User:Alexsheh/hp

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genomics

from DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling

  • mrna is about 10% of total RNA in bacteria
  • vast majority of mRNA not polyadenylated
  • How much total RNA is obtained from HP extractions?
  • E. coli genome size 5.1 to 5.3 Mb
  • B. subtilis genome size 4.2 Mb

From QIAGEN's RNeasy Bacteria Protection Mini Kit

5 x 10^8 E. coli in Minimal medium yield 25 μg of Total RNA

5 x 10^8 E. coli in LB yield 70 μg of Total RNA

1 x 10^8 B. subtilis in Minimal medium yield 8 μg of Total RNA

1 x 10^8 B. subtilis in LB yield 15 μg of Total RNA

  • HP's genome is about 1.6 Mb and we are using Brucella broth. Assume it's like LB... hmmm E. coli's yield is about 5 times the total yield of B. subtilis, but it also uses 5 times more bugs.

So let's say about 5x10^8 5Mb bugs yield 70 ug. A260 of 1 ~ 40 ug/ul of RNA RNA is single stranded. let's say a nucleotide is 300 daltons and a dalton is 1.650x10^-24 grams. so a nucleotide is 4.95x10^-22grams.

Alright so 70 ug / 4.95x10^-22 grams should give me 1.41x10^17 bases, well that's 141,000,000,000,000,000 bases or 141,000,000,000 Mb per 5x10^8 bugs each with a 5Mb genome. Considering that 5 x 10^8 bugs at 5Mb each = 2.5 x 10^15 base PAIRS (it would be double in bases but we're dealing with RNA), so i should be getting 5.66 times more RNA bases for each DNA base pair. Makes sense that there is more because DNA is for storage.

So back to the pylori story and running with these numbers... 1.6Mb * 5.66 = 9.05x10^7 bases. That is 4.48 x 10^-14 grams/bacteria. Supposing a gerbil stomach has between 10^4-5 bacteria, we get 4.48x10^-10 grams/stomach using the more conservative estimate. That's about 0.448 ng per stomach. I believe the desired quantity for RT is 2ug which would bring the desired number of gerbils per array to 4464.286... talk about an impossible experiment.

Actually since only 10% of total RNA is mRNA... that would make it 44000 gerbils... there must be something wrong here.

Helicobacter pylori

From Dunn et al. 1997 in Clinical Microbiology Reviews "Helicobacter pylori"

Where it is found in the stomach. Not in gastric mucosa but in the mucus layer overlying the tissue.

Culture time of isolates. Needed longer than usual culture time (5 days vs 3 days) to see colonies.

Diseases HP is linked to:

  • inflammation in the gastric mucosa (chronic superficial gastritis)
    • normally asymptomatic. Not a disease per se.
  • PMN infiltration (chronic active gastritis)
  • duodenal ulceration
  • gastric ulceration
  • peptic ulcer disease (HP is major cause)
    • used to be considered idiopathic, but HP eradication leads to cure
  • adenocarcinoma of stomach-DISTAL/lower not proximal/top. World’s most important gast. malignancy.
    • Associated strongly with development of ATROPHIC GASTRITIS (precursor lesion)
    • Gastric cancer is second leading cause of cancer death worldwide
  • Also affects the lymphoid system
    • gastric non-Hodgkin’s lymphomas
    • gastric mucosa associated lymphoma tissue (MALT) lymphoma
  • Many of these can be corrected with antimicrobial therapy
  • Menetrier’s disease (gastric folds are hypertrophic) is believed to have some correlation

Epidemiology

  • Developing countries: 70-90% of population. Infection acquired b4 10 yrs age.
  • Developed countries: 25-50%. Lower as less kids infected.
  • “Submerging” disease as it used to be close to universal and now less than 10% of kids get infected.
  • A similar disease but milder gastritis is caused by H. heilmanii

Transmission

  • No known reservoir outside the human stomach.
  • Three putative routes
    • Iatrogenic: doctor’s endoscopes
    • Fecal-oral: Most important but fecal isolation is uncommon (is shedding intermittent? Or VBNC?)
    • Oral-oral: premastication of food

Morphology

  • Spiral, microaerophilic, gram-negative bacteria
  • Shape varies depending on environment
    • Seem bluntly rounded on ends in biopsy specimens. Rod-like in solid media culture (few spirals).
    • After prolonged culture, will become more coccoidal.
      • U shaped bacilli joined by a membranous structure
      • Coccoids are metabolically active but can’t be cultured in vitro
  • Size is 2.5 to 5 microns long and 0.5 to 1.0 microns wide.
  • Has 4-6 unipolar sheathed flagella, each about 30 microns long and 2.5 nm thick.
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