Microbome epigenetics

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  • Created by: Sarah
  • Created on: 18-05-19 17:44
the microbome incdeces epigenetic changes which affect what?
host immunity, intestinal cells and health of host
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what does the human gut harbor?
its a very complex ecosytem has approx 1 trillion microbes
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what are microbes mostly?
prokaryotes
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how many species of microbes are there in the gut?
around 5000
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what is the gut ecosystem called?
microbiome
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whats mroe the total number of these microbes or the number of human cells?
more microbes than human cells of host
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why is this (so many microbes)?
prokaryotic cell is really small and eukaryotic cells are much larger
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what are gut microoragnisms important for?
normal digestive function and gut health, they co evolved iwth host but actions spans further than just digestion control
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2 questions to study microbe?
1) how to undertsnad which species are present in healthy conditions and how this changes in disease 2) how to study this complex ecosystem- how to model role of microbe in health
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what technique is used to see which species are present?
next generation sequencing (inappropriate for such a large number), or metagenomics
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what do the most common strategies in next gen sequencing involve?
generating a library of DNA fragments and then sequencing short reads either from a single end or paired ends
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what do you do after that?
using bioindormatical approaches aligning the resulting reads to each other and to a reference genome
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what will the reference genome tell us?
see which kind of mutations are in this particular patients if we're sequencing patients samples or genomic rearrangements and any info withint he DNA/RNA
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Why is this strategy difficult with the microbome?
because it uses a reference gene but there's more than 5000 species which makes the approach hard
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what problems do you get when sequencing the composition of microbiome species with the reference genome?
don't know what reference genome to align it to when you get a read
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what is the problem with the numbers of species?
so many genomes- some from underrepresented species are present in low nums in the microbiome not represented by different reads
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what is the problem with highly conserved genomes?
genes are highly conserved so if genomes overlap but not fully is that because a mutation or is it coming from a different species
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what is the practical problem?
identification and removal of contaminants- 5000 species, contaminated by 1 hard to say if its a contaminant or real species
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what are the 3 main approaches for overcoming these problems and analysing the complex complex specieis composition by sequencing?
1) analysing marker genes 2) grouping sequences into different taxonomic groups and analysing composition based grouping of sequences 3) de novo sampling sequences into distinct genomes
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what marker genes which are taxonomically informative are mostly used?
16S RNA
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when else is metagenomics used?
when people do probes of soil or water to understand the composition of the species based on the DNA
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how does analysing marker genes work?
a marker gene would be present in all different species (or most) and this is used to assess the diversity and the taxonomy of the population
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what is the main drawback of this method?
it relies on 1 of several markers which will not necessarily be present in all of the species
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why is it sometimes not useful for distinguishing evolutionarly close related species?
genes present in all species are really important for function they'll be similar between closely related species so you won't be able to disntinguish them
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what happens when a less evolutionary or functionally important gene is used as a marker?
will probably be more diverged between closely related species so they are not likely to be present in other species so finding a marker gene which covers the whole range of microbes is challenging
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what is the binning approach?
can be done by aligning to pre-defined known sequences of the known genes or can be done based on CG content of genome
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why can binning be done based on the different CG content of the genome?
different species have different CG content of frequency of different repeats
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what is binning using prefined known sequences or CG content used for?
to build a taxonomy to understand which kind of specieis and what is the relevant amount of different species present
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why is binning much more computationally challenging?
it deals with a lot of different seqs which has to be aligned to diff things
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what is the most reliable approach to understanding what species are present and the only one that allows you to identify new species?
assembling de novo full genomes
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what does binning and using taxonomic markers rely on?
all species being known
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why is de novo sequencing full genomes better?
its more accurate, identifies new species, identifies which species are present and in which relative amounts
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what is the most computationally challenging technique?
de novo sequencing- relies on really deep sequencing so that all genomes even if they're underepresented are present
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what mice were used to model the role of the microbiome?
germ free mice
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what did they expect to get from germ free mice? (why they created them)
thought if we got rid of all germs we'd live forever without disease
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what did they actually find?
the mice died
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how were germ free mice originally created? why?
by aseptic c section as a lot of microbes are transferred from maternal animal to pups during birth
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how are mice kept in these experiembts?
in a sterile germ free environment and researchers dont have direct contact with the animals
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how can you see the impact of different compositions of different species?
germ free animlas can be supplied by donor microbioata or synthetic combinations wgucg was created by pooling different species of microbiota in the lab
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what did this provide a lot of knowledge about?
how microbes regulate mice's heath and disease
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what did they learn from the germ free mice?
idealistic vision of no disease and living long was wrong- without germs won't be healthy and live long
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animals that are raised germ free have what problems?
dofferent behavioural changes including cognitive defects and altered anxiety, neural function changes, stress hormone sig defects
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what were the changes in neural function?
alteration in synapse related gene expressions and altered neuronal activity
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what was the defects in stress hormone signalling?
altered GR, GR signalling is affected by germs
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what were the gut microbiome shown to control
stress, brain function and neuronal activity
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what do microbiota supply to the host?
produce and supply the host by releasing vitamins B22, B6, and B12 and folate
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what members of microbioata are known to do this?
bifobacterium and lactobacillus genera
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what are 1C metabolism products supplied by?
diet and microbiome
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what is SAM?
the key donor of methyl groups required for DNA methylation
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without supplemetation of food with folate what serves as the main sourc of folate?
microbiome
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what metabolites do gut microbiota produce to alter the epigentic code?
short-chain fatty acids and polyphenols
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examples of short chain fatty acids?
butyrate, acetate propionate, succinate and lactate (all the ate)
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what are short chain fatty acids for?
incorporated by the intestinal epithelial cells or can diffuse across epithelium into the underlying intestinal lamina propria and enter bloodstream
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why can they get into the bloodstream?
because they are tiny diffusible molecules
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what polyphenols are there?
phenylacetate and pheylbutyrate
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what are polyphenols transformed into in the gut?
aromatic short-chain fatty acids
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what is the difference between short-chain fatty acids and polyphenols?
polyphenols have a aromatic addition to it
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what is the most known short chain fatty acid produced by the microbiome?
butyrate
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what does butyrate do?
is an inhibitor of HDACs- histone deacetylases
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what has butyrate been linked to?
anti-cancer activity, activation of prop-apoptotic genes and activation of cell cycle inhibitor P21
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why would it be anti cancerous?
beacause it activates the cell cycle inhibitro P21 and that prevents the cell cycle which prevents tumour growth and tumourgenesis
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what happens to P21 expression in cancer cells
P21 is known to be epigenetically silenced in cancer cells
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what signalling pathway is butyrate involved in?
wnt signalling
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why is butyrate contorlling wnt signalling going ot be anti-cancerous?
wnt signalling is constiutively active in most corectal cancers so inhibiting the signal by butyrate caqn prevent cancer growth
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why is butyrate suggested to be involved in anti-obesity?
it increases expression of PGC1- alpha and that modulates wnt signalling, dietary intake of butyrate prevents diet induced obesity and insulin resistance in mice models
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what happens to PGC1 alpha expression in calorie restriction?
SIRT 1 deacetylase activate PGC1 by deacetylating PGC1 alpha and it increases mitochondrial biogenesis activity (more energy produced)
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what happens to PGC1 alpha in high calorie excess?
activates acetyltransferase which inhibits PGC1 alpha function so no mito biogenesis less energy production
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how does butyrate supplementation increasing PGC 1 alpha expression prevent obesity?
same portion of protein is acetylated/deacetylated, the proportion of protein in calorie excess not acetylated would be higher even with excess more PGC1 active and increase mitochondrial activity and fat release
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how could you test this?
looked at butyrate supplementation altering the amount of acetylated verus total PGC1 alpha protein
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functions of butyrate?
1) anti-cancer 2) anti-obesity 3) anti-inflammatory
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how was it shown to be anti-inflammatory?
supplementation of butyrate following the inhibition of deacetylases that follows increases expression of FoxP3 in naive peripheral CD4 T cells
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what does increasing expression of FoxP3 gene do?
results in an increase in circulating T regulatory cells and these cells reduce local inflammation
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what does this tell you about this small diffusible butyrate molecule?
butyrate produced by gut bacteria, acting and inhibiting the deacteylase enzyme change multiple phenotypes which aren't were the gut bacteria are eg neuro epigenetic changes
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which mouse model was used to study cognitive function and behaviour changes caused by butyrate?
BTBR mouse model
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what is the BTBR mouse a model for?
mouse model of autism
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what is interestin about this BTBR model?
its not a single mutation and the genetic nature of this model is unknown its an inbred line of mice
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what does the inbred line of mice mean for the genotype?
they all have the same same genotype but multiple changes in comparison to normal mice
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what phenotypes do they display?
display some phenotypic traits of all diagnosed symptoms of autism
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what did the authors look at with the BTBR mice?
they supplied different concentrations of butyrate to these mice and looked at how their behaviour changed
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what behaviourdid they look at thats linked to autism?
sterotype pattern of behaviour of marble burying, the mice given small objects they try to bury it in different places
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what did butyrate do to this action?
butyrate supplementation significantly reduced time spent on the stereotypic actions
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what did butyrate supplementation do to acetlation?
increased total acetylation of histone 3
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what was this increased acetylation associated with?
differential expression of groups of different genes
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what were the genes whose epxression was affected to do with?
transmission of nerve signalling, cell-cell signalling, genes involved with all these processes
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what did the authors conclude?
that butyrate supplementation modulates the excitation/inhibition balance in the prefrontal cortex in the brain which has positive effects on social behaviour in the autism mouse model
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what kind of microbes produce polyphenols?
bacteroides and clostirdium species
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where do the polyphenols come from?
they are supplied in the diet by plant polyphenols eg plant or veg food that have dif polyphenols that's broken down into smaller polyphenols produced by gut microbes
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what do polyphenols do?
inhibit HDAC
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why is the inhibtion complex?
the degree of inhibition varies from one polyphenol to another, diff microbes break down same plant polyphenol into diff compounts so hard to predict effect on HDACs
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what else do some polyphenols inhibit?
DNA methylases
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example of polyohenol contained in different fruits and nuts
ellagic acid
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how does supplementation of ellagic acid alter microbe ecology?
decreases ratio between 2 diff species because one species is disadvantaged and can't metabolise ellagic acid so one that can metbaolise it benefits and survives
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what happens to microbes that can metbaolise ellagic acid?
they produce different metabolites from ellagic acid with different activities,
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what will the final result of breakdown of ellagic acid in the gut by microbes depend on?
intake of ellagic acid in nuts and fruit but also the composition of microbiome which is affected by intake of ellagic acid
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how do ellagic acid and butyrate (short-chain fatty acids and polyphenols) act in similar ways?
both inhibit histone deacetylase enzyme
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what does the inhibiting DNA methylase and HDACs by polyphenols mean for activities?
gas a wide range of consequences an wide range of activities int he prevention and elevation of disease
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what diseases does butyrate affect?
obesity, cancer, cognitive function and behaviour
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what do members of microbiome produce?
produces vesicles out of their surface, both gram positive and negative bac produce them
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what were the vesicles found to contain
various RNAs
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what is the difference between positive nad negative gram bacteria?
release of the membrane vesicles is either from outer M or outer inner M
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what were the RNA in the vesicles suggested to function as?
micro RNAS
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why were they suggested to act as micro RNAs?
they get in the host cell are released and regulate expression of host genes in the same pathway micro RNAs use
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how big are the secreted RNAs? what are the enriched in?
generally smaller than 60 nucleotides, they are enriched in various non coding RNAs and are very distinct from bac IC RNAs
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what does the RNAs secreted from the gut bac being very different from the bac IC RNAs raise qs of?
how these are secreted, where they evolved from
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what did these RNAs matched when aligned to a human genome?
when these microRNAs were aligned with a human genome majority mached regions representing DNAase I hypersensitivity clusters and intronic regions with elevated transcription levels
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what genes were aligned when they sequenced them?
epithelial apical-basal polarity or cell adhesion which might link to integrity and function of the gut epithelium but unknown functional relevance
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2 main uses to apply the knowledge of gut microbiome to human health?
1) probiotics 2) stool transplant therapy
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what happens in stool transplantation therapy?
feccal samples are transplanted from a healthy subject to an unhealthy subject
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what did the research show with immunotherapy for cancer that didn't work in some cancer patients?
stage 2 clinical trials feccal tranlats from patients who responded well to cancer immunotherapy improved the response to those who were unresponsive originally
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Card 2

Front

what does the human gut harbor?

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its a very complex ecosytem has approx 1 trillion microbes

Card 3

Front

what are microbes mostly?

Back

Preview of the front of card 3

Card 4

Front

how many species of microbes are there in the gut?

Back

Preview of the front of card 4

Card 5

Front

what is the gut ecosystem called?

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