Revision Cards Flashcards
What are some of the biggest killers?
- Diabetes (dysfunctional glucose homeostasis)
- Heart Disease (plaque build-up in arteries)
- Chronic lower respiratory disease (airflow blockage)
- Cancer (uncontrolled growth and spread)
- Neurodegenerative disease (loss of nerves)
- Anxiety and depression (poorly characterised)
What is a model organism?
Non-human species which is studied as an in vivo model to understand aspects of human disease.
Why are specific model animals used?
- Gene conservation
- Mechanisms of gene action are conserved
- Easy genetic manipulation
- Transparent models allow real-time visualisation
- Help identify life-course genes/events
Why are animal models used?
- Understand variation between changes caused by disease progression and life course changes over time
- Genetically-tractable organisms that exist in large numbers and can be environmentally influenced (Gene X Environment)
- Sequencing and GWAS to understand mechanism and disease susceptibility [limited]
- Effect of new therapeutics in development
- Whole organism or system analysis
What are the limitations of GWAS?
These studies don’t show when or where the gene is expressed, how gene is translated to protein or how the gene/protein might trigger, contribute or exacerbate the disease.
What do transgenic reporter lines enable?
Can allow for the real-time visualization of gene expression in a living organism in health and disease, or after drugs, genetic manipulation, environmental influence or insult. Follow expression over time and combine with cellular behavioral studies.
What can FACS do?
Fluorescence-activated cell sorting enables individual cells to be dissociated from the tissue and sorted according to their fluorescence by electrostatic deflection.
What are mice, drosophila and zebrafish usually used for?
- Identify molecular pathways in normal cells, tissues, organs
- Identify normal and aberrant cellular outputs
- Identify disease progression: early events and consequences
- High throughput screens for drugs and toxicity
What are mice and chicks usually used for?
Experimental embryology gain-of-function approaches
Informed regenerative stem cell medecine
What is Diabetes?
Metabolic disorder in which the body is unable to carefully control the levels of blood glucose in the body. Hyperglycemia due to insufficient insulin supply and/or insulin resistance.
What normally occurs in glucose homeostasis?
Stomach converts food substances to glucose. Glucose enters the blood stream. B-cells of the pancreas secrete insulin which encourages skeletal muscle tissue to take up excess glucose to restore normal blood glucose levels.
What is the difference between Type 1 and Type 2 diabetes?
- In T1D, patients don’t have enough B-cells, make or secret enough insulin. Glucose doesn’t get taken up.
Usually congenital.
-In T2D, patients have an normal initial mechanism of insulin production and secretion but body is resistant to its effects. Over time, B-cells become depleted.
How does insulin resistance cause loss of B-cells in T2D?
5-10 years before symptoms present, the body becomes resistant. Insulin levels rise to try and combat the increased glucose. Excessive levels of glucose and insulin cause the loss of B-cells. Insulin levels drop and hyperglycemia presents.
How is Type 2 Diabetes classified according to obesity?
Abdominal obesity along with two others factors: elevated BP, low HDL, elevated triglycerides or impaired fasting glucose.
How are T2D and obesity linked?
Obesity is highly associated with T2D. Unclear how it links because so many tissues are involved. Obesity is known to cause increased free fatty acids and insulin resistance but it is unknown which comes first.
How does changes in FFA and insulin resistance contribute to T2D?
Increased FFA and insulin resistance lead to increased levels of Apolipoprotein B and Hepatic Lipase. This leads to increased levels of triglycerides and small dense LDL and decreased HDL.
What causes hyperglycemia in T2D?
- Pancreas can no longer secrete sufficient insulin
- Dysfunction in liver glucose production
- Peripheral tissues are unable to uptake glucose
How does obesity cause the loss of adipocytes?
Obesity leads to the accumulation of fat in ectopic tissues such as the liver and skeletal muscle instead of in adipocytes. Imbalanced accumulation leads to a loss in the adipocytes.
How can mice be used to model the lack of adipocytes in obesity?
GM mice with a lack of adipose tissueare characterised by hyperphagia, hyperglycemia, insulin resistance and Type 2 Diabetes. Lack of adipose tissue means the mice are Leptin deficient. Suggests that primary cause is linked to brain dysfunction.
How can reporter lines be used to identify the cause of hyperglycemia?
Cross 3 reporter lines to make a fish in which you can see the pancreas, liver and adipose tissue. Feed high-fat diet and see which tissue falters first.
What is the biology behind reporter lines?
Depends on the fact that genes are deferentially transcribed in different tissues depending on their promoter/enhancer and all the cell-specific transcription factors.
How do you create reporter lines?
1) Cut off the coding part of the gene from the control regulatory elements.
2) Fuse liver-specific gene promoter to the coding sequences of genes that code for proteins that fluoresce when UV light is shone on them.
3) Make a transgenic mouse or fish in which this extra gene is incorporated. The reporter is only expressed in the liver at the time when the endogenous gene is expressed.
How can you measure proliferation in cells?
EDU or BrdU labelling intraperitoneal injection for body, intracerebroventricular for brain. Both are analogues of thymidine that have be modified so that they can be used to visualise proliferating cells.
- BrdU labelled with an anti-BrdU antibody
- EDU labelled with a click chemistry kit
How does EDU visualise proliferation?
1) Incubate cells with excess EDU
2) EDU is incorporated into the DNA after semi-conservative replication
3) Detection of newly synthesised DNA with a ‘click’ chemistry reaction that attaches EDU to the DNA within 30 minutes.
How to make a tissue-specific knockout?
1) Genetically modify the specific gene you want to KO so that it is flanked by ‘lox’ which are recognised by Cre recombinase. Make a transgenic line.
2) Identify a gene that is only transcribed in the specific tissue, clone the coding sequence for Cre recombinase downstream of the gene’s promoter.
3) Cross these two lines. Whenever Cre is expressed, it will cut out the gene at the lox sites, no expression of the gene.
What is the current new idea for therapy for Type 1 and 2 Diabetes?
Both in type 1 and late-stage type 2 diabetes there is a depletion in the number of B cells. If you can restore the number of B cells, you could create a curative approach that increases life expectancy. This could be a human version of BefA but further study is needed.
What is the link between microbiota and B cells?
Hill et al found that a conserved protein from certain gut bacteria BefA is necessary for the pancreas to populate itself with a robust no. of B cells.
How did Hill et al (2016) establish a link between microbiota and B cells?
Hill et al (2016)
ZEBRAFISH
-Created a Tg(Ins:GFP) line in zebrafish to count the number of B cells. Found that after hatching, B cell numbers increased.
-In microbe-free environment, the number of B cells stayed the same as pre-hatching
-High levels of ‘whole glucose’ was found in MF
-Exposing MF fish to certain bacteria restores the MF B cells levels
-Using EDU, BefA protein causes B cells to proliferate and could restore B cell levels
How were rodents reveal the regenerative capacity of B cells?
Experimental ablation of B cells by chemical treatment or partial pancreatectomy in rodents displays significant recovery of the B cell mass.
This regenerative capacity could be exploited therapeutically.
How did Andersson et. al. (2012) test the drugs that could restore the numbers of B cells?
Andersson et. al. (2012)
Double transgenic Tg (ins: CFP-NTR) and Tg (ins:Kaede). CFP-NTR (nitroreductase enzyme) is cloned downstream of insulin promoter. When B cells are expressed, nitroreductase is also expressed which converts Metronidazole into a cytotoxic product, killing the B cells.
Then double-transgenic fish treated with MTZ were placed into 100s of wells to test over 7000 compounds. After washing away the MTZ and 2 days of recovery, B cell regeneration is easily quantified. Compound NECA showed a major increase in B cells.
How are body-brain interactions affected in poor glucose homeostasis?
Likely that there is either abnormal signalling from the peripheral tissues like the gut, liver, endocrine pancreas and adipose tissue to the brain, indicating that there is insufficient fat increasing food intake OR abnormal signals from the brain to the tissues.
What is Leptin?
Hormone produced by adipose tissue to signal to the brain that status of the body’s energy content. This tells it to eat more/less and exercise more/less.
How did Cohen et al (2001) link the leptin receptor to obesity?
Cohen et al (2001)
Mice lacking leptin are obese, diabetic, infertile and hypoactive. Immunohistochemistry shows that many tissues in the body express the leptin receptor. You can conditionally knockout the leptin receptor in different tissues. In a liver leptin receptor KO, the mice are normal. In a brain leptin receptor KO, obesity is induced. Intracerebroventricular administration of leptin in ob/ob mice causes reduction of body weight and food intake.
What are 2 major leptin-responsive neurons?
Animal model studies showed that within the brain the LepR neurons were in the hypothalamus in the arcuate nucleus.
- Leptin binds to Neuropeptide Y neurons and inhibits them. This coordinates action to stimulate food intake and reduce energy expediture.
- Leptin binds to Pomc neurons and stimulates them. Pomc is a neurohormone which acts to reduce food intake and increase energy expediture.
How are Pomc and NPY neurons generated?
Hypothalamic stem cells (Tanycytes) which have a radial glial character. Cell body lies at the 3rd ventricle and a lateral projection that is used as a scaffold. Tanycytes can divide to produce more of themselves or differentiated cells. The differentiated cells use the scaffold to migrate to the ventricular position.
It’s likely that NPY and POMC neurons develop and differentiate to anticipate or respond to changes in glucose homeostasis.
What experiments were done to find that NPY and POMC neurons developed from stem and progenitor cells?
- Identify different populations that develop over time through markers. Stem cells express pea3, Glast1 and FGF10. Progenitors express Islet1.
- Lineage-trace the cells
- Identify the genes that maintain each cell.
- KO these genes at look at daughter cell differentiation
- Use the promoters of these genes to make cytotoxic transgenes to elemiate particular cell populations
How did Nasif et al (2015) link Islet1 to Pomc?
Nasif et al (2015)
Islet1 is well-known to be involved in motor neuron differentiation. Islet1 was seen to be expressed before Pomc because it is a progenitor marker. After that you see Islet1 and Pomc coexpressed.
Islet1 binds in vitro and in vivo to critical homeodomain binding DNA motifs of the Pomc enhancer. Without these binding sites (mutation in enhancers), there is no expression of Pomc. If you conditionally remove Islet1 from Pomc neurons, you impair Pomc expression and get hyperphagia and obesity.
How to perform lineage-tracing in an adult stem cell?
Cell-specific promoter identified and Cre-recombinase expressed downstream (Cre-ERT2) to create a transgenic mouse. This Cre is genetically modified to be fused to ERT2 which is a mutant oestrogen ligand-binding domain so that it is only activated when you add Tamoxifen.
Make a second transgenic mouse where a stable reporter is downstream of a constitutive reporter but seaparated by a floxed STOP sequence.
Breed mice and add tamoxifen at any stage of life to cut out STOP, allowing the reporter to be expressed in the cell and in all daughter cells.
How was lineage-tracing in adult stem cells used to find that tanycytes give rise to NPY and POMC neurons?
Identify that Glast1 is only expressed in tanycytes. Make Glast1:CreERT2 transgenic line and cross it with a RosaSTOPLacz trangenic line. Allow the mice to develop into adults and add tamoxifen at 6-8 weeks. Cre is activated, lox sites are removed and Lacz/GFP is expressed.
Using UV light or anti-GFP antibody you can visualise cells expressing Glast1. Leave for 9 months you cna see that a=tanycytes give rise to POMC and NPY neurons.
What did Scarlett et al (2016) find about intracerebroventricular injections FGF1 in ob/ob mice?
Models of T2D. You can see sustained diabetes remission unlike in intraperitoneal FGF1 infusion. Effects did not include changes in weight loss, hypoglycaemia, basal glucose production or glucose tolerance. Establishes a functional link between activation of 3rd ventricle tanycytes and diabetes remission induced by FGF10.
What is multi-morbidity?
Presence of two or more chronic non-communicable conditions like cardiovascular disease, cancer, chronic respiratory conditions and T2D. Whereby risk factors that affect one disease often affect other diseases too independent of age, nationality or class.
What increased risks do diabetics have?
Increased risk of CV disease in a Type 2 diabetic compared to a prediabetic or someone with normoglycaemia. Cardiometabolic risk factors tend to cluster and all effect one another.
Increased risk of breast and bowel cancer in a prediabetic.
What is insulin resistance?
Impaired response to the physological effects of insulin (Including those on glucose, lipid and protein metabolism) and the effects on vasular endothelial function.
What contributes to insulin resistance?
Some people have a genetic susceptibility to insulin resistance which can contribute to diabetes onset. Environmental factors like nutrition, obesity and physical inactivity can also contribute.
How does hyperinsulinemia contribute to poor vascular endothelial function?
Endothelial dysfunction refers to a maladapted endothelia phenotype charcaterised by reduced NO, increased oxidative stress, elevated expression of pro-inflammatory and pro-thrombotic factors and abnormal vasoreactivity. Insulin exerts vasodilator and vasoconstrictor actions which cause imbalance.
How can metabolic dysfunction lead to cancer?
1) Oestrogen - after menopause, oestrogen made by adipocytes can make cells multiple faster in the breasts and the womb, increasing risk of cancer.
2) Insulin and growth factors - excess fat can cause levels of insulin and other growth factors to rise, which tells cells to divide.
3) Inflammation - macrophages release cytokines which encourage cells to divide
Why is insulin linked to the cancer/aging?
Animal model studies show that the insulin pathway is an ancient, conserved signalling pathway fundamental to life and aging. Receptor and ligand have been conserved from hydra to mammals as well as the whole signalling transduction cascade. FOXO is the transcription factor at the end.
What is an environmental insult?
Environmental insult can be from the outside (like a mutagen) or can be within the cell or among the tissue. In cancer, it could be the environmental insult of inflammation or a genetic insult within a cancer cell.
How was Int1 discovered?
1000s of years ago ‘fancy mice’ were bred by the Chinese. Inbred mice were kept in Jackson labs in 1900s. In 1930s, female mice were found to be highly susceptible to mammary tumours.
In 1983, there was a retrovirus discovered in the mice that interfered with the mice’s DNA machinery to insert itself upstream of a proto-oncogene and upregulate a gene. This gene was clones and found to be Int1 which was thought to be a secreted protein due to it’s signal sequence. Very difficult to purify.
How was Wg discovered?
Nusslein-Volhard and Wieschaus performed a mutagenesis screen in Drosophila identifying patterning genes including segment polarity genes. One of these was Wingless.
What is Wnt1?
Wg had the same sequence homology to Int1. Thus, Wnt1 was born, which has adhesive, signalling and stem cell communication properties. Wnt signalling pathway was elucidated 1980-1995 using mice, drosophila and xenopus. Found that Wnt binds to Frizzled which causes B-catenin to become stabilized. Its accumulation in the nucleus allows it to activate target genes.
How can Wnt1 mutations lead to cancer?
Overexression of Wnt1 led to the development of mammary tumours in the mice. But in humans, 2 kinds of mutation. Activating mutations in protooncogene components of the Wnt pathway which mean that B-catenin is always stablised in the nucleus or when both copies of a repressor are lost and there are no breaks on the signalling, B-catenin is always in the nucleus.
Mutations are implicated in a variety of human cancers. Mostly commonly mutations in APC lead to colorectal cancer.
How can B-catenin be visualised?
You can make anibodies to detect stablised B-catenin in the nucleus. Animal models have allowed the identification of a ‘biomarker’ to detect an early-stage cancer (adenoma). Now used in humans to detect cancer. More stablised B-catenin will precede any change in morphology.
How have transgenic mice been used to trace Wnt-responding cells?
Gene called Axin is usually turned on by Wnt signalling. Generating mouse transgenic lines whether by Axin promoter is fused to LacZ, means when Axin is expressed so is LacZ. LacZ blue can be visualised by anti-LacZ stain to visualise Wnt signalling. Can be used to trace Wnt-responding cells and examine their origin and fate with or without insult.
Found that most cells responding to Wnt are stem cells involved in growth and development.
How have transgenic mice been used to track CBC stem cells?
Use Cre-ERT2 to lineage-trace Lgr5+ CBC cells shows that the Lgr+ CBC cells are stem cells (self-renew, multipotent).
What did Clevers and Nusse (2014) find out about the intestinal stem cell niche?
Clevers and Nusse (2014)
One of the most well-defined stem cell niches is the mammalian gut crypt. At the distal end, there are stem cell residing, and differentiating cells at the proximal sustained by Notch. Lgr5 is a G-protein coupled receptor expressed by crypt base columnar (CBC) cells which are pluripotent and long-lived stem cells. They give rise to all differentiated cell types of the intestinal epithelium. Trans-ampliying cells emerge from the stem cells and differentiate over 2-3 days into one of the four principal epithelial cell lineages of the intestine.
How can genetically modified mice be used to aid in human cancer research?
- Identify oncogenes and tumour suppressor genes and how they control signalling and act in cancer
- Predict which genes might give rise to cancers if mutated
- Impact on diagnostics and treatment
- Understand how stem cells can go out of control
How are stem cells linked to new therapy for breast cancer?
Cancers are present in mixed populations of stem cells and progenitors either of which can overproliferate. Some breast cancers are resistant to commonly-used anti-oestrogen therapies. New rationale is to treat these patients with a combination of anti-oestrogen to target progenitors and anti-Notch/Wnt/Hh to target breast cancer stem cells.
Can we replicate human diseases in animals, particularly CV disease?
No, but we can model some disease processes and elements involved.
There appears to be enough shared mechanisms in zebrafish to justify their use in CV disease. Can cure CV disease in zebrafish but this has not yet translated to humans.
What is congenital heart disease?
Common form of heart disease (1% of all live births, more common in spontaneously aborted fetuses).
Septal defects, aortic or pulmonary stenosis, AV canal defects, Fallot’s tetralogy abnormalities and single gene defects are the most common.
Often a feature of genetic diseases through single gene mutations and chromosomal abnormalities but not generally known.
Can reduce life expectancy.
What are the features of human cardiovascular diseases?
Many ‘preventable’ elements.
Usually manifest in 40s, 50s, 60s.
Maternal factors like nutrition have an effect on CV development.
Low birth weight associated with a risk of heart attack, diabetes and hypertension in adult life.
Adult ‘lifestyle choices’ programmed in childhood.
How can normal parts of the heart go wrong?
MYOCARDIUM (leads to heart failure)
Reduced ability of heart to pump blood sufficiently. Caused by prior MI, hypertension, valve disease, alcohol, genetic disease. Symptoms of breathlessness and swelling.
VALVES (leads to valve disease)
Stenosis or leaking/regurgitation of heart valve caused by deposits of calcium. Congenital or acquired. Some genetic contribution.
CORONARY ARTERY DISEASE
Biggest cause of death. Blockage of coronary artery caused by progressive atherosclerosis leading to thrombosis, visualized using injections of X-ray contrast in angiograms.
RHYTHM DISORDERS
Assessed using electrocardiogram to measure Q-T interval. Normal cardiac cycle tightly controls atrial and ventricular contraction (depolarisation). Genetic predisposition.
What are the disadvantages of current CV disease models?
Often mice. Relatively expensive. Only partial success in modelling disease, good for artherosclerosis, moderate for hypertension, poor for valve disease. Difficult to genetically manipulate. Very difficult to image the vasculature.
How to visualise the vasculature when looking at animal models of CV disease?
In non-transparent organisms, a technique is needed to see blood vessels. Angiography involves the injection of x-ray opaque dye followed by an MRI. This takes 4 hours, often kills the mouse and you can’t see the capillaries.
Confocal/fluorescent microscopy in transgenic lines can image blood or endothelium. Digital motion angiography shows the sum of subtracted frames. Much quicker and allows for investigation of physiology and anatomy.
What CV diseases have been modelled in Zebrafish?
Beis et al (2005)
Genetic dilated cardiomyopathy - easy to model, identify common familial mutation, KO in zebrafish. Informs human screening.
Cardiac defects - Fish have simple single circulation. But patterning signalling are similar.
Coarctation of aorta - narrowing of aorta. Rare condition. Look for genes in aortic formation. KO for phenotype.
Heart regeneration - After a MI, damage is permanent, but adult fish can regenerate their hearts.
Thrombosis - Can label thrombocytes to assess clotting.
Drug-induced long QT syndrome - Identify genetic KO that predispose to defects in heart rhythms. Submerse zebrafish in different drugs.
What are the advantages of the zebrafish?
Cheaper, smaller, genetically tractable.
Able to oxygenate via diffusion.
Vascular visualisation is much easier.
What are the weaknesses of Zebrafish?
Small size limits some techniques.
Single circulation.
Genetically less similar to humans than mammals.
Rapid development - moving baseline.
No spontaneous cardiovascular diseases similar to humans and can’t give them CV disease.
How is inflammation linked to progression of disease?
Cancer Research (2013) Initially Rudolf Virchow
Inflammation is thought to increase the progression of cardiovascular disease, cancer and Type 2 diabetes.
What did Myant et al (2013) find that showed that inflammation lead to malignant colon cancer?
Myant et al (2013)
Constitutively active Wnt signalling leads to increased active Rac1, leading to hyperproliferation. Rac mediates this effect via ROS and NF-kB, indicating a role for inflammation in progression of a benign to a malignant tumour.
NF-kB is pro-inflammatory factor that positively regulates the expression of proinflammatory genes including cytokines and chemokines.
ROS are key signalling molecules that play an important role in the progression of inflammatory disorders.
How does inflammation encourage tumour growth?
As a tumour grows. its demand for nutrients, oxygen outstrips its supply. Cancer cells begin to secrete proinflammatory signals, including cytokines which will attract more inflammatory cells.
Macrophages invade the tumour, and begin to secrete even more cytokines that kick start angiogenesis (growth of new capillaries). Inflammatory signals and cells also help break down extracellular matrix, to promote metastasis.
How was Cyclopamine found to antagonise Smo?
1940-50s: Pregnant sheep eating corn-lillies gave birth to cyclopic lambs.
1960-70s: Cyclopamine identified.
1970-80s: Hh gene identified that when mutated would make drosophila curled and spikey.
1980s: Vertebrate homologues Shh cloned and characterised - expressed down the midline.
1990s: Hh/Shh pathway was characterised. Ptc inhibits Smo, activation of Smo needed for activation of Shh pathway.
1990s: Shh -/- KO leads to holoprosencephaly in mice and humans.
In the 1990-2000s how did scientists’ view of Shh’s role change?
It was seen that shh signalling is usually at high levels suring building of the tissue but when its finished there are low levels negatively regulated at many end stages.
2000s: Key idea emerges that although initially involved as a patterning protein, many of the genes activated by Shh signalling are genes involved with proliferation and stem/progenitor cell renewal e.g. PTCH1, CycD1, Myc, Bcl-2, NANOG, Sox2
