Start up Ideas

Probiotics and Prebiotics

They are living microorganisms which confer a health benefit on the host, when administered adequately. It's a targeted modulation, but it depends on the gut microbiome of the patient since the bacteria can adapt or not and be expelled. Probably in the future will be new probiotics, but now they can be ineffective or even toxic.

After antibiotics, probiotics are suggested for the recovery of the microbiome, but they colonize the gut, lowering down the recovery of the personal flora. Compared to spontaneous post-antibiotic recovery (few weeks), probiotics induce a markedly delayed and persistently incomplete indigenous stool/mucosal microbiome reconstitution.

Prebiotics

They are substrates selectively utilized by host microorganisms conferring a health benefit. They aim to confer a selective advantage to beneficial members of the microbiota. More effective since they feed specific bacteria. Example: impact of short-chain galactoligosaccarides on the gut microbiome of

Lactose-intolerant individuals. The high purity prebiotic GOS resulted in adaptive shifts in the microbiome and correlated with improvement in clinical symptoms. After a trial of 45 days, the intolerance was lowered and many patients started to drink milk, because prebiotics allowed the growth of bacteria that digest lactose.

Diet: Eating onion, garlic or banana there's inulina that is a prebiotic. If bacteria aren't fed they start to eat the mucosa, that becomes thinner and the integrity of the wall is disrupted. If this happens, the wall is no more blocking bacteria from going in the blood streams causing inflammation, allergies and a lot of chronic diseases. A group of bacteria help the epithelial cells to maintain the mucosa adease, so eating vegetables prevent the leaking gut.

Fibers are fermented by bacteria, producing short-chain fatty acids, that will be energy source, modulate the gene expression and activate PRRs signaling.

Drug metabolism:

1. Activation: gut

• Microbes can convert prodrugs into active drugs.
• Gut microbes can encode enzymes that detoxify drugs.
• Gut microbes can bind directly with drugs, compromising their bioavailability.
• Some metabolic pathways share steps between host enzymes and those encoded by gut microbes. Intermediate microbial products can lead to pathogenesis.
• Gut microbial metabolites compete with drugs for enzyme-binding sites, leading to alterations in the drug efficacy or toxicity.
• The gut microbes can impact drug efficacy by stimulating the host immune system.
• The liver processes many drugs by adding glucuronic acid, to detoxify the drug and tag it for transport to the intestine and elimination. Gut microbes produce the enzyme β-glucuronidase, which enables them to scavenge the glucuronic acid and reactivate the drug, leading to toxicity.
• Gut microbes can alter gene expression.

hepatic expression of key host enzymes involved in drug metabolism. Research trend: - Microbiome modulation - Treatment Monitoring - Species isolation and characterization - Mechanistic studies, beyond association - Studying the microbiome Milestones: - 1675: Antoni van Leeuwenhoek invented the microscope and discovered bacteria. - 1953: discovery of the structure of the DNA by Watson, Crick and Franklin. - 1975: discovery of 16S rRNA by Carl Woese. - 1977: first generation of DNA sequencing → Sanger sequencing. - 1983: invention of PCR by Katy Mullis. - 2001: definition of microbiome by Joshua Lederberg; "the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease". - 2005: next generation sequencing and bioinformatics. 07/03- 16S-NGS - A way to study microbiome is the culture dependent method, allowed to study the first microorganism. It's limited since most of

Bacteria don't grow in standard conditions and require a special environment (like the human gut). The DNA sequencing it's a culture independent method, divided in Sanger and NGS. It starts from the PCR, and in the Sanger sequencing it must be cloned. The sequences must be compared to the database so it's possible to identify the microorganism.

16S rRNA: RNA present in the ribosome, it's the marker of choice for the identification of prokaryotes.

Pros:

• In bacteria and archaea.
• Part of the small subunit of the ribosome.
• 1542 bp long.
• Variable (sequencing) and conserved (primers) regions.
• Updated databases (Greengenes, Silva, RDP).
• BARCODING GENE: allows identification of the microorganism.

Cons:

• Variability in the number of copies of the gene.
• Intragenic variability (even up to 20%!).
• It's just a gene in a genome.

Illumina sequencing

1. Sample preparation.
2. Cluster generation.
3. Sequencing.

doesn’t work (no plot/visualize more than 3 dimensions). In any case, I can always calculate the distance, for every number of dimensions.

Dimensionality reduction: a new coordinate system or data representation that reduces dimension, for complex data. To find a way to say the same thing but using less data. It eliminates redundant or useless systems and see the pattern in our data visually.

PCA (Principal Component Analysis) and PCoA (Principal Coordinates Analysis), that allows to visualize high dimensional patterns in fewer dimensions.

In reality the importance of a dimension can be reduced, but can’t really eliminated. To do this kind of analysis researchers use QIIME and Mothur software tools.

Protocols problems

The 16S technique is useful to compare microbial communities, not for absolute analysis, just relative.

At each step errors are introduced:

• Sampling (Do I have to do replicas? Do I have to sequence DNA or RNA?).
• Nucleic acid extraction (Which kit? Which pre-treatment?

Are there any spores?).- Amplification and construction of the library (Which primer? Contaminants? Quality? Which provider do I choose?).- Sequencing (Which platform? Illumina or Ion torrent? Quality?).- Analysis (Which software? Which database?).

Different parameters can be chosen to determine an OTU (threshold: 97% or 99%?). 16S is not always providing the species.

· 16S-NGS limits:

• Each protocol step is a variable that can return different results.
• It is difficult to compare results produced with different methods.
• Sequences do not always allow species identification.
• Bacterial species contain bacterial strains that often perform different functions.
• The analysis of the 16S is always to be coupled with other carefully selected techniques and markers.

· Beyond 16S-NGS

The analysis of the 16S rRNA is just at the beginning:

• Metagenomics:
• Identification of Eubacteria, Fungi, Archaea, protozoa.
• Genes and genomes.
• Metabolic pathways.

Metatrascriptomics: active metabolic pathways, starting from RNA.

Metabolomics: metabolites.

Metaproteomics: proteins.

Sequencing and beyond: integrating molecular 'omics' for microbial community profiling.

Lab- Lab results -

1 uL ruler = 0.5 ug DNA, each band has different quantities, present in different %. Ex. 500 bp is 15% and so 75 ng of DNA.

(to divide by how many uL put in the gel to have the concentration of the PCR)

Group 3. No sample. Controls are good. Extra sample is too much (they told us wrong concentration) and maybe it can have gone also in the ctrl - because of the quantity (very faint band). Cloud at the end of the gel = primer residual.

Group 2. Sample higher than controls (ctrl - maybe contaminated by + control).

Group 8. Reload of ctrl -: nothing in this second run, probably is the ES that has bloated and invaded the ctrl - lane.

Controls are necessary.