better for the patient! Blood contains cfDNA from dead and recycled cancer cells - a very promising field Availability of biopsy services is more limiting for tissues than blood (any clinic can draw blood)

types of microscopes used by cell and molecular biologists

Bright Field Microscopy Phase Microscopy Differential Interference Microscopy Darkfield Microscopy Polarizing Light Microscopy Confocal Microscopy Fluorescence Microscopy Electron Microscopy

Plants as mRNA Factories for Edible Vaccines"

CHO (Chinese Hamster Ovary -below) and HeLa are the preferred cells for the clinical productions of mAbs Univ Of Ca. Riverside researchers are trying to address the current problem of Moderna and Pfizer's requirement of keeping the current mRNA vaccines cold/frozen.

Chinese use Nanomedical editing techniques (CRISPR-Cas9) on pre-implantation Human Embryos

CRISPR-Cas 9 was discovered in 2008 intended for lab use but...the team attempted to modify the gene responsible for β-thalassaemia, a potentially fatal blood disorder, using a gene-editing technique known as CRISPR/Cas9. Concerns now that Eugenics may happen in the future

Vital Dyes used for Fluorescence Microscopy

Calcein-AM (green) Propidium Iodide (red)

A rough start to Tissue Engineering

Called the "Vacanti Mouse" given Charles Vacanti created it in 1997

biomarkers

Can be in the form of a molecular marker (e.g.protein), circulating tumor cells, cell free DNA, exosomes or microvesicles that indicate the status of a possible or pre-existing disease state sampled via "liquid biopsy" (blood, urine or cerebrospinal fluid)

Tissue Engineering for Cancer Research: Pancreatic Tumoroids

Can be used in 3D screening to identify drugs that might target pancreatic cancer or secreted biomarkers

Immunomodulators -

Cancer cells have a "don't kill me" signal that prevents our immune system from killing tumor cells. New drugs now block this don't kill me signal.

1872

Carl Zeiss makes the compound microscope, first optical microscope company in the world

Types of Cell culture

Cell Strain Cell Line Telomerase-immortalized Cell Line/Strain

Cell Strains - Leonard Hayflick and the Hayflick Hypothesis (Limit) Proposed in 1961

Cells in culture have three phases (see below) Human somatic cells can divide a maximum of 50 times and then they die There is an indirect relationship between the donor age and the maximum number of potential doublings

How Migrant Cancer Cells Sleep for Years before Reawakening

Cells that leave an original or 'primary' tumor can remain in a non-cancerous, 'dormant' state for years before awakening to generate tumors at other locations in the body—a stage in cancer progression called 'metastatic cancer'. The authors show scattered cancer cells remain in a dormant state preventing the recurrence of metastatic tumors if the environment of these cells is enriched with Type III Collagen - an extracellular matrix protein. coats the cell-not metastatic-. when the cell stops synthesizing When the scientists increased the amount of Type III Collagen around cancer cells that had left a tumor, cancer progression stalled and the disseminated tumor cells maintained a dormant state. But when the tumor cell stops making Type III Collagen it converts from dormant to metastatic. So how can we use this information as a means to target dormant cancer cells? That is the "million dollar question."

cell biomarkers can be

Circulating Tumor Cells (CTCs) and Cancer Stem Cells

Macular Degeneration

Clinical Trials with RNAi was the first medical application-as of 8/19, 150 clinical trials underway/completed using RNAi

Anthony Atala's Group

Clinical implants of tissue engineered neovaginal organs using biodegradable scaffolds, autologous muscle and epithelial cells - Mayer-Rokitansky-Kuster-Hauser syndrome

Teloyears

Company dedicated to Reading your Telomeres and Comparing your "cellular age" to your chronological age. From TeloYears website: Some people look and feel older than they really are, while others look and feel younger. This can be attributed to what's known as cellular aging. Your diet, lifestyle, your levels of stress, your exposure to toxins—even how much you sleep—can affect your cellular age. And TeloYears uses leading-edge technology to look deep inside your cells to read your cellular age. We then use that information to help you develop a personalized gameplan, based on cellular information found in your DNA to help you Because the length of your telomeres has been shown to associate with how long you'll live and how healthy you'll be along the way.

Old mice grow young again in study. Can people do the same?

David Sinclair's lab at Harvard Medical School has shown that aging is reversible in mice (see below)! It can be accelerated or reversed. Aging is driven by epigenetic changes to the genome due to age dependent double-stranded DNA breaks (epigenetics is the non base change in DNA function such as DNA methylation, Histone phosphorylation) This loss of epigenetic information accelerates the hallmarks of aging These changes are reversible by epigenetic reprogramming By manipulating the epigenome, aging can be driven forward and backward

biotechnology

Diverse set of disciplines designed to develop new medical devices, drugs, biomarkers, engineered cells, etc. for the purpose of improving human health and welfare CPSI Biotech is located in Owego, NY Recently developed a new clinical cryosurgical device that will be in the clinic soon. Works on super freezing target tumors such as pancreas

dolly and cloning

Dolly was born at Edinburgh University in 1996 - 27 yrs ago Dolly was euthanized at age 6 due to lung disease and advanced arthritis but why? But other identical clones from the same cell line (Debbie, Denise, Diana and Daisy) are fine! But is human cloning legal or ethical? No federal laws against it yet but some states have laws forbidding it such as NYS (2019-2020 Legislative year!). Using discarded human embryos for stem cell therapy is legal in US and in clinical trials worldwide (therapeutic cloning)

Fully Functioning Artificial Human Heart Muscle Developed

Duke University developed a beating patch of heart tissue (see youtube video below). from iPSCs (induced pluripotent stem cells) This means that in the future you could have this done from your own cells - no immune rejection. Once heart tissue is dead it can't regrow from the same heart tissue.

Cell Separation Requires ?

EGTA and a Protease such as Trypsin (research only) or Liberase (cell therapy such as pancreatic islets transplantation) step 1: tissue dissociation = getting single cells dissociated from tissues -EGTA: calcium chelator, disable Ca2+, dependent cell adhesion molecules -protease (like trypsin, collagenase, liberase: cleaves proteins, extracellular matrix edmonton procedure: transplanted pancreatic islets for diabetes step 2 - cell separation/purification

Dateline: 1/27/2021 - REGEN-COV™ ANTIBODY COCKTAIL IS ACTIVE AGAINST SARS-COV-2 VARIANTS FIRST IDENTIFIED IN THE UK AND SOUTH AFRICA

Eli Lilly's monoclonal antibody, called bamlanivimab, received an emergency use authorization from the US Food and Drug Administration in early November 2020. Less than two weeks later, the agency granted an EUA to Regeneron's monoclonal antibody cocktail, made up of two monoclonal antibodies, casirivimab and imdevimab, and called REGN-COV2. It was given to President Trump when he got infected. According to the FDA, monoclonal antibodies should be given as soon as possible after symptoms emerge and a person tests positive for infection. And, because of limited supply, the authorizations are limited to high-risk patients, such as people 65 and older, those who have a BMI (body mass index) of 35 or greater and those with other health conditions like diabetes, cardiovascular disease or chronic kidney disease How do mabs work in this regard? They bind to the spike protein preventing the virus from binding to the ACE2 receptor

Elizabeth Holmes

Elizabeth Holmes, the former CEO and founder of failed blood testing startup Theranos, was found guilty on four charges of defrauding investors, capping off the stunning downfall of a former tech icon. Holmes began courting the press with claims that Theranos had invented technology that could accurately and reliably test for a range of conditions using just a few drops of blood taken from a finger prick. But it was all fraudulent. She started Theranos at the age of 19 after dropping out of Stanford and was able to get outside parties to invest $950M. But a whistleblower who was a former employee said that the data was fraudulent. She was sentenced in November, 2022, to 11 years in prison

Abbe's Rule now Overcome

Eric Betzig, Stefan W. Hell and William E. Moerner are awarded the Nobel Prize in Chemistry 2014 for having bypassed a presumed scientific limitation stipulating that an optical microscope can never yield a resolution better than 0.2 micrometres. Using the fluorescence of molecules, scientists can now monitor the interplay between individual molecules inside cells; they can observe disease-related proteins aggregate and they can track cell division at the nanolevel.

Success of Tissue Engineered Human Bladders reported in April, 2006

Examples human bladder: TEC spina bifida: bladder is defective transplants: auto grafts ELADS extra corporal liver assist devices C3A cells: hepatoma cell line 50% cytochrome p450 activity cell line → grow locs carticel cartilage - autographer other models in vitro toxicology cardiac patches cardiac bandaids lab grown tissue engineered device is being tested with people who have specific cardiac infarctions clinical testing in europe not the US tho

Fighting Cancer with a Pinch of Parsley and Dill

Extracts from parsley and dill seeds are a new source of a precursor of GVA - an anti-mitotic drug now more easily synthesized GVA inhibits the growth of tumor cells by halting cells in mitosis and is not cytotoxic to normal cells CellInsight CX5 High Content Screening (HCS)

Methods-Cell Separation, FACS, a type of Flow Cytometry

FACS = fluorescent activate cell sorting

FRAP

Fluorescence Recovery After Photobleaching Can Reveal Exchange Rates or Membrane Fluidity relies on photobleaching lateral fluidity of cell membrane protein other applications green on outside

nanomedicine

From nanoparticles to Immuno-oncology

FRET

Förster Resonance Energy Transfer -Can signal when two molecules are within close proximity -looks at protein-protein interaction, 1-10nm protein -relies on select membrane of the GFP family to signal protein-proein binding to GFP

human pig embryos

GFP labeled human cells are tracked introduce human cells into the pig blasocyst, use GFP to tell what protein it is humanize pigs to make liver cells that have more human cells in them

Fluidically linked Blood-Brain Barrier and Brain Organ Chips offer new method for studying the effects of drugs and disease on the brain and its blood vessels

Goal is to make 10 different "lab on a chip" human mimetic "organs" and hook them up together so that they can work for 4 weeks They reported a blood brain barrier (BBB) 3 chip system (below) that can be used to study BBB functions

Microcarrier Beads - Designed for

High Cell Density

Personalized Medicine Requires Protection to Human Subjects - Informed Consent

History: Henrietta Lacks and HeLa cells - 1951 GINA: Genetic Information Nondiscrimination Act - 2008. Enacted after Venter sequenced the human genome for the first time in year 2000 but before the $1000 genome was available. States that your genetic information can't be used as a criterion for employment or medical insurance HeLa: unknowingly gave up her cancer cells we need to protect people's rights and their genomes downside: company knows your genetic sequence so insurance is shitty info can be used against you

Column Chromatography

Importance of NaCl

DIC

Important for Single Cell Electrophysiology and Patch Clamping

The Process of Generating Monoclonal Antibodies wasn't Patented!

In 1975 Kohler and Milstein gave a presentation of the process of making mABs with a paper pending in Nature Vickers realized its importance and tried to patent the process before it was published in Nature but the UK patent process was too slow and the paper was published thus preventing patent filing This case was a big concern with Prime Minister Margaret Thatcher getting involved as well who was a chemist by training.

red

J-aggregates = high PMF

resolution of TEM

Lambda = 12.3/sqrt(V) in Angstroms 100,000 Volts = 2.4A

longeveron

Longeveron - Cell Therapy from Young Healthy Donors - now in Clinical Trials Longeveron is a life sciences company developing biological solutions for aging and aging-associated diseases through the testing of allogeneic human Mesenchymal Stem Cells (MSCs) that are derived from the bone marrow of young, healthy donors. The Longeveron-grown Mesenchymal Stem Cell (LMSCs) product is manufactured in Longeveron's cell processing facility located in Miami, Florida Currently there are no FDA-approved allogeneic mesenchymal stem cell treatments in the United States. LMSCs are currently undergoing rigorous testing for safety and efficacy in Phase I and II clinical trials. Longeveron hopes to be the first to offer stem cells as a safe, effective, FDA-approved treatment for some of the world's most difficult chronic and life-threatening conditions such as Aging Frailty, Alzheimer's disease and Metabolic Syndrome metabolic syndrome, faulty, alzheimer's if you volunteer, they get your stem cells by doing a lipid suction take out fat and purify stem cells

Time Release Microparticles Could Deliver "Self-Boosting" Vaccines

MIT bioengineers have created microparticles containing vaccine that could be implanted under the skin and "time-tuned" to release several "shots" in a process called "self-boosting vaccines" They are given only once and designed to "boost" spontaneously at specified times. They resorb over time like degradable sutures. One important advantage of this technology would be for childhood vaccines in regions where people don't have frequent access to medical care. And yes, this is in the world of cell and molecular biology as well

Dateline: GEN; 8/31/21 "Self-Injecting Pill Could Allow Oral Delivery of Monoclonal Antibody and Other Protein Drugs"

MIT engineers, in collaboration with scientists from Brigham and Women's Hospital and Novo Nordisk, are developing a drug capsule technology that could allow the oral delivery of monoclonal antibodies, or other large protein-based drugs that normally have to be injected, for diseases ranging from cancer, to rheumatoid arthritis, to Crohn's disease. The new technology is a type of self-injecting capsule, called a liquid-injecting self-orienting millimeter-scale applicator (L-SOMA), which is swallowed, and then effectively injects the liquid medication directly into the stomach wall. The pill, about the size of a blueberry, has a high, steep dome inspired by the leopard tortoise. Just as the tortoise is able to right itself if it rolls onto its back, the capsule is able to orient itself so that its needle can be injected into the lining of the stomach. Works in animals with insulin and mABs!!

Is cell and molecular biology at a point where we can understand and stop/reverse aging?

Maybe - Many factors may be involved one of which may be the sirtuin genes (referred to as the longevity genes). Overexpression increases max lifespan of fruit flies to round worms by up to 50% Stem cell theory of aging (clinical trials - Longeveron) - Young folks sourced stem cells are better and more plentiful But other factors (mitochondria age and health, epigenetics) are probably involved as well.

CellSearch by Veridex

Method for Detecting low number (1 in a billion) Circulating Tumor Cells (FDA approved) other systems e.g. Veridex, CellSearch FDA approved for counting CTCs works by using antibodies coupled to iron nanoparticles that are sorted out and collected using magnetism

Microfluidic Flows Enter Medical Mainstream

Microfluidic devices that can capture CTC exosomes are being developed and tested.

Liposomes and Nanoparticles - Critical to ?

Moderna/Pfizer mRNA Delivery

CellSearch Data

Monitoring a Patient's "Tumor Burden" also called "Tumor Load"

Ultrastructural Tomography - Life in 3D from 2 D

Montage of a computer based 3D reconstruction of an EM tomogram with a LM-fluorescence image of the cells (green) on a carbon coated EM grid (left) Also, Immature HIV Virus (right)

plant-based biomanufacturing

Most "biologics" (drugs that are either made in cells through genetic engineering or harvested from native plants) are generated in animal cells But now many like Pfizer's Elelyso to treat Gaucher disease is now made in plants Dept of Defense (DARPA) currently has a $100 M grant program to support plant biopharmaceutical technology

Adult Stem Cells

Most popular for medical applications are adipose (fat) derived mesenchymal stem cells (adMSCs) now in more than 1000 stem cell therapy trials worldwide

governmnt hierarchy

NIH --> NSF --> DoD

nanoparticle slips suicide gene into pediatric brain cancer cells

Nano particles and gene therapy are now joining forces to combat cancer In the past nanoparticles have been shot directly into tumors and then infrared light heats them up and kills the tumor. More recently a group at Johns Hopkins has been able to package suicide genes into biodegradable nanoparticles and once cancer cells take in these particles cancer cells die due to the activation of these suicide genes. Advantage: Improvement over viral delivery systems that cause problems in pediatric patients due to their fragile immune system Advantage: Rather than a single small gene currently used in viral gene therapy larger genes and a group of several genes can be packaged together

Velocity Sedimentation

New Microfluidic Devices Being Developed cell separation system that relies on differential speed of cells through a gravitational field- cells have to be a different size

Lab (Organs) on a Chip - Advances in Microfluidics and Bioengineering

New microfluidic devices called "Labs on a Chip" or "Organs on a chip" promise to improve and change the field of disease diagnostics such as cancer cell detection Now being used for drug toxicity testing called "in vitro toxicology" among many other things

3D Imaging Technique Visualizes Lung Tissue from COVID-19 Patients

New multi-scale phase contrast X-ray tomography is a new X-ray microscope technique as of 8/2020 The scientists visualized large areas of lung tissue embedded in wax blocks like CT scans but the latter simply doesn't have the required resolution Rather than using constructive and destructive light interference to generate contrast they used different propagation velocities of X-rays to generate the intensity pattern seen here. Image is pulmonary alveolus with hyaline (membrane) from an autopsy sample showing deposits that reduce gas exchange - major problem with COVID-19. This technique can also map the 3D distribution and density of lymphocytes infiltrating the diseased tissue.

Can we prevent mitochondrial diseases in newborns using nuclear transfer and three parents?

Now legal in the UK but not US. FDA reprimanded New Hope Fertility Center (Dr. Zhang) in 8/17 for doing this nonetheless

HeLa Cells

One of the most commonly used cells to generate "biologics" Established by George Gey in 1951

Can RNAi be used in a combination cocktail to more effectively treat cancer?

P-Glycoprotein (also called MDR1 - Multi-Drug Resistant Protein) is a cell membrane protein that can pump toxic compounds that include cancer drugs out of cells Cancer cells have very robust p-glycoproteins but siRNA directed against p-glycoprotein might be a means to block p-glycoprotein.

palm microscopy

PALM microscopy uses photoactivatable fluorophores to resolve spatial details of tightly packed molecules Once activated by lasers, fluorophores emit for a short period but eventually bleach. The laser stochastically activates fluorophores until all have emitted.

Decelluarization

Preserving the extracellular matrix while removing cells = Scaffold for complex organs such as the pig heart and pig lung

Telomerase-Immortalized Cell Line

Pros/Cons -cell strain pro: most in vivo like con: limited # of divisions -cell line pro: unlimited # of cells con: not similar to their in vivo counterpart -telomerase immortalized cell line/strain immortal but in vivo normal characteristics like its parent cell strain

Plate reading spectrofluorometers

Quantitative assessment of fluorescent probes not a microscope

inherited diseases

Research by cell and molecular biologists have resulted in genetic tests that can predict disease states and health issues. There are over 75,000 different genetic tests with the number of tests sold doubling every year for the past year. But not all are FDA approved and many are direct to consumer which can be problematic given that the lay public is now interpreting the results without physician guidance and counseling.

Photosynthetic Mammalian Cells

Researchers have generated photosynthetic human cells by transplanting "nanothylakoid units (NTU)" (engineered mini chloroplasts) into them. When transplanted into degenerating cells in mice with osteoarthritis and illuminated disease progression is prevented. How does it work? The NTUs produce ATP and NADPH (more energy and reducing agents) that may also be important to prevent aging as well as diseases

Old Blood Found to Contain Factors That Induce Aging in Young Animals

Researchers report that when young and old mice were surgically joined (heterochronic parabiotic mice) such that they shared the same blood circulation for three months the old mice did not significantly benefit in terms of lifespan In contrast, however, the young mice that were exposed to blood from old animals had significantly decreased lifespan compared to mice that shared blood with other young mice. Isochronic (same age) parabiotic mice served as the controls Conclusion: Old blood may have factors capable of inducing aging in young animals

1655

Robert Hooke - "cells"

SARS-CoV-2 Infection Modeled in 3-D Stem Cell Culture Model

SARS-CoV-2 access the lung via the ACE2 Receptor An alveolar tissue engineered model has been developed to model the disease Image shows alveolar stem cell marker (red) and ACE2 protein (green)

Scanning Tunneling Microscope (STM)

Scanned Proximity Probe Microscope first to visualize DNA major and minor grooves

Heart Attacks Mimicked on a Chip

Scientists at the University of Southern California have developed a "heart attack on a chip" One big problem with heart attacks is to better understand what happens in the heart at the no oxygen/with oxygen border or interface in the cardiac tissue. This two part chip device can model this interface. In contrast this can't be studied in real time in animal models Will be useful to identify new drugs to treat heart attack after the infarct and to better understand how the cardiomyocytes respond to this oxygen/no oxygen border

Purpose of project edible vaccines

Show that DNA coding for mRNA vaccines can be successfully delivered to plant cells Show that plants can produce enough mRNA to rival a traditional shot Figuring out the correct dosage They are inserting genes into spinach and lettuce chloroplasts, not nuclear genome Advantage: Grow your own mRNA vaccine in your garden

Non-ionic Detergents are Used for?

Solubilizing Membranes

Microporous Membranes

Supports the differentiation of kidney and intestinal cells as well as engineered skin Microporous membranes can be used to study transcytosis of proteins including maternal antibodies from blood to breast milk

HVEM: high voltage electron microscope

Theoretical limit of resolution is better because accelerating voltage is 1000kv, not 100kv as in typical TEMs best used for imaging thick sections

Two Photon Microscopy

Thicker Images, Lower Phototoxicity, in vivo Applications in Real Time virtues- fluorescent images, deeper penetration, less heath, less phototoxicity, diffraction limited

genesight

This company uses DNA samples to predict how the efficacy of select medicines used for depression can be influenced by your own DNA - very important in this COVID age! Doc collects cheek swab, it is sequenced at Genesight, then results are sent to Doc. Known pharmokinetic genes (see patient report to the right) that process medications are analyzed for variations that can alter the medicine efficacy and breakdown products in each patient.

3 applications of using cells to build living animals

Three applications: (1) Understand why some very early stage embryos fail to develop in utero. (2) Source of synthetic human organs for transplant. (3) Understand the key genes involved in brain development by using gene knockout stem cells as the building blocks of this mouse construct Group is now working on human synthetic embryos

Organoid Culture to Match Human Small Intestines

Tissue engineered intestinal organoid developed in the Netherlands that now includes "Paneth" cells Not previously included in intestinal organoids, Paneth cells defend the intestine from pathogens with secreted proteins called "Defensins" Defective or missing Paneth cells occurs in diseases such as inflammatory bowel disease So this model better represents a tissue model that can be used for testing new drugs for intestinal diseases and could be included on a lab on a chip as well

TIRF

Total Internal Reflection Fluoresence

SIRT 6

Transgenic mice that overexpress SIRT 6 results in a reduction in frailty and an increase in lifespan by 30% compared to non-transgenic mice. Why? Could be that the transgenic mice are better at generating energy from stored molecules like fatty acids. Also, the transgenic mice exhibit fewer age-dependent cancers and blood disorders Resveratrol stimulates human sirtuin genes but not practical as a drug

GEN- US anti-aging pill

U.S. Military (U.S. Special Operations Command) will conduct clinical trials of a pill designed to block or reduce many degenerative effects of aging. Start: 10/1/2022 It is a NAD+ enhancer ( may work like SIRTUINs) developed by MetroBiotech in Worcester, MA - Generates more ATP in mitochondria "MIB-626 may also be useful to treat amyotrophic lateral sclerosis (ALS)

Gene Therapy

Using gene transfection technologies to cure disease

Cancer Stem Cells

Very low in number and don't respond to standard cancer treatments. They can be quiescent for years. How do we target these cells?

Microfluidic Extraction of CTCs from Liquid Biopsies

Vortex - An improvement over Cynvenio system? Relies on unique physical characteristics of CTCs to capture them in a microfluidic vortex - similar to eddies in a river WBCs and RBCs "move down the river faster" leaving CTCs behind. They are collected and counted. No antibodies are used to count CTCs This technology can be referred to as "Nature Inspired" because the vortex is naturally occurring in rivers, etc.

Can we use stem cells to build living animals?

We are getting close.... At University of Cambridge 3 types of stem cells were used to develop a mouse construct that has a beating heart and brain tissue.

Genetically modified/engineered T cells -

When injected into Chronic Lymphocytic Leukemia patients honed in on the cancerous B cells inside the bone marrow killing them. Now being tested at many venues - "Immuno-oncology". issue: many cancer cells have a "don't kill me T-cell" protein where even if binding occurs the cancer cells won't be killed

GFP Can Reveal Protein Expression in x as well as cells

Whole Organisms

personalized medicine

Whole genome sequencing can now be done for less than $1000 as of 2012/2013 - "$1000 genome" 23 and Me Inc. capitalized on this market and developed an ancestry and health predisposition report currently as of 2022 that costs $129 But FDA told 23 and Me to stop selling their kits in 12/13 because they went from ancestry to include diagnostics, health reports, etc. 2022 - 23 and Me has complied with the FDA and now offers: 65 Health Predisposition reports This includes "carrier status" reports and "health predisposition" reports based on simple observations 4 possible outcomes: drug not toxic and beneficial, theyre cured; see picture sequenced a genome: cost 100k → wanted to set a price point at 1k. arose in part bc someone found a cheaper way to do genome sequencing- 23 and me

are all microscopes governed by Abbe's equation?

Yes but there's an outlier: super resolution microscopy: not diffraction limited

Do we know enough about cell and molecular biology to make an artificial organ or tissue?

Yes, it has been done! Tissue Engineering - a sub-discipline of Regenerative Medicine Building tissues in the lab that can use human embryonic stem cells or human fetal cells - controversial... Fetal tissue

Atomic Force Microscope (AFM)

a Scanned Proximity Probe Microscope no lenses image nuclear pores

cryostat and frozen section

a mid alternative freeze tissue is like infiltrating with wax then section is as frozen tissue disadvantage is that you get poorer tissue morphology

densitometry

a process by which you can scan the density of a band (gel electrophoresis, Western blots, autoradiograms) and convert them to a number

C-1

a vital mitochondrial dye

Four Categories of Stem Cells

adult fetal embryonic induced pluripotent

EpiDerm function

all done in a diffusion chamber as follows using a test substance, radioactive water, and meadure radioactivity leaking through EpiDerm

liquid chromatography

all proteins from cell membranes - separate the proteins liquid chromatography: column, beads, preserves the native activity of proteins ion exchange chromatography - beads are either positive (DEAE ion exchange chromatography) most commonly used because most proteins have overall negative charge. CM is using negative ion chromatography elute the proteins with NaCl gradient 0-1 M NaCl

FPLC

an improvement on conventional ion exchange chromatography

10 microns

animal cells

liposomes

artificial bilayers encapsulate molecule of interest

limitations of MDCk cells

artificial environment genetic drift proper phenotype e.g. MDCK not all cells divide in culture e.g. Hepatocytes substrate has to be defined hESCs function can be lost over time

affinity

attraction of an antibody to an antigen

1 micron

bacteria/mitochondria, use LM

specificity

bind to the antigen, not x-react

antibody

bivalent

Botulinum toxin

blocks the release of acetylcholine used in cosmetic surgeries, treats facial dystonia

reticular theory

brain is more like a reticulum/blood vessels (think of endoplasmic reticulum), you'd be able to see individual cells by then and you couldn't

neuronal theory

brain tissue consists of neurons , had to evidence to support at the time

Organoids

can be generated from a variety of stem cells

Fluo3-AM

can measure intracellular calcium concentrations in living 3 cell functions 1.) mitochondria -several changes in mitochondria activity -examples: Rhodamine 123, JC-1 2.) life/death (live-dead assay) -2 dye system: -calcien-am → fills living cells and is green -PI - not membrane soluble -PI comes in an stains nucleus 3.) changes in [Ca2+] -Flu3-SM -intensity of emission reflects Ca2+

is our Protein of integral membrane fluid within the plane of the membrane?

can this (the arrow near the word living in the drawing) go back and forth or is this stationary lazer zap → black hole about 50% of the integral cell is stationary in fluid

1838

carl sehleizen + Schwann - cell theory

Substrate Requirements

cell culture dishes most common -microcarriers beads that carry bnd cells large batches "batch cultures" purify "biologics" eg mAb picture roller bottles cell culture/asserts nanotubes vasculature neurospheres

Fluourescence Immunocytochemistry can reveal ?

cell polarity

Dynabeads

cell separation selective surfaces panning beads/nanoparticles with selective surfaces like DynBeads and Sppedbeads

Regenerative medicine

cell therapy gene therapy tissue engineering tissue engineered construct

double label fluorescence microscopy

cells are labelled alive and can be tracked over time

gene for GFP is tagged onto the gene of interest, making a ?

chimeric gene

neurosphere

clusters of neural stem cells that can differentiate into neurons, astrocytes (glial cell) and oligodendrocytes (glial cells). Soon to be in clinical trials to treat CNS diseases

photo-activated localization microscopy (PALM)

collect thousands of images, in each of which only a few GFP molecules are excited GFP excitation distribution each GFP molecule emits photons in a Gaussian distribution for which the center can be calculated and used to create an image

Harvard's Personal Genome Project

collecting genomes and any health issues u have for a public database to see what genes may be related to what illnesses. 5k participants which isn't a lot

adult stem cells pros and cons

cons: Not as easy to procure as hESCs But probably less pluripotent pros: Many sources (fat, hair, teeth, etc.) Can be autologous match Fewer ethical issues

Fetal Bovine Serum

contains growth factors but serum-free medium (Defined medium) is preferred for clinical applications

in super resolution microscopy: x is a problem bc theyre mostly water in the cell

contrast

monoclonal antibodies

control/select mAb mashed on specificity and affinity clones (hybridms) can be cryopreserved/bio to then thawed and propagated

hESCs

derived from human embryos (blastocysts) and are now in stem cell therapy clinical trials- Requires the destruction of human embryos. Dickey-Wicker amendment: bans the use of federal research funding that uses or destroys human embryos, gotta get private funding OK for medical applications shows disconnect between clinical trials and government hESCs: discarded from IVF clinic (can't make them), let them develop until they get inner cell mass, can grow then inject into people or anything else

direct or indirect techniques

direct: one antibody linked to fluorochrome indirect: 1º, 2º antibody

myriad genetic

distributes a number of kits for predicting breast and prostate cancer including Polaris for prostate cancer - ran into problems in 6/2013 with US Supreme Court GenomicHealth now distributes OncotypeDX that is a genetic test used by prostate surgeons that can predict the aggressive level of individuals with prostate cancer. Helps patient determine if "watchful waiting" or "resection" would be the best treatment. developed a test for breast cancer 1 & 2 provides genetic testing for genes that could be defective and become prostate or breast cancer can make a suggestion to you ab the line of treatment u should exercise

Guava

doesnt do cell separation and sorting, but counts FL-labeled cells Flow cytometry but using microcapillary tubing = small samples

Verve Therapeutics

dosed its first patient with a drug that will edit bases in the patient's DNA as a path to treat the patient's Heterozygous Familial Hypercholesterolemia VERVE-101 is a base editor mRNA that makes a single Adenine to Guanine change in the DNA genetic sequence of gene PCSK9 which increases the number of LDL receptors thus lowering cholesterol The LDL (low density lipoprotein) receptor endocytoses LDL (the "bad" cholesterol) into the cell where it is degraded so that the ratio of HDL (the "good" cholesterol) to LDL is now in the healthier range. This is the first human ever treated with any base editing medicine and represents a new and important medical frontier that will be exploited in the future.

photosynthesis

efficiency is less than solar panels. range often quoted is 0.1% to 2% not perfect

electroporation

electricity (pulsins) to make transient holes in cells

antigen

elicits an immune response

Feeder Layers (inactive mouse embryonic fibroblasts) and hESCs - hESCs form "x" in vitro

embryoid bodies

tissue engineering: 3 parts

engineering cells: cell source, iPSCs, adult stem cells, genetic tools, CRISPR-Cas9 engineering materials: novel chemistries, growth factors, biomechanics engineering tissue architecture: decellularized organs, #d printing, self assembly

protein purification

enrichment ion exchange chromatography and many more functional on structural analysis differential centrifugation pellet has intact cells and nuclei, centrifuge more to get mitochondria, lysosomes, peroxisomes, then centrifuge again to get microsomes microsomes = small bodies smooth vs rough microsomes

In Situ Hybridization for Detecting HPV

example HeLa cells are cancer cells (cervical carcinoma), HPV is a select cervical carcinoma cell transformed in cancer cells because of a select HPV virus? unknown- sample positive control: HeLa cells negative control: GMK cells (green monkey kidney) can be very diagnostic

stimulated emission depletion (STED) microscopy

excitation laser point surrounded by donut-shaped "depletion beam," making area excited much smaller

Tumor microenvironment

extracellular signaling can keep cancer cells in non proliferating form or can trigger proliferation and metastasis

spinning disk confocal microscope

faster lower laser intensity less photobleaching less heat dynamic interactions in living cells can reveal changes in endoplasmic reticulum in living HeLa cells

Katalin Kariko

first suggested synthetic mRNAs to try to address disease Problem- mRNA causes an immune reaction and could be degraded ALSO How do you get naked mRNA into cells so that it could synthesize the target protein of interest and secrete it? Solution - Change a few of the bases (modified mRNA) Critical Application - Pfizer/BionTech and Moderna ("ModRNA") took notice And then COVID hit with a vengance

what type of microscopy requires you to kill the cells?

fluorescent microscopy

cell strain

freshly isolated from tissue limited # of divisions (20-50x) Hayflick limit

western blots

gel electrophoresis blotting technique, uses monoclonal antibodies to identify and quantify a protein of interest separated on a gel gel electrophoresis, usually SDS and not 2D put gel on its side and apply it to transfer paper add antibody plus secondary antibody to get one of the bands labelled with protein of interest once its developed, u can only see that particular protein western blots are qualitative (u can determine MW) and its quantitative

isoelectric focusing

gel electrophoresis, tubular gel, separates proteins based on isoelectric point, pH gradient end to end on the gel, resolution = 0.01 pH unit

structured illumination microscopy (SIM)

generate interference patterns that can be computationally reconstructed to create an image

genetic drift like HeLa cells, cells lose ?

genes over time and is true of all cancer cells not all develop the proper phenotype

GFP

green fluorescent protien, came from jellyfish

cell culture

growing cells in an artificial medium (bacteria, plant cells, yeast, mammalian cells) monoclonal antibodies in 2025 will be $400 billion mammalian cells that are used are CHO and HeLa

Embryonic Stem Cells

hESCs (human embryonic stem cells). Now in clinical trials since 2010

darkfield microscopy

has a dark field, generally looks in eukaryotic cells can illuminate "small structures" i.e. mitochondria, lysosomes, etc. microbiologists because it's really good at increasing the contrast of small particles diatoms are the abrasive component of toothpaste

neural retina

horizontal cell, could see the cell you're working with

mass spectrometry

identify proteins with resolution one amino acid, key parts: laser, "chips" - reactive surfaces (based on either -/+ or antibody), ToF

cell line

immortal telomerase → repair telomeres sources -embryonic cell lines e.g. 3T3: mouse embryo fibroblasts -cancer cell lines derived from a tumor (HeLa) transfect cell strain with oncogene

Telomerase

immortalized cell strains/lines are immortal compared to the parent cell strain from which they were generated

Golgi stain

in vitro = cell culture, in vivo = in the living organism, in situ = in place history -1900 - brain tissue in a cell culture dish. found that brian tissue would develop axons to a blood clot in a petri dish -George Gey = John Hopkins 1951. Henrietta lacks - cervical carcinoma cells, HeLa cells now established -Leonard HayFlick - limit cells and cells strains, finite number of divisions

confocal ADV

increases practical limit of R by 50% optically sections a cell stereoimages multiple labels

freeze-fracture

interior of cell membranes, cell membrane proteins metal replica/uses platinum needs a carbon coat for stability needs TEM

facial dystonia:

irreversible contraction, very uncomfy, in face, the motor neurons and muscle cells have issues

how can a donor kidney be repurposed?

it can be "decellularized" then seeded with new stem cells as the starting point to build a tissue engineered kidney

MDCk

kidney cells, around 1958 they grow really well in simple set up, can grow a lot of them they dont have a kidney like function the cells when grown on a biological scaffold (microporous cell membrane insert) looked completely different, they were polarized and transported things

Tissue Engineering

lab grown tissues that can be used to treat diseases

Cell Bioreactors such as Millipore's Mobius CellReady system grows what

large numbers of cells on microcarrier beads

how do lasers work

lasers confocal pinholes computers displays image dot by dot straight image is reduced practical limit resolution increases by 50% optically section use multiple dyes/stains 3-4 problem? most images in confocal use fluorochromes once they fluorescence → photobleaching = drop over time in light intensity options: change dye or make a different microscope

constituitive protein

little variation

phase microscopy

looking at living cells cell microbiologists use it

cell fractionation

lyse the cells, enriched fractions of the organelle of interest differential centrifugation density gradient centrifugation sonication: destroy all organelles detergents: dissolve cells, destroy all organelles trituration: mechanical shearing, organelles are preserved

He Jiankui's crime

made 3 gene edited babies sentenced to 3 years in prison and fined $430,000

Cynvenio Biosystems

makes a LiquidBiopsy device for the immunomagnetic (antibodies) and microfluidic capture of CTCs

patch clamp

measure the electrical current going through a single membrane cavity

bright field microscopy/compound light microscopy

microtome cuts fixed tissue designed to look at fixed and stained tissue (this is what a pathologist does) steps: -take tissue of interest (tissue X) → fix it (use a preservative to keep it in a lifelike state, use formaldehyde because it cross links proteins which means the cell will stay intact) → dehydration, replace the water with ethanol, then replace ethanol with Xylene → infiltration, rely on the fact that its disssolvable in Xylene so use paraffin/candle wax = solid block of tissue where all the water has been removed and now it's all wax -we do this bc tissue is very soft and malleable then cut 10-15 micrometer sectionrs → remove wax, go backwards, infiltrate with Xylene, ethanol, and water, then stain it

green=

monomers = low PMF

liposomes are

nanoparticles, not common

gel electrophoresis

native gel electrophoresis - separates proteins on a polyacrylamide gel )sieve) based on charge and size most proteins have a negative charge, so they will travel from the negative end to the positive end compare their activity to standards which you know the distance SDS electrophoresis - the proteins are denatured first. Urea compromises the H-bonds and B-mercaptoethanol compromises the disulfide bonds. Boil SDS. disadvantage is denaturation, advantage is resolution

detecting cell death

necrosis: pathological cell death like mitochondrial poison when cells explode apoptosis: programmed cell death, chemo triggers this annexin V-FL + PI used for chemo

ELISA - Enzyme Linked Immunosorbent Assay- Direct, Indirect and Sandwich Applications

not a microscope technique, tells us how to measure your protein of interest use a substrate that turns color or be fluorescent, the more the color the more the protein of interest colorimetric (E): substrate has no color, color with antibody fluorescence : substrate has no fluorescence, becomes fluorescent fluorescence imaging technology = qualitative (image) elisa = quantitative (MEASURE)

Super Resolution Microscopy

not diffraction limited (does not adhere to the equation) e.g. PALm Breaking Through Abbe's Diffraction limit (Nobel Prize 2014)Z-stack image

what's the basis of aging?

older blood has agents that can cause precocious aging in younger individuals

virtues

one amino acid resolution, small sample size of about 1 microliter

epitope

part of an antigen to which an antibody binds

density gradient centrifugation

particles reach a point of equal density with the medium, stop moving

Cell culture inserts/microporous membranes improve ?

phenotype of MDCK and other epithelial cells

The major enzyme (RUBISCO) sometimes uses oxygen rather than carbon dioxide that results in glycolate that is toxic - a wasteful process called

photorespiration This glycolate reduces photosynthetic efficiency by 50%! Researchers from the USDA and U of Illinois made transgenic tobacco plants that process glycolate in only one cellular compartment rather than throughout the entire cell. (Tobacco plants are "model organisms") As a result photosynthesis improved and plants grew 40% more biomass

100 micrometers

plant cells

hESCs pros and cons

pros: Easy to procure More pluripotent (greater ability to differentiate to virtually any type of cell) cons: But ethical issues are a concern Not autologous match - (self to self)

Galleri test

qualitative, next-generation sequencing-based, in vitro diagnostic test looks for DNA methylation patterns using cell-free DNA (cfDNA) isolated from human peripheral whole blood. DNA methylation is a natural process used by cells to regulate gene expression. Certain DNA methylation patterns can serve as a signal of cancer and provide information about the origin of the cancer signal approved in the UK It works by analyzing cfDNA shed from tumor cells. $1250/test with a 2 week turnaround time. Can reveal 50+ different types of cancer. Must be done via health provider. Grail, whose work is focused on detecting cancer early, is backed by investors including tech billionaire Bill Gates and Amazon founder Jeff Bezos.

polyclonal antibodies

rabbit, inject antigen, increase antibodies

Research team used CRISPR to ?

replace defects in heart tissue of Duchenne Muscular Dystrophy patients Tissue engineered heart tissue now contracts better

reporter molecule:

reports back to us if a gene of interest is present

gel filtration

separate proteins based on size, beads and holes used with bead path much longer

cell separation

separating out a cell of interest in a tissue FACS, velocity sedimentation, selective surfaces, others

single cell intracellular injection

single cell intracellular injection e.g. lucifer yellow SCNT = somatic cell nuclear transfer -remove the original nucleus from the egg -introduce a somatic nucleus one cell at a time

Orphan Diseases - Fibrodysplasia Ossificans Progressive (FOP)

soft tissues to transform permanently into bone.

In Situ Hybridization using FISH (Fluorescence In Situ Hybridization)

technique that can localize and identify mRNA or genes in cells using a complementary probe probe transfect the probe into the mRNA or gene of interest, it binds and fluoresces for detecting mRNA or Gene Sequences

Tissue Engineered Constructs are used for ?

testing and optimizing thermal ablation medical devices

put chimeric gene of interest in a living cell, when its green it means?

that the protein is being expressed possibilities

even though there's not much in cells...

the different parts of the cell have different refractive indices in organelle-specific refractive indices

synthetic biology

the engineering discipline that encompasses the synthesis or creation of complex, biologically based or inspired systems which display function that do not exist in nature using cell/molecular biology tools and techniques example: engineered antibodies called bispecific, trifunctional antibodies that are now being used to treat cancer They bring tumor cells together with killer T cells and Accessory Cells so that the T cells can kill the cancer cells

Researchers at the J. Craig Venter Institute (JCVI), a not-for-profit genomic research organization, published results today describing"

the successful construction of the first self-replicating, synthetic bacterial cell. The team synthesized the 1.08 million base pair chromosome of a modified Mycoplasma mycoides genome. The synthetic cell is called Mycoplasma mycoides JCVI-syn1.0 and is the proof of principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome.

laser capture microdissection

tissue section overlay with ethylene-vinyl-acetate laser = melt the film

100 nanometers, cant use LM, viruses and ribosomes →

transmission electron microscope

3 types of microscopes in 1950s-1960s

transmission electron microscope (TEM), resolution = 2.4 A scanning electron microscope - resolution is about 100x less than TEM STEM - scanning transmission electron microscope

best for Moh's surgery

treat skin cancer

Bispecific antibodies

trifunctional and generated using "quadroma" cell lines. They have two different Fab ends - Used for treating cancer The mechanism of action of a bsMab (bispecific monoclonal antibody), exemplified by catumaxomab, representing the first FDA approved bispecific trifunctional antibody

UNOS

united network for organ sharing demand outspaces the supply 3D tissue engineering may alter the transplant landscape

3 options to use super resolution microscopy

use dyes colormetric: absorb light and transfer to a different color example: Hematoxylin (stains nucleus) and eosin (stains cytoplasm) fluorochromes emit light, diagnostic manipulating light no dyes rely on other factors computer image enhancement super resolution microscopy

fluorescence immunocytochemistry

use of antibodies to detect and visualize a protein of interest in cells

autoradiography

use of radioisotopes to track or follow a selected cell process e.g. thymidine (DNA synthesis), methionine and leucine (protein synthesis) track cell processes over time radioisotopes to track a process e.g. gel electrophoresis identify newly synthesized proteins, label S35 methionine do gel electrophoresis then autoradiography

Stem Cell Therapy

use of stem cells from different sources to treat diseases

vivascope

used by dermatologists, not cell biologists hand held confocal imaging system skin anomalies (skin cancer) non invasive

polarizing light microscopy

used by neurobiologists looking at living neurons - microtubules muscle cell biologists view highly ordered parallel structures in cells OR outside of cells collagen fibers microtubules actin filaments/myosin just see individual fibers fibrosis: deposition of scarred tissue

Deconvolution and Confocal Microscopy - A new view

uses an algorithm to create a 3-D view of fluorescently stained cells

viral transfection

viral particles

what qualities are required for intracellular fluorescent dyes?

vital dye fluorescent membrane insoluble diffuse easily throughout the cell

Special cardiac stem cells

were modified so that they beat like a heart but are on two sides of a tail causing the fish to "swim" in cell culture media up to 100 days. The autonomous cyclical action that causes "swimming" mimics the function of the human heart

artificial environment doesn't represent ?

what is typical of in vivo

image analysis systems

what proteins are phosphorylated in human fibroblasts in response to insulin grow fibroblasts label the cells by adding radioactive phosphate 32PO4 goes into the cells then becomes radioactive ATP lyse the cells gel electrophoresis autoradiography see lots of phosphorylated proteins in the control, in the experimental (insulin) you will see the same set of proteins but also see an additional one that is insulin

2 Questions about selecting a microscope

which microscope do i use (theyre all different bc theyre designed to answer different questions) specimen preparation -living cells -fix cells (kill them but preserve them in a life like state so we can process and look at them, stain them)

photoreactive amino acids

with UV light, they generate a covalent bond with other proteins with which they're associated. what proteins bind to X? cell culture and photoreactive amino acids control is no UV light and experimental is to add UV light

power of cell culture

working with a single cell type control the environment (cell culture media - add etc) grow unlimited number of cells (cell line) recombinant DNA - biologics some kind of useful molecule that comes from a living source basic gene function unique cells with reporter molecules cell therapy - stem cells therapy tissue engineering organs on a chip cell division/differentiation

Can we use stem cells to build organs?

yes, some day. hESCs were used by a group at Brigham and Women's Hospital in Boston to build a functioning human kidney organoid. Why? Will allow us to use this construct to test for new therapies to treat kidney diseases. One of nine people globally suffer from kidney diseases.

during the aging processes:

your dsDNA breaks. non-base changes