MMG111 - MT1 TA NOTES

Describe various “scales” of microbes as compared to macroscopic organisms how such scales in microbe-host interactions and ecology contrast with ecology in macroscopic forms of life

1. Difference between absolute and relative abundance

2. Microbiome diversity differs depending on the part of the body, both in overall amount as well as microbial compositions

   1. Additionally, a lot of diversity is unknown

   2. Composition can also differ for the same part of the body between different individuals, yet still maintain the same function

3. Microbial physiology influences where a microbe can be found

   1. Resources it can utilize, conditions it does well in, etc…

   2. Also means the environment influences microbial composition

4. Microbes have high metabolic diversity relative to humans

   1. Faster generation times, microbial ecosystems have many niches with different characteristics, and potential for gene sharing contribute to this

5. Understand whether an attribute of an ecosystem is abiotic or biotic

   1. Abiotic 

      1. Physical environment or setting/host

      2. Chemical gradients (e.g., oxygen)

      3. Biomolecules (carbohydrates, sugars, lipids proteins, metabolites)

      4. Cycling or “recycling” of nutrients

   2. Biotic

      1. Organisms - species, populations, diversity, relative abundance of taxa

      2. Interactions, food webs, networks

      3. Producers/consumers/symbionts/viruses

      4. Structured environments - biofilms

Explain some types of cognitive flaws or biases in scientific reasoning and how these may impact microbiome research study design and interpretation

1. Questions that can improve microbiome interpretations

   1. Association interpreted as causation

      1. What alternative causal variables remain?

   2. Selective use of evidence

      1. Were all outcomes and analyses reported?

   3. Post hoc patterns as predictions

      1. Was the hypothesis specified before the analysis?

   4. Circular reasoning or false dichotomies

      1. Was dysbiosis independently defined and measured?

   5. Generalizing beyond the evidence (over-claiming)

      1. Does the result replicate in another population or study?

   6. A strong study should make methodologies, alternative explanations, and limitations visible (as well as the claims)

1. Understand common logical fallacies and be able to identify them

   1. Causal fallacy: interpreting correlation as causation

   2. Confirmation bias: having an expected result influence the investigation

      1. Cherry picking/p-hacking involve selective use of data that supports an expected result 

      2. Failing to test alternative hypothesis

   3. Post hoc patterns: claims being presented as hypothesis

      1. Indicates significance of a trend was applied after observation, meaning the experiment was not designed with that trend in mind

   4. False dichotomies: assuming results stem from one of two possibilities

   5. Circular reasoning: premise and conclusion are the same and lack outside evidence

   6. Over-claiming: insufficient scale and evidence for how generalized the presented claims are

      1. Needs to increase sample size, sampling duration, and sampling method

Describe the process of hypothesis “falsification”

1. Scientific experiments need to rule out hypotheses that are clearly wrong

   1. A hypothesis must be testable and can be falsified

   2. Experiment should either reject, retain, or revise the hypotheses

      1. Hypotheses can be falsified but not proven

Define the elements and purposes of sections of a scientific paper

1. Hypothesis: a testable explanation for an observation

2. Prediction: an expected result if the hypothesis is correct

3. Evidence: observations and analyses used to evaluate the prediction

4. Claim: the conclusion supported by the evidence

5. Reasoning: why the evidence strengthens or weakens the claim

6. Assumptions: conditions required for the reasoning to remain valid 

Contrast anabolism and catabolism and how they can be linked

1. Catabolism breaks down various compounds to generate ATP by phosphorylating ADP with an inorganic phosphate (polymers → monomers)

2. Anabolism uses ATP and raw materials to produce various products (monomers → polymers)

   1. Necessary for cell differentiation and growth

   2. Compounds include: Polysaccharides, lipids, RNA, DNA, and proteins

Describe the primary composition of life (elements, biomolecules, etc.) and relative ratios of components

1. Rules of a Cell

   1. 1: Cells are composed of four main biomolecules

      1. Protein (50%)

         1. Ribosome

      2. RNA (20%)

      3. Polysaccharide (10%)

      4. Lipid (10%)

      5. DNA (3%)

   2. 2: Cell composition is conserved

      1. Central metabolism is conserved

Define minimal essential components for cellular growth 

1. Carbohydrates

2. Amino acids

3. Vitamins

4. Inorganic Salts

Explain general differences between various ways microbes make ATP (catabolism) 

1. Glycolysis is a big module in ATP production

   1. Same as TCA cycle

   2. Pathways include more than one enzyme

2. Aerobic respiration: organic donor/O₂ acceptor OR inorganic donor/O₂ ACCEPTOR

   1. Higher ATP yield per cycle

3. Anaerobic respiration: organic donor/acceptor not O₂ OR inorganic donor/acceptor not O₂

4. Fermentation: organic donor/acceptor

   1. Lower ATP yield per cycle, but at a faster rate than cellular respiration

   2. Alcoholic fermentation produces ethanol + CO₂

   3. Lactic acid fermentation produces lactate

Compare aerobic respiration and anaerobic respiration (electron donors, acceptors, products, substrates, ATP yield)

1. Understand the role that redox reactions have in metabolism

   1. Glucose as an electron donor gets oxidized

   2. Oxygen as an electron acceptor gets reduced

      1. Fermentation uses organic compounds as electron acceptor

1. Aerobic respiration: organic donor/O₂ acceptor OR inorganic donor/O₂ ACCEPTOR

   1. Higher ATP yield per cycle

2. Anaerobic respiration: organic donor/acceptor not O₂ OR inorganic donor/acceptor not O₂

3. Fermentation: organic donor/acceptor

   1. Lower ATP yield per cycle, but at a faster rate than cellular respiration

   2. Alcoholic fermentation produces ethanol + CO₂

   3. Lactic acid fermentation produces lactate

Explain the impacts of oxygen on metabolism in various types of microbes

1. Oxygen relationships of microorganisms define where they can be seen inhibiting, and what type(s) of metabolism they use

   1. AEROBES

      1. Obligate

         1. O₂ required

         2. Aerobic respiration

      2. Facultative

         1. O₂ not required, but growth better with O₂ 

         2. Aerobic respiration, anaerobic respiration, fermentation

      3. Microaerophilic

         1. O₂ required but at levels lower than atmospheric

         2. Aerobic respiration

   2. ANAEROBES

      1. Aerotolerant

         1. O₂ not required, and growth no better when O₂ present

         2. Fermentation

      2. Obligate

         1. O₂ harmful or lethal

         2. Fermentation or anaerobic respiration

2. Oxygen and hydroxyl radicals are toxic

   1. Aerobes have enzymes to detoxify (anaerobes lack these enzymes)

      1. Catalase

      2. Peroxidase

      3. Superoxide dismutase

      4. Superoxide dismutase/catalase in combination

      5. Superoxide reductase

3. General names of enzymes that degrade polymers to a monomer

   1. Sucrase

   2. Protease

   3. Lipase

Contrast fermentation and respiration (electron donors, acceptors, products, substrates, ATP yield)

1. NAD+ is reduced to NADH when it accepts an electron and NADH is oxidized to NAD+ when it drops off those electrons

   1. Constantly undergo turnover to maintain balance and when NAD+ cannot be generated by the ETC, it can use fermentation instead, turning pyruvate into lactate or ethanol

Compare and contrast different types of fermentations with respect to substrates and products

1. Fermentation can use diverse substrates (cellulose and hemicellulose, mucin, sugars, fatty acids, proteins) to make diverse products (fatty acids, alcohols, aldehydes)

Compare/contrast methods for identification of microbes (growth, microscopy, taxonomy, and phylogeny)

1. Selective enrichment technique is a method that either directly increases a specific microorganism or inhibits other microorganisms

   1. One way or another, growth of the desired microorganism is favored

   2. Requires an understanding of a microbe’s biochemical properties such as what nutrients it requires and what conditions it can handle

2. Morphology, motility, metabolism, physiology, cell lipid chemistry, cell wall chemistry, and other traits are characteristics used to classify microbes

3. Know common problems for discerning evolutionary relationships between microbes

Describe rationale for use of ssu rRNA as a “proxy” for organism phylogeny

1. Community sampling: amplification of ssu rRNA (snapshot)

   1. Group-specific primers based on conserved and variable regions of ssu rRNA

Define steps in ssu rRNA analysis of a single microbe

1. Ssu rRNA analysis

   1. Isolate DNA

   2. Make copies of rRNA gene

   3. Sequence DNA

   4. Align rRNA gene sequences

   5. Generate a phylogenetic tree

Describe rationale for cultivation-independent approaches in general

1. Necessary because being able to culture microbes already requires some understanding about them

   1. Can determine type of microbes and microbe abundance

      1. Community sampling: amplification of ssu rRNA (snapshot)

         1. Group-specific primers based on conserved and variable regions of ssu rRNA

      2. Environmental genomics: genome sequencing (identifies all genes)

         1. Can also predict

Compare and contrast alpha diversity and beta diversity

1. Alpha diversity (species diversity) is within sample variation

   1. Metrics include richness, evenness, Shannon diversity index, and Simpson diversity index

   2. Typically illustrated using rarefaction and Shannon boxplots

2. Beta diversity is between sample variation (comparing samples)

   1. Typically illustrated using PCoA plots

      1. Should understand the axis of a PCA plot and how to calculate the total amount of variation a plot accounts for

   2. Metrics include Bray-Curtis dissimilarity (0-1 of how dissimilar two samples are)