User:Alexsheh
Background
Current affiliation: BE Class of 2005
Advisors: Schauer/Fox labs
Project: Working on Helicobacter pylori stuff
Ugrad: Cornell '04
Home Country: Costa Rica
Project description
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Quick work notes
9/12/06 HP SS1 swam! SS1 has slower growth kinetics but it is more motile even in the fresh media. portant. Furthermore, his new lab has reproduced the reports. His main concern was strain specificity, mentioning that 26695 is not that great. G27 is good and SS1 known for its colonization would be good too.
9/7/06 KMK's 1st day of school
8/30/06 Meeting with Roman notes
- Problem with HP motility could be due to
- Passage related problems! My bacteria were 20+
- Time of culture (try to get them in mid log… 15 hours post inoculation)
- pH of the media, should be acidic according to Merrell_Goodrich_FALKOW
- Lack of viscosity
- Also want to test viscosity of methylcellulose in water which has a viscosity of 1cp or a dynamic viscosity of 10-6 m2/s
- Need to contact Jeff Aristou about the viscometer. Prepare solutions close to 25-50 cP as determined from the chart from Sigma Aldrich. And use blank water as a control.
- Centrifuging bacteria to collect them to change the media done at 2000g for 5 min. This is normally done 3 times in the Stocker Lab.
- Expt 1 just get trajectories/movies to get motility and an idea of the speed.
- Expt 2 Vary viscosity
- Expt 3 Use T shaped microfluidic device and study variations like viscous+hi pH vs. less viscous+low pH.
Methyl Cellulose Specs on Sigma's website
8/04/06 Lax week… 008 is begun in duplicate taking od600 readings and plating out the bacteria. Obtained new samples for 009 which is the continuation of 005.
7/28/06 005 tried to ethanol precipitate sample so I can resuspend it.
006 need to measure pH in culture as paper by Merrell, Goodrich et al. 2003 says acidity really affects motility and prepare plate samples and liquid culture samples for Roman. Took samples to Tanvir and he imaged them. Unimpressed by the swimming.
7/27/06 006 saw pretty good motility for 1 day culture with staining.
005 Had new sample from TIGR and tried cleaning it up. Got much better a260 than before but still too low, and not near to what they wanted.
7/26/06 006 worked on liquid culture and the plates. Stained and saw them. They all looked viable but the liquid culture due to being overgrown seems to have form more clumps.
005 Giving up on RNA from initial extraction as yield may be too low due to ethanol usage.
7/25/06 lab fishing trip
7/24/06 Worked on a short RNA -> DNA procedure with a 5-hour incubation. Took 2 hours to prepare it as I was working to dissolve the pellet and clean it up with the RNEasy kit and then I had to get the spectrophotometer reading. Used the equation 38 x A260 x dilution factor. It’s ½ of what the spectrophotometer said the real concentration was for my sample. Tried with old and new samples and they were pretty low using the calculation. REALIZED PERHAPS THAT SOLUBILITY IS GREATLY AFFECTED WITH OVERDRYING AND I DID THAT! Stopped it after the kit, before the drying of the cDNA and the labeling with Cy dyes.
Also worked on RNA extraction following the TRIzol protocol with two ½’s of a plate and 700 ul of TRIzol.
Also looked at the microscope and am now ready to do some work for Roman. Emailed him about the next step.
Also tried to prepare the liquid culture the way the DCM protocol states, using 150ml brucella broth + 5% FCS using 1 ml of broth with bacteria as inoculant. I used ½ a plate.
7/21/06 Began reading the Sethu et al. 2004 paper on microfluidic device for erythrocyte lysis.
David meeting 7/21/06 · Scope training (key from Nancy Guillen)… look at cells on Monday and get ‘trained’ then.
o PEEVES
§ Getting oil on dry objective when switching
§ Not focusing on low and going to high.
§ Forgetting to turn off the mercury bulbs.
· Develop protocol for Motility and scope? David suggested a system from 0 to 3 where 0 is non viable. 3 is very motile. · Thought I’d work on streamlining processing time, testing several incubation times, perhaps temperature of media (would help motility I believe). Maybe take the bacterial jar to Roman’s. Don’t vortex. Page 33 in Lab book. Different conditions, different strains. · Gerbil vendors were discussed. Harlan Barrier 265I in Indianapolis seems most promising as they are rearing a clean colony. No other place has these. Discussed possibility of using antibiotics but it is very stochastic and would be hard to do ethically as many gerbils could die.
Methylcellulose… didn’t bring this up.
· Space in –80C for RNA storage. RNA extra from extractions that will be archived should be stored this way but for stuff used within weeks it is fine to use the –20C.
· For some reason during the 2 photon talk I was very interested in using it to see stomachs… DIDN’T BRING IT UP. DOESN’T SEEM SO USEFUL.
· Printing in color upstairs? Follow up with Sue or someone.
Search pubmed for microarray and microfluidics… bacteria and microfluidics… HP specific AB… or LPS specific AB
Purchases: salmon sperm, air duster and slides. When can I use Pylori grant? David will check with Dr. Fox about the way I’m going to be funded.
Need bevin’s paper…got the Spek et al. papers… need to read them.
Go through the protocols for RNA… mark possible problems. Believe that the major problem is desalting. Skipped the cleanup step. Will try again next week. Salt is possibly affecting the pmol dye incorporation. Concentration of RNA too low to begin with? Possibly the ratio of AAdUTP is off? I will focus on the extraction steps now.
Look thru trizol one. David mentioned chaotropic agents such as guanidine hydrochloride (which is the active ingredient in RLT of the Qiagen kit). This is often complimented with beta mercaptoethanol which breaks up disulfide bonds. RLT denatures proteins. This should help stop the RNAses set free. TRIzol separates the RNA. Third method mentioned by David was immediately freezing scrapings using isopentane and liquid nitrogen. Using very high tech method to analyze sample but using very low tech method to collect it… the main problems I see are speed (how fast can I get the sample fixed/frozen/processed before the transcript changes while conserving as much of it as I can), get high number of bacteria (Colonization of gut numbers) and sort these out from the mucosa and other either bacteria or host cells… Feel like bacteria are few in the gut as long as my procedure is clean in extracting the stomach then it should be fine (scraping or some way of lysing the whole stomach).Propose creating a device that can either take tissue lysate or stomach scrapings and quickly (seconds!) separate the bacteria (perhaps utilizing the LPS on Gram negative pylori using an antibody or perhaps an HP specific AB) from the mucosa and the rest… two selections? How much time? Then it would follow the trizol procedure and inject trizol, pump up and down to mix and lyse and spit it out for incubation and further processing… hopefully a small device… non microscope dependent which can be taken to the necropsy room. Sethu et al. 2004. Could roman help with this? Lots of physical interaction, lots of time, not as uniform if done the old fashioned way, but more control in troubleshooting. Diffusion problems?
Vacation ok for several weeks in August.
Talked about the gerbils. Previous literature has shown gerbils infected with SS1, G1.1 (Nashville collection, same place where B128 comes from), ATCC 43504, clinical isolates. Normally about 1 ml with concentration of 109CFU/ml. The only way I’ve seen to count the numbers of H.pylori in a gerbil is to do it at necropsy by cutting the stomach in half David suggested that I should work on proposal type things and give it to them surrounding particular experiments to get used to writing background, methods and aims.
Other possible problems mentioned in previous lab meeting, meeting with Drs. Fox and Schauer and other times · 25-30% specificity of microarrays to other helicobacter species… problems with cross-contamination. · Size of primers spotted (70mers) · Enrichment and amplification. Can it be done? Specific primers for each possible gene have been used before in TB. · Where are the spots? From the top (opposite end of barcode) and the sides, the spots are 4mm away. · What is the colonization rate vs how much RNA would you need for a microarray? Calculate how many stomachs. One paper said 102CFU/g, not sure if of stomach or of mucosal scraping. · Think about the mechanics of being in the necropsy room if the number of animals is feasible.
July 20, 2006 · Read the Peterson et al paper on the effect of CSP (competence stimulating peptide) in Streptococcus pneumoniae and how it upregulates a variety of genes that make Strep positive during exponential growth phase (quorum sensing related). Mostly for the RNA extraction protocol using hot phenol acid. · Tracked down HARLAN folks and they do have a small gerbil colony being raised to be helicobacter free! But it’s not on inventory yet. · Compiled papers and tried to think of ways to tackle project. Future directions?
July 19, 2006 · Looked at microarray slides again. Realized that the slides are not up to spec. TOO SHORT. Assessed space in cold storage. Now have space in the –80, -20 and 4C. Found goat serum from before. Asked Katie to order coverslips, air duster and salmon sperm. Read the Bergin et al paper on eradicating helicobacters in gerbils which led to death by enterotoxemia by C. diffile. However the survivors were helicobacter free 3 weeks post the treatment.
July 18, 2006
· 1st entry: another attempt at digitalizing my work for easier future referencing. · Attended the HST Methods in Bioengineering conference at Kresge for the 2nd and final day. Notes on that follow.
o 7/17/06 1st day of conference
o Ron Tompkins, Harvard Med, MGH, Shriners Burns
§ Traumatic injury and how that affects gene expression in humans. How long does it take to fully return to preinjury transcriptome? (2-3 months… weeks?) Trauma affects many structural proteins which are often sensitive and they may overreact.
§ From tools (microarrays, proteomics) to interactome to modules to cell to systems. Tompkins spoke mainly about the glue grant that he chairs at MGH dealing with inflammation and how the body responds.
§ Genomics core takes blood samples and studies the leukocytes to understand response and see if there are markers present. Take blood and remove the buffy coat (band of leukocytes). In the future the hope is to separate this into the components of the innate response like T cells, monocytes and neutrophils. Later on look at T regulatory cells. Take all this data and use bioinformatics. 10000 genes upregulated in the buffy coat transcripts and are upregulated for months and in the very sick even up to a year. 1000 genes have been seen to be very good predictors of the outcome as well as 600 proteins.
§ Most people use PAXgene which lyses the blood and stabilizes its RNA. Problem is that globin (I suppose hemoglobin) dominates this set so we see almost a single band when running out the RNA. That’s an artifact! It’s just so much that there is very small signal for the others. Taking the buffy coat focuses on the leukocytes and when run out on a blot it smears…. Showing different transcripts.
§ Mentions that the variance is very small! And it cannot be detected by spotted microarrays because in the best case they have a 0.9 sensitivity that it would not be able to pick up the slight difference.
§ Also spoke of the HUMAN LPS model where volunteers are injected with LPS, the bacterial endotoxin, to see their reaction. It is a good NFKB model and potent upregulator of the immune system (Tompkins in Nature 2005). 1 day post injection, the patients have flu like symptoms.
§ VERY INTERESTING TO SEE that NEUTROPHILS increase in quantity in that day and the lymphocytes and monocytes actually decrease.
§ Using microarrays they also noticed a 5% difference in the control groups between people that were kept in bed for the day and those that walked around for the day.
§ LPS group was monitored at 2 hr intervals. More info on gluegrant.org. Take all the microarray data and used a proprietary database of transcripts (better than KEGG) called Ingenuity… which is used by pharma.
§ Also mentioned the usage of microfluidics to lyse the blood cells and compared to ammonium chloride. Downloaded some of their protocols under gluegrant protocols folder. Registered on their site as alex_pub array706
o 2nd speaker was Joachim Kohn talked about Combi Chem used to make lots of drugs in the 60’s how something like that could be used to make lots of polymers using parallel synthesis, better screening methods and modeling using Artificial Neural Networks (ANN), etc.
o Matthew Becker, Matthew Libera and Doyle Knight were next. The 1st 2 of this group were very polymer heavy so I lost interest. Concepts like bulk erosion vs surface erosion. Materials that can be metabolized, using PEG to prevent water damage. Designing in a way that your product breaks at a slower rate than what the recovery rate is. Temp is key! Knight is more a computational guy like people in our dept. Talked about surrogate models (like QSAR, QSPR, Decision Tree, ANN, Partial Least Squares… key because they are nonlinear). Too many descriptors lead to overfitting the data and making it irrelevant. Then a slew of tissue engineering people came… doing some work on stuff like Al Grodzinsky and perhaps silks and stuff. David Kaplan and Gordana Vunjak-Novakovic who couldn’t make it so a grad student spoke.
o Irene Georgakoudi from Tufts had a very imaging oriented background but was working with David Kaplan in tissue engineering stuff. Talked about using natural fluorescence of silk and was able to show the structure of the material without staining using the endogeneous sources of contrast. The advantages of the optical methods to detect stuff is that it’s non-invasive, hi resolution and can obtain morphological and biochemical information.
o She gave a good simple overview of microscopy. Light matter interactions are basically scatter (elastic and inelastic), absorption and fluorescence. Elastic scatter is single (returns immediately like organelles and nuclei) or multiple (bounces off many things caused by collagen, elastin fibers, organelles). This helps you see the structure and morphology. Absorption like hemoglobin and betacarotene helps you understand some of the physiology. Fluorescence in NADH, FAD, porphyrins, collagen and elastin help you see some of the biochemistry as well. Thick specimes scatter more light and become fuzzy as there is more outside the plane of focus that disturbs your view. Confocal microscopes fix this problem by using a pinhole that does not allow light from other levels to come in. The problem is that the sample is still getting photobleached at the same level, but your image doesn’t show it. 2 photon on the other hand sends to weaker beams that only excite stuff where they meet because that’s where the intensity is enough. Less photodamage is done and there is no fluorescence from other planes so no pinhole is needed. She also mentioned second harmonic generation which I’m not sure what it was supposed to be. Silk scaffolds and stem cell viability were observed. Also wanted to look at artificial tissues as models of cervical cancer induced by HPV as more of the variables could be controlled.
o Jason Burdick (Upenn) spoke of his photocrosslinkable polymers to entrap cells and Jeff Morgan (Brown) spoke of making non adherent spheroids of cells using a special hydrogel (agarose).
· 7/18/06 notes
o Martin Yarmush (MGH/Harvard Med) was the keynote speaker and talked about real time genomics for analyzing dynamic cell and tissue processes due to inflammation. Local inflammation occurs all the time but systemic inflammation is not as addressed until recently. This is when the body marshals resources and gets into defensive posture (INNATE). Trying to find good markers in the trauma patients it has been found that c-reactive protein and serum amigloid A increase and are good markers of systemic inflammation. Albumin in the body also goes down. Inflammation occurs because of many things in the body (environment, trauma [major killer in US for under 44], anaphylaxis and has now been related to many diseases like osteoporosis, asthma, cirrhosis, cancer, psoriasis, crohn’s disease, etc.). ROS/RNS damage cells that proliferate more causing mutations. Yarmush focuses on tissue perfusion and cell culture using the liver. When a wound is inflicted it secretes glucose and alerts inflammatory cells which secrete IL-1B, IL6, TNFa and alert the nervous system and the endocrine system and secrete more stuff that get the liver going. The liver also secretes IGF-B1 which makes skeletal muscles release amino acids to feed it. Inflammation mediates hepatocytes’ altered gene and protein expression. 1st day after a trauma there is an EBB in the response due to loss of blood and shock. Several weeks there is the hyperactive metabolic state. Systemic Inflammation Response Syndrome (SIRS) is chronic inflammation. Can lead to multiple organ dysfunction and death.
o Characterizing dynamic cell response to dynamic stimuli has a need for a higher tech way to do it quick. Pipetting is slow and perfusion has been serial so they created a parallel microfluidic perfusion. Better than microarrays as it is OUTPUT focused and we need to limit the inputs. RTPCR leaves you with too many plates. Yarmush developed a GFP fluorescent living cell reporter system where the cells respond to signal that activates a receptor that triggers a transcription factor of interest which causes it to express GFP. Have to create reporter colonies for all the TFs but can then expose them to many signals in parallel in a microfluidic device. Reproduce the results seen in FACS using the microfluidic PDMS device. Using h35 liver cells to make the reporters. Can easily get clones and replicates using this system. Valves horizontal and vertical prevent cross contamination from cell species and/or stimuli. Can take more time pts easier than microarrays just using microscopy and can also see effects not seen in microarrays by following longer, as in NFKB is a strong early responder but other TFs are activated later on. Have also combined strong single stimuli and have noticed that combinations yield low response… probably not enough energetics. Can use it to make signal gradients as well. Used it to study steatosis by causing the cells to be extra fatty which could lead to cirrhosis or carcinoma. Can also give them hypoxia conditions…
o Mehmet Toner (MGH, director of BioMEMS Resource Center). Talked about the applications of MEMs devices… sorting. Useful in animal work because you can take small volumes without affecting the physiology and do more time points. Toner mentioned that we are using methods from the 30’s and 40’s to collect the samples to put into our state of the art analysis. Mentioned Sethu et al 2004 using microfluidic lysis mechanisms. Faster! Pure lysis without separation of cells in blood gives microarray data that is very similar to neutrophil alone as they dominate but doesn’t show that monocytes and leukocytes are downregulated.
o William Rodriguez (partners aids research center) talked about what is important in designing a point of care device to detect aids.
o Joel Voldman (MIT EECS) FACS can’t separate cells based on location or dynamics so he developed an electrical field that causes a dipole in the cell and cages them. Doesn’t work well with bacteria.
o Rashid Bashir (Purdue) very interesting work with bacteria which I will follow up on. Says that isolation of bacteria is rate limited by culture on petri dish. Said something about using impedance microbiology as bacteria become ionic to conduct them. Use dielectrophoresis to separate the bacteria. Can I use this on my project to separate from mucus and collect RNA? He used antibodies to the bacteria to select them and grow them in place on the chip (petri dish on a chip). Still does that help growth kinetics?
o Sangeeta Bhatia was the last but Bashir had caught my attention… Bhatia is like Linda Griffith but using different scaffolds.
Previous notes that went unposted From Brock’s Biology of Microorganisms 7th edition Bacterial properties · Average size 1 x 3 um (E. coli) although there are exceptions · Flagella are too small to see unless EM is used · Haploid ~ single copy of DNA so mutations occur much faster · Surface Area to volume ratio is higher b/c of small size so metabolic processes are much faster! Nutrients and waste pass in and out faster. · The cytoplasmic membrane is about 8nm. · Prokaryotes lack the sterols that eukaryotes have. Sterols are rigid and planar and stabilize the membranes. Can’t endure same stresses but this makes prokaryotes not susceptible to polyene antibiotics which target sterols. · GRAM Stain is due to differences in the cell wall
o Positive looks purple. Cell wall mainly composed of thick layer of peptidoglycan and has no LPS layer. 90% of cell wall is peptidoglycan ~20 layers.
o Negative looks red. Cell wall has little peptidoglycan but has extra outer membrane composed of lipopolysaccharide (LPS), lipoprotein and other macromolecules. From the outer surface of the cytoplasmic membrane to the inside of the LPS layer is the area known as the periplasm. 10% of cell wall is peptidoglycan ~1 layer at times.
· Peptidoglycans give strength/rigidity & are made of N-acetyl-glucosamine & N-acetylmuramic acid, L,D alanine, D glutamic acid, lipids, DAP. Only in bacteria. · LPS layer is a 2nd lipid bilayer with polysaccharide and protein. Toxic/pathogenic to animals. Many porins in Gram negative on LPS layer. · Periplasm is 12-15nm –gel-like, has enzymes, binding proteins, chemoreceptors for chemotaxis. · Lysozyme in tears, saliva, egg white, hydrolyze peptidoglycan so cell wall weakened and water rushes in lysing the cells. Use a buffer or sucrose to create a gradient to prevent lysis. Protoplasts are the microorganisms that have lost their cell walls. · Mycoplasmas are natural protoplasts as they don’t need the cell wall so they are hard to kill. · Flagella or gas vesicles are used by prokaryotes to move. Flagella can be 20nm thin. Polar flagellation, polar lophotrichous (tufts of flagella like HP), peritrichous (all over like long cilia). · Flagella are made of flagellins (as opposed to microtubules in eukaryotes) and have a couple ring like proteins that turn them and a hook. Gram negative bacteria have more rings. · Average velocities from 20 to 80 um/s · Chemotaxis movement of organism toward chemoattractants or away from a repellent. · Proteins to sense absolute concentration of proteins are the methyl-accepting chemotaxis proteins (MCP) or transducers. E. coli has 4 and they are transmembrane proteins ~ each sensing a variety of compounds. Use methylation and demethylation to govern flagellar rotation. MCP is demethylated by CheB and methylated by CheRà CheWàCheYàmotor. CheZ demethylates CheY. · Fimbriae and pili are structural but not used in motility. Fimbriae might help in adhesion to surfaces. Pili are longer, normally 1-2 only and are used for mating and attachment of some pathogens. · Prokaryotes also have CAPSULES/SLIME LAYERS composed of glycocalyx which may help resist dessication and recognition by immune cells. · Prokaryotes also often have GRANULES and other INCLUSIONS. · Ply-Bhydroxybutyric acid [PHB] (very common) polymerizes to form PHA (poly Bhydroxyalkanoate) to store carbons and energy. Thought to be a possible biodegradable polymer as it’s not in Euks. · Other stored things are glycogen, polyphosphate, sulfur, magnetosomes (allow magnetotaxis), gas vesicles to confer buoyancy – once collapsed it cant be reinflated. · Endospores resist heat (very resistant) and chemicals to help survival. They are spores formed within the cell. At the “core” lies the original bacteria with the wall, membrane, nucleoid, etc. right outside it has a cortex of loose peptidoglycan. On that it has a spore coat and then the exosporum. They remain dormant but can be activatedà germinationà outgrowth. Many genetic changes occur. · DNA in prokaryotes
o Naked molecule arrayed in circle which is well folded and twisted (supercoil)
o Haploid
o Gene exchange by
§ Conjugation ~ cell to cell contact
§ Transduction ~ genetic change mediated by viruses
§ Transformation ~ free DNA is involved ~ one cell lyses and other uptakes.
H. hepaticus infects the liver, cecum, colon and affects dendritic cells, macrophages, T reg cells.
In the 90’s, DCs (monocytes) found to be professional presenters, better than macrophages. Sit in lamina propria, put out dendrites and sample the environment thru the epithelial cells and find the antigen and traffic it to local lymph nodes. Innate response, neutrophils, macrophages, nk cells vs. immune. Marker on the DC cells might be able to sense LPS. Marker also might bring address weather. Sample environment talks to B cells to and can mature. DC CD80 and 86 only gel activates with something senses LPS. IF not CD28 on T cell not done and you get anergy/tolerance. T reg suppressor and helps tolerize. CD4, CD25 and Foxp3 (TGFB causes expression. T cell T cell contact, CT1L4 or alter cytokine environment at site of inflammation. TGFB and IL10 are anti-inflammatory at antigen presentation, shutdown (macrophages, stop proliferation of T cells, anergic T cells, macrophages also emit cytokines that help T cells).
Hh increases immune and tolerize by making T cells and help inflammatory?
