Human micropath Exam 2

What is microbial census?

Identify which microorganisms occur in a sample and estimate their abundance.

What does qualitiative microbial census look like?

What is present in the census.

What does quantitiative microbial census look like?

How much is present in a census.

What do culture-independent tools detect?

These detect organisms that are difficult or impossible to culture.

Describe the 16S rRNA gene sequencing approach.

Bacterial community identity and phylogeny using conserved primer sites and variable sequence regions that discriminate taxa.

Describe the qPCR/quantitiative PCR approach.

Specific DNA targets or organism abundance (need to know what you are looking for!!!); fluorescence rises with amplification; lower Ct = more starting target (inverse relationship).

Describe the metagenomics approach?

DNA/genes from the entire community, including unculturable members; can indicate genetic potential and relative community composition.

Describe the transcriptomics/RNA-seq approach.

Expressed RNA and changes in gene transcription. Dual RNA-seq can examine both microbial and host transcripts.

Describe the proteomics approach.

Protein abundance/identity and expreses functions. Digest proteins into peptides, identify by mass spectrometry and database sequencing matching.

Describe the metabolomics approach.

Small-molecule metabolites, cellular activity and physiological state; mass spectrometry or NMR.

What is 16S rRNA used for in microbiology?

A marker gene for identifying bacteria and studying bacterial community composition.

Is 16S rRNA a bacterial or viral marker?

Bacterial (and archaeal). It is not a viral marker.

What are conserved regions of the 16S gene?

Streches of sequence nearly identical across almost all bacteria.

What is the function of the conserved regions in 16S sequencing?

They allow universal primers to bind, so the gene can be amplified from nearly any bacteria.

What are the variable regions of the 16S gene?

Streches that differ between taxa.

What are variable regions useful for?

Taxonomic distinction: telling one bacterial group from another.

Which part of the gene lets you amplify everything, and which lets you tell things apart?

Conserved regions amplify; variable regions distinguish.

How do you identify community membership from 16S data?

Compare your sequence to reference databases (ex. SILVA, Greengens, RDP)

What does "Community membership” mean?

Which taxa are present in the sample (and in what proportion)

What do you infer after aligning 16S sequences to study relationship?

A phylogenetic tree

What does the root of a phylogenetic tree represent?

The shared common ancestor of all taxa on the tree.

What does branch length often represent?

Evolutionary distance, amount of sequence change.

On a tree, what does a longer branch usually indicate?

More divergance from the node it branches from.

Wht can't 16S sequencing detect viruses?

Viruses have no ribosomes, so they have no 16S gene, and no single gene is shared by all viruses.

What would you use to study viral communties instead of 16S?

Shotgun metagenomics.

What markers are used for fungi/eukaryotes instead of 16S?

ITS or 18S rRNA.

WHat does 16S sequencing tell you and what can it not tell you?

Tells you who is there and how they are related; it doesnt directly show function.

What does qPCR add compared to standard PCR?

Real-time, quantitative detection of amplification as it happens.

How is standard PCR typically read out?

At the endpoint, giving qualitiative result (product present or absent)

What does Ct stand for?

Cycle threshold

WHat is the Ct?

The cycle at which flourescence crosses the detection threshold.

What does a lower Ct mean?

Higher internal gene abundance (more starting DNA)

What does a higher Ct mean?

Less starting DNA (lower initial abundance)

Sample A has Ct=18 and sample B has Ct=27. Which has more starting template?

Sample A, because it crossed the threshold in fewer cycles.

Is the relationship between Ct and starting amount direct or inverse?

Inverse, lower Ct means more template.

What are the two kinds of controls in qPCR mentioned?

Positive controls and negative controls

What is the purpose of positive and negative controls?

To detect assay failure and contaminations.

What does positive control tell you?

Whether the assay is working. If it does not amplify the assay failed.

What does a negative control tell you?

Whether contamination is present, if it amplifies something is contaminated.

Qualitative or quantitive: standard PCR vs. qPCR?

Standard PCR is qualitative (endpoint) and qPCR is quantitiative (real time)

What signal does qPCR track to monitor amplification.

Fluorescence, measured each cycle.

If you want to know which genes are present in a community, which method?

Metagenomics

You want to know which proteins are being produced, which method?

Proteomics

You want to know which metabolites are present, which method?

Metabolomics

You want to count one specific species, which method?

Species specific qPCR

What does metagenomics tell you?

Which genes are present (the community’s genetic potential)

What do RNA-seq and microarray tell you?

Which RNA genes are up- or down- regulated (gene expression)

What does proteomics tell you?

Which proteins are produced

What does metabolomics tell you?

Which metabolits are present.

What does species-specifc qPCR tell you?

The abundance/count of one particular species.

‘genes present’ points to which method?

metagenomics

‘RNA genes up/down’ points to what method?

RNA-seq or microarray

‘proteins produces’ points to which method?

proteomics

‘metabolites’ points to which method?

Metabolomics

‘one species count’ points to which method?

Species specific qPRC

Which two methods both measure RNA-level changes?

RNA-seq and microarray

Which method targets a single species rather than a whole community?

Species-specifc qPCR

Order these from DNA to RNA to protein to small molecules by method.

Metagenomics (DNA), RNA-seq/microarray (RNA), proteomics (protein), metabolomics (metabolites).

What does SDS-PAGE seperate proteins by?

Principally size

Does gel electrophoresis amplify proteins?

No. It only seperates them. Proteins cannot be amplified the same way DNA can.

What are the steps of a western blot in order?

1. Seperate proteins (gel)

2. Transfer to a membrane

3. Primary antibody binds binds the antigen

4. Labeled secondary antibody enables detection

What does the primary antibody to in a Western Blot?

Binds the target antigen (protein)

What does the labeled secondary antibody do?

Binds the promary antibody and provides the detectable signal.

What is the purpose of a Western blot?

To detect a specific protein in a mixture, AFTER seperating by size.

Which method combines gel seperation with antibody detection?

Western blot.

What does immunohistochemistry do?

Uses antibodies to locate antigens ot pathogens in tissue selection.

What kind of sample does IHC use?

Fixed tissue section, often paraffin-embedded.

What does IHC show that Western blot does not?

Where in the tissue the antigen is located. (spatial localization)

What does ELISA stand for?

Enzyme-linked immunisorbent assay.

How does ELISA produce a signal?

Antigen-antibody binding and enzyme subtrate reaction.

How does mass spectrometry identify proteins?

By peptide passes (mass/charge) and fragment spectra, matched against refrence databases.

What does m/z stand for?

Mass to charge ratio

What does MS/MS need in order to assign an idenity?

A refrence database to mathc the spectra against.

Which method identifies proteins without antibodies?

Mass spectrometry.

Western blot, IHC and ELISA all rely on antibodies.

Separate proteins by size, no detection of a specific one?

Gel electrophoresis

Detect a specific protein after size separation?

Western blot

Locate antigens in fixed tissue?

Immunohistochemistry

Antibody + enzyme color signal?

ELISA

Identify proteins by peptide masses/spectra?

MS/MS

What are the 5 main functions of the microbiome?

1. Colonization resistance

2. Nutrient sand metabolism

3. Production of vitamine and metabolites

4. Mucosal and immune development

5. Signaling in the gut brain axis

What is the common theme of the microbiome functions?

The microbiome supports host protection, nutrition, development and communication.

What is colonization resistance?

The ability of the resident microbiota to prevent pathogens from establishing themselves in the gut.

Which function helps protect against pathogen invasion?

Colonization resistance

What does the microbiome contribute to nutrient metabolism?

It helps break down and process nutrients (ex. food compoents that the host cant digest on their own)

What does the microbiome produce that the host benefits from?

Vitamins and microbial metabolites.

What does the microbiome help develope in the host?

Mucosal and immune sevelopment (the gut linning and the immune system)

What is the gut-brain axis?

Two-way communication between the gut (and its microbes) and the brain

Which function involves communcation with the nervous system?

Signaling in the git-brain axis

What creates a direct signaling connection between gut and brain?

The vagus nerve

What does the vagus nerve connect?

The gut and the brain

What function does the vagus nerve connect to?

Gut-brain axit sisgnaling

Keeping pathogens from taking hold?

Colonization resistance.

Digesting and processing food components?

Nutrient metabolism

Making vitamins and small molecules the host uses?

Vitamin and metabolite production.

Shaping the gut lining and immune system

Mucosal and immune development.

Communicating with the brain

Gut-brain axis signaling.

What does SCFA stand for?

Short-chain fatty acids

Name the 3 main SCFAs

Acetate, propionate and butyrate

How are SCFAs produced?

By microbial fermentation of dietary fiber. (which the host cannot digest on their own)

Who makes SCFAs: the host or the microbes?

The microbes, though fermentation