Writing Seminar 10/14/08

For those of you that missed Peter Sterling's writing seminar yesterday, I've included my notes below. The take-home points are summarized at the bottom. I found his talk to be very straightforward and immensely helpful. Although none of it was earth-shattering, he highlighted a number of common problems people face while writing papers and grants. His website is: Peter Sterling's Website. On the website is a link to his powerpoint presentation which is pretty self explanatory and a link to a paper on writing that he recommends.

Another resource that he recommended is the book Style: Ten Lessons in Clarity and Grace by Williams (not the abridged version).

Notes:

Writing a paper or a grant is, in essence, telling a story.

Parts of a story:
Context (background)
Something happens
Moral

He uses the example of Sleeping Beauty to demonstrate how a story works. You know the story.

Important point:
Length of these sections- something happens is the longest section; make sure the context (introduction) and moral (discussion) are not longer than the results

In science, he recommends to begin writing the paper and creating the figures as soon as you get the first real result. Why? Because it will help you build the story and imagine the appropriate controls and experiments and identify early on what is missing.

When you start writing:
List 3-5 key observations (results)
Sketch the figures
Arrange them in compelling order
Write a strong lead for each legend to tell the story (this will help you avoid repeating it in the text)

Write the title BEFORE the story
-this will remind you of the story from the beginning
-if the story drifts, you either revise the story OR the title

Designing the title
-make the biggest claim that you can support (modified big claims if you have to ie. evidence that vesicles on the synaptic ribbon can be rapidly released; doesn't prove anything, but got published)

Title example:
BAD- Differential expression of chloride transporters in ON and OFF bipolar cells
-end of title is most important to reader, but it's the most specific and least likely to be interesting for most readers
-differential expression- what does this mean? not only does this tell us very little, but this is an example of nominalization (when you make a verb into a noun), action verbs are GOOD
BETTER- ON and OFF bipolar cells express different chloride transporters
-makes a definite claim, uses fewer words, uses a strong verb, appeals to wider audience

Write the context (introduction)
-identify the issue/problem, don't use "little is known" or similar phrases. Clearly, SOMETHING is known and by saying that little is known, you negate the work done in the field and look either stupid or ignorant.
-explain current status
-explain approach
-3 paragraphs is perfect
-Don't summarize results

Outline the story in modules
-a module should match the readers attention span (3-5 paragraphs)
-label each module so you and the reader can keep track
-they require a specific temporal order; DON'T go back and forth in time (ie. anesthetizing the animal after surgery)

Diagrams and figures
-make them intrinsically comprehensible; label everything so that you need the legend as little as possible; no one wants to read any more than they have to

Seek criticism
-don't take it personally but acknowledge that it hurts but is helpful
-it's hard to get constructive criticism (few people can identify and articulate problems)
-goals; maximize your understanding of the problems so that you can revise, minimize time for critic (they are busy too), leave the critic feeling good
-don't explain why you wrote it that way; you are just talking to yourself

Sentence structure
-avoid wordiness
-exploit stress positions (beginning should be context and end should be the important part of the sentence- the result)
-keep subject and predicate together
-avoid weak verbs with nominalization; use strong verbs!

Examples:
Weak sentences: It is our goal to test the hypothesis that the HNF3 proteins have an important function in endoderm development and hepatic specification. We have generated a mutation in the gene encoding HNF3g via gene targeting as a first step towards this goal.

Stronger sentences: Apparently, the HNF3 proteins affect endoderm development. To test his, we mutated …
-why is this stronger? shorter, stress positions utilized, trim wordiness, active verbs

For consecutive sentences:
-the stress position in sentence one introduces the topic position of sentence two
-use parallel construction and words to tie things together

Paragraphs:
-topic sentence is like a title
-one idea per paragraph, ~5 sentences
-last sentence leads into next paragraph

TIPS:
Use the singular, use the positive and avoid double negatives.
Use past tense in results and present tense after the first stage of the discussion.
Use shifts of direction (however, yet, but) at the beginning of sentences to tell the reader where you are going.
Know who is on your study section or who your reviewers might be and give them credit in your grant/paper.
Balance of space: introductiondiscussion
Avoid using "little is known" statements; tell what IS known.
Get to the punchline/moral before the reader loses interest.
When a key piece to your story is missing, take the time to find it or collaborate to get it done; it's easier to do than to defend a story with a missing piece.
Don't use text to describe a figure.
When a paper is not accepted, keep reading; oftentimes it can be resubmitted once the comments are addressed.
Science is already hard to understand; don't complicate things with confusing writing. Keep sentence structure simple!
Avoid excessive use of "we"; you are not the important part of the paper, the results are.
Avoid metadiscourse; TRIM IT!

Parasitology Gordon Conference - June 2008

SESSION I: Hijacking the Host Cell
David Sibley (WashU), Herb Tanowitz (Einstein), Dirk Dobbelaere (Bern), Vern Carruthers (Michigan). [Victor Nussenzweig had to cancel, as he did for his planned Philadelphia visit.]

Intro (David Sibley)
- DS will talk about ROPK virulence
- VN was to talk on CS entry into hepatocytes
- HT to talk about T.cruzi modulation of APCs via parasite thromboxane
- DD to talk on Theileria immortalization of host cells (Dan and I were discussing ways that we might want to build on this in our lab)
- VC to talk on Toxo egress from host cells (based on K+ sensing, leading to increasing Ca)

David Sibley (WashU, St Louis) -- ROPKs and virulence
1. ROPK
- genetic scan for phenotypes by Sonya Taylor & Chunlei Su fm IxIII cross IDs chr 7a, high type I polymorphisms, 100X expression of ROP18 in type I (off in type III)
- express type I allele in type III restores virulence
- note parasite ROPKs v diff fm host kinases
- have structure for ROP8 (Hui, Toronto) used to model ROP18 ... killing kinase kills virulence.
- N-terminal extension wraps around active side ... regulatory?
- ROP18 has conserved DKN (predicted active), but many inactive. PPE rather than APE for substrate binding site ... function unknown, but conserved even in kinase dead mutant.
- expressed as recombinant protein shows autocatalytic Plation of N-terminal region; mutation ablates autophosphorylation
- what is happening in host cells? express in COS cells ... target back to PV ... IP active vs dead ROP18 ... see Plation of many prots in parasites & host
- don't see diffs in Xcr in host cell (unlike ROP16 expression) ... must act at protein level?
2. Age of Toxo
- old work of Chunlei said 10K yr to MRCA for types I/II/III
- global scan by Asis Khan: v diff diversity in S Am: some clonal, some more diverse; MRCA 1-2 Myr
- genetically diverse strains share common ROP18 allele.
- type III underexpress, type I overexpress and are virulent; type II variable ... corr with virulence (Xc: Castelles * P89 virulent by low type III expression; 1 low virulence but high expression
- some handwaving about pN & pS for ROP18 ... thinks under strong positive expression
- overall ... pretty nice review, but little really new

Herb Tanowitz (Einstein, NY) - Chagas disease as a vasculopathy
- endothelin is a 21 AA peptide: powerful vasoconstrictor (like thromboxane), inv in hypertension etc
- increased expression in Chagas ... could this be responsible for cardiomyopathy (wall thinning)?
- think works in part by decreasing adiponectin, blocking the role of this cytokine in reducing inflammation
- TXA2 (short-lived) synthesized from arachidonic acid. Infected endothelias cells (HUVEC) show high TXA2 ... from parasites, comparable levels to platelets
- Tc TXA2 is biologically active (CHO cell assay); can inhibit with receptor blocker
- Is parasite TXA2 responsible? test in TXA synthase KO mice ... 80% from parasite
- More interesting is TXAR KO ... find increase in parasitemia
* (isn't this the reverse of expectations?)
- does aspirin help recovery? no ... increases parasitermia ... maybe aspirin decrease in COX1 means parasite cannot scavenge PGA2
- somehow think that this must be involved in modulating parasite infection/virulence
- Q (Ira Blader): does TXA2R promote invasion/replication? apparently not
- bottom line: something interesting seems to be going on here, but I don't understand it. Might be nice to look at chimeric animals with receptor KOs in heart only?

Dobbelaere talk -- Theileria
- gp34 GPI-anchored surface antigen in Theileria
- stage-specific: expressed only in transformed form (even in same cell
- express ectopically in mammalian cells ... targets to centrosomes (assoc with suroraB)
* cool idea: try siRNA/cDNA/small molecule screen for inhibitors of spindle localization ... do mutants affect Theileria replication?
- focusing on centrosomes, look at plk1 (polo-like kinase), important for replication ... associates with schizont surface
* are there DN mutants of PLK1?
- gst-gp34 pull-downs show assoc with Plk1, auroraB, gamma-tubulin (but not alpha/beta tubulin)
- have mapped binding sites for Plk1 and gamma-tub (diff sites, both in N-term
- polo box domain responsible for binding to parasite surface
- overexpress gp34 (tagged with V5) induces host cell binucleation (typical of interfering with plk)
- parasite DNA synthesis occurs during G2/M ... is this regulated by host cell kinases, e.g. plk1, cdk1, other?
- Q: is it really GPI-anchored? dunno
- Q: see plk Plates gp34
- Q (Sibley): parasite plk1? dunno ... but seems v different
- bottom line: very nice system ... would be fun to think about exploring mutants ... and possibly exploiting for studies on mammalian replication, e.g. with Mike Lampson

Vern Carruthers -- Toxoplasma egress
- perforin-like protein in Toxoplasma ... involved in egress?
- nice intro: know a lot abot attachment, invasion, and intracellular replication; less about egress ... not just escape from host cell, but also stuff assoc with PVM
- screen secretory products ... ID PLP1 (chr 7a, 1151AA, SP+, MACPF domain)
[nb: ToxoDB search IDs 2: 20.m03849 on chr7a & 59.m03629 on 8)
- others have looked at MACPF prots in Plasmodium ... PLP1 (SPECT2) in liver cell entry; PLP2 in RBC, PLP4 in Gct, PLP3 & 5 in insect
- KOs lose virulence in vivo (RH background)
- get trapped in 'spherical bodies" (bad name!)
- treatment with A23187 to stimulate Ca influx ... do not see egress; striking videos (respond to ionophore to move, but do not egress.
- note that this is diff from Mike Black's egress mutant, which does not respond to A23187
- WT trans-complements within same cell, but not different cells
- 'spherical bodies' derive from multiply infected cells: 1 leaves, other blocked (can't get a foothold?)
- what is integrity of PV like during egress?
- expt1: cytD+PI (stain host cell permeability)+ A23187+3'37•+fix+stain for GRA & SUB1. See in wt, A23187 -> MN secretion, then host permeability than MN prot dispersal. in mutant, block early
- expt2: xpress secYFP ... 1 min postA23187 released from PV; in KO, so release
- Q (Soldati): why doesn't it affect during invasion? note: know processed, maybe this regulates?
- summary: v nice work ... dunno how working ... what is the block to host cell

Session II Epigenetics & Gene Expression -- Intro by Gloria Rudenko
- Apicomplexans use normal eukaryotic marks ... talks by Deitsch & Lopez-Rubio
- Mirelman to talk on Entamoeba
- Tryps transcribe differently ... silencing of VSGs (Rudenko)

Rudenko talk:
- how are silent VSGs kept off? Introduce FP-labelled transgenes
- minichromosomes also silenced ... do 177bp repeat arrays silence?
- test candidates by RNAi. Find SWI2/SNF2 family protein ISWI (picked up from work by ??? at Hull, by binding to 177bp repeats in minichrs) ... derepresses VSG expression, in both bloodstream & insect forms
- does TbISWI affect other regions? VSG reporter also derepressed in other regions of genome (basic copy arrays), but VSG reporter only derepressed with VSG promoter.
- no binding of telomeres in BS, not insect forms
- conclusion: TbISWI expression correlated with repression in telomeres, minichromosomes, silent VSGs

Arthur Gunzl -- PolI transcription of VSG and procyclin in Tbrucei - 10M surface VSG on surface, all from single gene using PolI. How do they do it? Huge rate of transcriptional initiation inherent in PolI; PolII genes cannot do this
- structuraly different polI promoters: rRNA, procyclin, VSG; acquire cap via postXcr trans-splicing
- many paralogs of RBPs specific to PolI ... RBP5, 6 & 10 have paralogs; RBP10 has N-term extension
- pull-downs ID other proteins as well; RPA31 is essential by KD & reduces PolI transcripts; in situ coloc with RBP6
- also checked prots ID from EMSA in Cross lab
- CITFA-2(?) complex, copurifies by TAP, sedimentation ... tryp specific complex. 6 conserved hypotheticals, also DLC
- complex binds to purified VSG promoter

David Mirelman -- silencing & virulence in Entamoeba - amoebapores formed by specific pore-forming proteins ... insert into memb and form channels
- trying to engineer expression: some plasmids work to upregulate, others silence native gene
- silencing plasmid happened to include 140bp of 550bp SINE from upstream of AP-A ... SINE nec for silencing
- hard to study chromatin, since K9 not conserved, but H2meK4 is conserved ... most genes IP fine with anti K4, but not silenced AP-A
- are other genes silenced? Microarrays show AP-A & AP-B ... also several others. Also several genes up-regulated, incl Rab GTPase & several hypotheticals
- three families of virulence factors silenced
- strangely, only seen in one strain, and only in G3 stage ... dunno why

Kirk Deitsch -- silencing of var genes in Pf
- How to turn on/off var genes and knob display on surface of iRBC during infection?
- How and why is exactly 1 of 60 active?
- structure: polII promoter upstream of exon 1; single intron, also serves as promoter for 'sterile' RNA
- intron and sterile RNA seem to be important for activity of primary promoter
- can engineer selectable contruct by replacing exon 1 with blasticidin ... selec tion silences all; remove intron & exon 2 (i.e. sterile transcript) abolishes trans effects
- colocalizes with telomeres by in situ hyb
- can amplify plasmid as concatemer
- what are sterile transcripts doing? see both forward and antisense transcripts, know from PolII ... dunno

Miguel Navarro -- RBP7 in Tryps
- Stufy PolI & PolII in Tb using FLuc in VSG locus & RLuc in tubulin locus
- siRNA KD of TbRBP4 affects PolII not PolI
- TAP IP of RBP5, 6, 6z, 7 ...
- also colocalize

Luisa Figueredo (Cross lab) -- histone MeXferase in Tbrucei
- DOT1 methylates H3K79 in yeast and mammalian cells; two homologs in Tb
- DOT1B know to make H3K76 triMe in Tb, but not essential

Jose-Juan Lopez-Rubio (Scherf lab) -- H3K9me3 and var expression
- silent var2csa enriched in
- active var2csa
- is this general for all active/silent vars?
- all subtelomeric regions enriched in H3K9me3 (var, rifin, etc)
- internal regions enriched also contain surface ag families (middle of chr 4, 6, 7, others ...
* not chlear ... would like to look at data more closely
- PfSir2 = HDA, in silent promoter, makes H3K9as in vitro; inactivation affects pattern of H3K9me3

Sergio Schenkman -- acetylation in Tb and DNA repair genes
- characterization of chromatin marks in Tcruzi
- H4K4 modification ... by EM within dense chromatin areas; vs K10 & K14 at dense/light boundaries, coloc by IFA with polII
- see some stage specific and cell cycle specific differences

SESSION III: Developmental Parasitology - Steve Beverley Intro
- development is just another term for life cycles, usually assoc with cell cycle exit ... and a good chemotherapeutic target
- expression profiling has been disappointing for some organisms (kinetoplastida)
- focus increasingly on large-scale screens
Talks by Steve Beverley (Leishmania virulence), Andy Waters (Plasmodium gametocytogenesis), Keith Matthews (T. brucei life cycle reg'n), Daniella Barthoemeu (T. cruzi mucins), Scott Landfear (Leishmania transporters)

Steve Beverley -- Leishmania development & virulence
- Leishmania: digenic fm sand fly to mammalian host; can replicate metacyclogenesis in vitro
- drastic changes in midgut: LPG increases length, changes in lipi rafts, global changes in gene expression, histone modifications, cell shape change ... also decrease 10x in volume ... Why?
- what are mechanisms? autophagy? proteasomes? TOR kinases?
- Atg8 KO still shrink 10x ... although less virulent (delayed lesions)
- proteasome? ... hard to study (lack of specific reagents)
- 3 putative TOR kinases: can't KO TORb, can KO TORc ... do not develop lesions; add-backs restore (mostly)

Andy Waters -- Translational repression during gametocytogenesis in Plasmodium
- review of translational initiation (eIF4 complex binding cap etc ... orthologs of all in Pf
- can disrupt translation by binding to eIF4E by maskin/cup, bicoid, etc
- model of translational repression in Pf: P25 & P28 ... mRNA in Gct, protein only Xla in zygote
- what is basis? UTR constructs with GFP reporters to test ... imp 3' and 5' regions
- repressor complex, containing DOZI (devt of zygote inhib); KO mutants fertilize, but do not develop (female dysfunction), reduced p25 & p28 etc
- what mRNAs assoc? epitope tag DOZI pull-down shows eIF4E, 4EBP, Bruno, CITH (CAR1/TrailerHitch), PolyABP2RRM, others: Alba-domain, pb-A, pb-B, ,others
- also several Alba domain proteins: e.g. 862.00.0, and other RBPs et 805.02.0,
* should have arrays for DOZI pull-downs ... complete set of repressed proteins?
- KO pbA fertilize, no ookinetes, pbB males fail to exflagellate
- Bruno KO no obvious phenotype; pulls down lotsa stuff ... involved in many complexes?
Q: A and B in both, or diff in male & females?
Q: what mRNAs assoc? (dunno)

Keith Matthews -- triggering diff'n
- select for parasite line defective in differentiation: treat with cis-aconitate (good differentiation inducer) ... pass in mice to select
- find don't drop VSG or upregulate procyclin
- array shows deletion, upregulatetion in
- highly expressed in stmpy form (transmissible, downreg in procyclics)
- eight genes in tandem array; test using specific primers show some Xcr regulated during diffn
- seem most similer to carboxylate transporters ... do they transport citrate? Yes in XL oocytes
- made antipeptide ABs ... none in slender forms, both PAD1&2 in stumpy forms, PAD2 only in procyclics
- KD prevents differentiation
Q: what are substrates? diff for dif prots?

Daniella Barthoemeu -- T. cruzi mucins
- MASP proteins 2nd largest family N&X term cons (80% ID), center divergent (20% ID)
- 5' and 3' UTR also highly conserved ... probes show fairly tight band of mRNA ... maybe only a few o f MASP family expressed? find several, but bias towards one subgroup
- also some chimeras with mucin genes (largest family)
- don't know if expression differs between cells, etc ... plan to clone, more mAbs, check pt sera, etc

Scott Landfear -- Leishmania transporters
- amastigotes scavenge nutrients from the macrophage phagolysosome: hexose, AA, polyamines, etc
- look for complementation of glucose Xporter null
- die intracellularly in macrophages ... but continuous passage IDs suppressor strains over months ... what are they?
- can take up hexoses ... how? Amplified LmGT4 gene (low affy hexose Xporter

SESSION IV -- Organelles

Dominiue Soldati (Univ Geneva) -- Toxoplasma glideosome
- Glideosome, Assembly of the motor complex; unusual myosin, aldolase, GAP45&50. How to assess function? Pull-downs, with myosin LC, N-terminal GFP (cannot use C-term pulldowns as interferes)
- why link GAP45 via palmitoylation, since GAP50 already TM? Probably to permit dynamic association
- 11 candidate myosin motors in Toxo … is MyoA doing all of the work? Find MLC in different compartments
- MLC2 also in IMC … does it have a specific partner? Yes. Pull-downs show MyoD, MLC2, GAP60 complex …. But dunno yet what it does. MyoD not essential (can KO; higher expression in bradyzoites)
- What about actin? Motor activity provided by formins: many possible functions. 3 formin genes in Toxo … FH1-long-FH1-FH2. Expressed FH2, show binds to actin, assess actin polymerization … potent
- Introduce tet-inducible N-terminal tag by double X-over … see at periphery during invasion (note diff fm Jake Baum’s paper). Also try DN effect … dimerization deficient mutants in FH2 domain … also think assoc with periphery near PM
- Note: important open question … how to close off constriction upon entry?

Keith Gull (Oxford) – The trypanosomatid flagellar pocket
- Flagellar pocket is the only place for trafficking (rest of surface covered by VSG & underlain by MT
- Examine by tomography (resolution as low as clathrin triskelions)
- how are receptors anchored in the FP? Others, like glucose Xported delivered to flagellar membrane, others (like VSG) on surface … two boundaries: PM to FP, FP to FM
- bottom boundary (PM to FP): see ‘collarette’ and radial fibres
- think radial filaments the docking point for ???
- top boundary (FP to FM): dense collar; neck region quite interesting: outside of collar, but not outside cell. Assoc with proteins Bilbo & RCMP ... but don't know function, associations, etc
- look at membranes by freeze-fracture. Density of IMPs diff fm E/P faces. See ciliary necklace of bumps assoc with radial fibers

Boris Striepen – Apicoplast biogenesis and function
- what does the apicoplast do, which are important, how did gene transfer to nucleus take place, how to proteins target back, how does the apicoplast replicate?
- vesicles thought to traffic to the apicoplast. ID candidate Tic/Toc proteins: work on Tic20, Tic22. HA tag and target to the apicoplast. Use split-GFP assay to show target to the inner membrane (nice).
- Is this the translocon? Tet-inducible KO shows essential. By pulse-chase mutant loses ability to process over days, also loses ability to biotinylate ACC (mitochondrial biotinylation still OK), also ability to lipoylate PDH (lipoate-specific ab).
- What about membrane 2? Uwe Maier suggests ERAD system (ER-export system for … uses Der1, Cdc48, Utd. ID orthologs pred to go to both mito and plastid (3 plastid Der1, 1 Cdc48, 1 Utd1; 1 each for mt). Conditional KO for Der1-Ap … completely lethal, not even small plaques … blocks all of above phenotypes, even as early as day 1. Nice!
- No data shown on vesicles, fusion … snare? [Need to get Manami's paper out!]
- another topic: how does apicoplast divide? No FtsZ & Arc5. What is the machinery? Can see spindle, and MORN ring (“one ring to rule them all”).
- Two dynamin-related proteins (unrelated to Arc5 … 1 involved in secretory pathway, 1 in apicoplast fission. DN GTPase under control of DD system … without shield fail to divide the apicoplast … form ‘starfish’. FRAP shows still connected. Think elongation assoc with MTs.
- Nice 'starfish' shape; shows different


Adrian Hehl – Giardia secretion
- studying secretory structure and function … no classical Golgi stack structure
- much missing, few SNAREs, other stuff … ER directly to PM
- secretion of ECM during encystation: CWPs etc. Synthesized in ER and blocked … secreted in just a few minutes during
- look for functional homologies with ESV … small GTPases (e.g. Sar1, Rab1, Arf1), coat complexes,
DN Arf1 fail to secrete … make naked cysts! ESV with CWM remain. Overall:
ET> (Sar1/COPII, Rab, etc)ESV> (Arf1,dynamin)CW
ESVs seem to stay relatively fixed … don’t move. Instead, ESVs make long tubular extensions … exchange between ESVs
Postranslational processing of CWP? Expressed double-tagged reporter … look at CWP1 vs CWP1deltaC see diverge, while CWP1 and CWP2 remain colocalized

NOTE: Cell biology of protozoan parasites ASCB summer conf? Hehl, Warren, He, Striepen, Gull, McIntosh, Soldati,

Chris de Graffenreid (Warren lab) – Polo-like kinase in Tryp Golgi biogenesis

Ira Blader – siRNA screen IDs host MT as imp in Toxo invasion

Steve Matthews --

Paul Vutova (Barragan lab) – Transport of Toxo by DCs

SESSION V -- BIOCHEMISTRY

Meg Phillips – Polyamine metabolism in Tryps as a drug target
Identify targets, druggability, essentiality
AdoMetDC – heterodimer, requires pyruvoyl cofactor for catalysis
Hs enzyme activated by putrescine
kcat/Km of Tb form shows much lower activity than Hs (1000x)
But in blood form parasites ( … missing allosteric regulator?
Genome shows all tryps have 2 AdoMetDCs (unusually), both expressed
TbAdoMetDC1 processed, by 2 not. Mix enzyme boosts activity 1200 fold (stable heterodimer, Kd <0.5um) style="color: rgb(51, 255, 51);">Dan Goldberg – Plasmodium Calpain as a novel target
PfCalpain (Mal13p1.310) is unusually long (6.1kb CDS)
Contains typical catalytic domain for this Cys-protease; Ca binding domain similar structure … but enormous N-terminus
Is it essential? Try to KO with N-terminal X-over to create truncation. Never got integration unless engineer with a promoter … suggests essential (but doesn’t prove). Alternatively, engineered allelic replacement with active site Cys mutated to Ala or Cys (with upstream synonymous restirction site).
Alternative strategy: added destabilization domain … C-terminal X-over with GFP-FKBP. Selected in presence of shield. Very low abundance protein: cannot see on Nern or Wern … can detect by IP with antiGFP from 100ml.
Remove shield … shows growth reduced by 50%. Drop in parasitemia at ring/troph boundary. [not all that impressive]
Think localizes to nucleolus (!) … putative NLS in sequence targets GFP to nucleolus (in both mammalian cells). Controlled by di-palmitoylation as well(!)
Q: substrates? Dunno … looking by Px in DD constructs
Q: palmitoylation in nucleolus?! no … palmitate leads to PM localization; without, goes to nucleolus
Summary:

Paul Wyatt -- Gene family-based target discovery for T. brucei
- Dundee activities: (1) hit discovery facility, (2) drug discovery initiative (8.1M£/5yr). Mgmnt team: Julie Frearson, Kevin Read, Andrew Hopkins + 6 biol, 9 med chem, etc
- Taken portfolio approach: N-myristyl transferase, PLK, RNAligase, PTR1,trypanothione synth, kinases, etc [DSR: should get copy of portfolio]
- Kinases: known druggable targets, good libraries, evidence for essentiality in tryps, may be able to get around tox problems by dosing, etc. Define search space of ~5K compounds, >160 scaffolds,
tackle genetically-validated kinases in parallel, collaborate with expert biology groups, multiple assays in parallel
- ~190 kinase orthologs in each of triTryps, 5-8% non-catalytic ... focusing on CRK3/cyclin6, PK4, PK50, Tb glycogen synthaswe kinase, Tb polo-like kinase, Tb aurora kinase 1, Tb Vsp34 (PI3K), cTb asein kinase
- gene family approach:
- kinetoglow assay in 384
- PK50: 13 hits <13 style="color: rgb(51, 255, 51);">Bob Jacobs -- Synexis
- Synexis: D&D: medchem, hit-to-lead, lead opt, contract chem, ADME. Work on pharma contracts, HepC, DNDi collab on T. brucei. Coord with Tidwell consortium, STI, Pace, Anacor, Genzyme, STI, etc
- Review of anti-tryps. Stage 1 (BS) treatment with pentamidine, suramin (work ok, although old); CNS treated with melarsoprol (highly toxic: 5% mortality, emerging resistance?), eflornithine (hard to admin: 4 infusaions daily x 14d!)
- goals: active against Tbr & Tbg, resistant, cure <14 ml =" 1106," ml =" 400," cytotox =" 150," chemiinf =" 8" style="color: rgb(51, 255, 51);">Ajai Saxena (JNU, Delhi) -- Pf p25 & p28
- interested in structure of transmission-blocking vaccine candidates (Pfs25 & 28)
- form interesting packing in crystal ... maybe related to protection from gut environment? Also solved structure with antibody ... big conformational change, now exposed?

Utpal Tatu (IIS, Bangalore) -- Hsp90
- specific inhibitors of Hsp90, e.g. geldanomycin kill parasites (ring/troph) ... acting as a natural sensor of fever? vector/human transition?
- GA binds PfHsp90 in vitro (slightly tighter than human). PfHasp90 ATPase highly active, as in cancer cell, not normal host cell.
- PfHsp90 acetylated (diff sites than Hs though)
- looking at PfHsp90 chaperone complex by IP
- GA works against Pb in mouse model
- Also doing proteomics on patient sera ... want to release data in PlasmoDB

Alberto Gimenez (Inst Pharm, La Paz Bolivia) -- microbial natural products
- Evanta bark used in traditional medicine: lots of alkaloids
- see some effect on chronic inflammatory reaction in vitro (Marita Troye-Bolmberg, Stockholm)
- clinical trials in amazonia, 243 cases of cutaneous leish
- topical application for 10 weeks ... ulcer gets bigger, then heals.

Enock Matovu (Makerere Univ, Kampala Uganda) -- DMFO relapse
- lots of resistance to melarsoprol in Uganda (both Tbr & Tbg)l, starting in 1999, move to DFMO as first line treatment ... fund relapse in 26/150 pt, almost always in CNS.
- Conintued monitoring, also looking at ODC, transporters, etc Other markers?

GENOME SESSION (not blogged live)
- DSR talked about sequencing white papers, EuPathDB data mining and plans, new data types and integration for improved gene models, ortholog-based inference and TDRtargetsDB
- Dyann Wirth talked about Plasmodium diversity. Key points include: LD in Africa much less than in Thailand less than in Brazil (but what about impact of CQR sweep?), large number of diverse proteins
- Michael Barrett (Glasgow) talked about FT-MS metabolomics for ... very impressive
- Gerald Spath (Pasteur, Paris) talked about phosphoproteomics in tryps, by DIGE
- Igor Almeida (UTx El Paso) talked about GPI diversity in Leishmania: GIPLs contain diverse sugars and lipids

VECTOR SESSION

Jose Ribeiro (NIH)
- philosophy of genomic-based approaches, arguing that genome-driven research is fundamentally different from hypothesis-driven research
- talk ranging from Aristotle to Bacon to Maimonides!

Serap Aksoy (Yale) talked about Tsetse

James Valenzuela (NIH) about immunization using Leishmania saliva

Ken Vernick (Pasteur, Paris) -- population biology of Anopheles interactions with Plasmodium - alternative outcomes when a Plasmodium ookinete invades the midgut: oocyst, lysed ookinete, melanized ookinete ... probably other mechanisms of killing as well
- malaria parasite is a pathogen for mosquitoes, with fitness costs
- genetic screens: need good phenotype for bias-free identification of important factors. Best done in nature, with natural diversity
- A. gambiae mates only once, storing sperm in spermatheca for rest of life. collect in Mali and Kenya, 100 pedigrees, ~100 sibs from each. Analyzed 40 genetically
- phenotypes to score: # oocysts, # melanized parasites, map using genom-wide STR map with 10cM resolution
- ID QTLs from indep peigrees: Pfin3 on chrX, Pfin2 on 2R, Pfin1, 4, 5, mel2 & EAPfin1 close on 2L, Pfmel1 on3R. Major locus picked up both in Mali & Kenya (15 Mb, ~1K genes). 2 genes most promising to test by RNAi
- APL1 KD increases oocyst number, APL2 does not. Leucine-rich repeat protein (family includes plant pathogen resistance genes, TLRs, NODs, etc). Develop readout based on Rel1/cactus (NFKb/IKb): toll signalling Plates cactus releasing Rel1 Xcr factor for Xloc to nucleus
- Rel1 antimalarial protection is provided by APL1C
- also working on mapping fungal pathogenesis ... including strains that kill all mosquitos, only mosquitos that are infected with malaria, or only malaria in infected mosquitoes!
- Q: don't know about ultimate effector/mechanism
- Q: don't know about role of parasite genetics (what would happen if expt done when fed on a single gametocyte carrier? do see that these genes affect all parasite genomtypes

Mamadou Coulibaly (Bamako) - reproduction of M & S Anopheles
- 7 subspecies of A gambii / SS most dangerous ... divided into 5 subforms ... 2 forms identified molecularly (M (Mopti) & S (Savannah)

IMAGING SESSION

Markus Meissner
- Metchnikoff microscopy,
- light limitations (200 nm)
- cutting edge: TIRF, stimulated emmission depletion ... 25 nm resolution, etc
- nice review by Mike Roth: Nature Rev MCB 7:63-8
- expensive: confocal 500K, AFM 700K, STED 1.3M, 3D cryoEM 3M = 5.5M euro
- need new algorithms for pattern recognition (esp fm
- need automated analysis of images
- how to prevent mistakes (image manipulation ... intentional or not
- digital imges are not just illustrations; supplement with metadata (self-calibrating microscopes and calibration standards)
- how/where to store images? OME too complicated [talk to Google]
- frontiers: not technology, but data management

Ute Frevert -- Imaging Plasmodium in the liver and brain
- Plasmodium spz migrate along ECM of liver (on proteoglycans? until reaching Kupffer cell, then enter into hepatocytes, through several until settle down. Mutant mice w/ few K cells almost never get in. Spz suppress respiratory burst in K cells.
- growth within hepatocytes to v large size; schizogony produces Ms; released as packets (merosomes), containing host cell organelles ... mt as energy source? Memb eventually disintegrates. Thinks blocking lung capillaries enhances chance of RBC infection.
- No inflammation against liver stages ... but in semi-immunes, see CD8+ CTLs (Tc) ... but in CD8-, CD4 can mediate sterile immunity (in mice). Note: all liver cells present Ag: hepatocyes, Kupffer cells, stellate cells, even sinusoidal endothelia!
- Q: what is on surface of merosomes? how rec'd by CD4? also, how long viable?

Thomas Cremer -- Nuclear architecture
- what orchestrates cell type-specific expression in somatic cells? Genetics & epigenetics ... nuclear architecture?
- believe chromosomes organized into territories, with complexes for Xcr, Xla, Repl, Repair, etc
- tomography of in situs shows domains for HsChr18 (gene poor, on surface) & 19 (gene rich, interior) ... Alu-rich regions in interior [contradiction?]
- replicating chromatin at periphery (ciliates, mouse, human)
- syntenic regions in Hs & Mm conserved in position, even when chromosomes rearranged
- transcription ... loops in euchromatin? active BACs hyb to euchr; inactive in heterochr. ... but still quite highly compacted (~300x)
- nuclear regions for silencing? splicing speckles in interchromatin compartment (test by changing osmolarity changing condensation)
- goal: go from microtomography to nanotopography of functional compartments
- also see changes with development: from 8-cell embryo to blastocyst
- rod cells reverse: gene dense in periphery, gene poor in center (happens during terminal differentiation)

Bamini Jayabalasingham -- Degregation of Spz Diffn
- IMC breaks down during spz differentiation; quite dynamic changes ... disruption esp near nucleus, then eventually collapses into membranous whorl
- micronemes pushed to periphery, sometimes in multivesicular bodies?
[notes: would be good to follow with GFP-IMC in Pb! Also, would be good to look in Toxo spz formation]; Manami: what is status of atg8 work?]
- associated with autophagy? test with 3Me-Ad ... arrest in spz (maybe, not v convincing)
- looking at Atg3, 7 & 8 see all transcribed; focus on atg8, show interax with 3 by Y2H. Tried DN atg8 ... no go.
- localize (ab?) show mostly in cytoplasm, not spec assoc with mn or imc

????? - Plasmodium Spz development in skin!
- after bite, 50% in dermis, 15% in LN, 30% in blood to liver
... running out of power!

Overarching meeting topics:
- need for more (genome-driven) targets
- need for additional genomic query tools in kinetoplastida

Last Morning's talks

Last morning talks


I didn't have time to edit these, so here are my notes directly as I jot them down:

An excellent talk by Prezeworski
Looking for recombination events in order to determine whether males are more
data for 728 meioses with many more markers than
own heuristic approach to call markers
to infer recombination events in the father, from pedigree data
cummulative genetics
resolution at which could resolve crossover eventsm 24K crossover
hotspots id'd in analyses of LD (hapmap)
enrichment of crossover events in hotspots
also estimated the fraction of crossovers
looking at hotspots
no diff in the mean of "use of hotspots" between male and female
looked at LRT null: significnbt variation among individuals using a likelihood ratio test
how heritable is this hotspot usage? (given the known pedigree of the individuals)
so individuals are varying significantly in their use of hotspots, and that variation is heritable.
a similar proportion of crossover events occur in hotspots in males and females genome wide
along the genome, there is extensive variation in cross-pver rates at fine scales, supporting inferences based on analyses of LD.

this could in part explain the diff in hotspots btwn chimpanzee and human.

Used the same data to infer constraints on recombination rate in humans.
it's thought that at least one crossover per chr arm is necessary to ensure proper disjunction.
interesting if look at Lenzi et al. female meiosis is error prone. This analysis is impt for evol bio and human genetics.
How often does proper disjunction occur per gamete among viable individuals.
Modeled the distribution of no of crossover events on each chromosome

find that the proper disjunction can occur in the absence of any crossovers on a chromosome.
how strong would chromatid interference have to be to explain this?
alternatively,
is there a back up mechanism in humans as there is in Drosophila?
There's no known affect on relationship of paternal age and no of recombination events
viable offspring of older mothers have more crossover events; Possibly to overcome insults to the meiotic exchange over the 35-40 years of female's lifespan.

Dan Neafsey talked about widespread selection and frequent recombination in the genome of the Plasmodium falciparum malaria parasite
genetic diversity drives this disease: immune evasion, drug resistance, vaccine failure.
tackling diversity
112K known SNPs
1 snp per 200 bp
trying to capitalize on known snps
SNP avg call rate 91%
avg accuracy 91% and good call rate across samples
Showed that there is a geographic population structure (at the continent and sub-continent level)
ML, HKY+ gamma
LD reflects the local epidemiology
plotted LD for the different continental populations: senegal, thailand, brazil
LD up to 100Kb in brazil vs. much lower in senegal- suggesting a different effective population size
incidence of mult infections is highest senegal (more than one genotypic variety)
while recombination rate doesn't vary, outcrossing does since outcrossing requires multiply-infected hosts (mosquitos often only bites one, so not likely to happen there).

selection: pattern of divergence universal pattern. nonsyn > silent
selection is impacting the
freq in the spectrum isn't biasing nonsynonymous vs silent divergence (mapped avg DAF against proportion of SNPs with signiical Fst (P<0.05))
so what's the role of selection in this pattern?
patterns of div within populations (as opposed to btwn pop)
patterns indicates negative selection
purifying selection comes from derived allele frequencies (mapped for Senegal and Thailand) snp population frequence relative to percent of SNPs)
selective sweeps interrupted by crossover events
3 of 33 strains exhibit ancestral allele
but we also know that positive selection plays a role, too
selective sweeps are detectable
looked at the pfcrt locus, saw disparity as expected.
cq resistant
high diversity regions of the genome a
decoupling : (ie, reduced divergence in high diversity regions)
thinks this is the result of balancing selection: retarding divergence
malaria SNP diversity influenced by pos and neg sel
LD is short, del on local epidemiology
new genotyping tools, 75K SNP genotyping array and Taqman molecular barcode.
Jeffares asked if Neafsey is looking for conservative vs radical aa changes, as well as looking for functional enrichment along selection profile.

Then was a talk about the 17q21.31 microdeletion syndrome (see Genome Research May, 2008)
these events are deNovo, estimated that 1% unexplained mental retardation is probably related to this microdeletion.
All the cases identified so far has been european
The inversion is under pos selection; ind which carry polymorphism show inc fecundity and global recombination
relationship btween microdel and inversion?
tested 17 parents of children
looked for the inversion in non human primates using FSH.
They estimated a human duplicated gene inversion toggling
inverted H2 haplotype most likely represents the ancestral state of huymans
inversions represent a premutation state for large scale de novo microdeletion and disease, maybe because of structural variation of segmental duplications favors NAHR.
(all this in Kidd, Nature 2008)

In all, the meeting was VERY informative. Many posters were also worth looking at. I will have the abstract book available in the computational lab.

The special session on non-human biology of genomes

They tell me that in past years, the Biology of Genomes meeting didn't have such a focus on human genomics nor sequencing methodologies. well, this morning's session was titled "Genomics of non-human genomes"-- hence reduced to a single session, the first speaker (Barsh) had to defend the activity of doing genetics and genomics in non-human species... and I'm thinking, how crazy is that?? This IS Biology of genomes, afterall...

Anyhow, talks of note below--
Work done by Carlos Bustamante et al:
dog human and cow genomes reveal extensive human reorganization of domesticated genomes.
Prompted by large scale projects to document genome-wide variation in many species, they've taken up the CanMap (genetic diff among domestic dog breeds) and GSK Propgen projects (but he didn't talk about the GSK project).
The CanMap project is to genotype 850 dogs and 200 wolves using affymetrix v2.0 array, and use the data to understand demographic history of dogs.
Since each breed fixed for specific phenotypes in a small number of genes, this is easier to delineate than if selection occured independently. Perhaps those same genes can be implicated for the phenotypic changes we see across 'racial' or 'ethnic' lines.
They use joint association and selective sweep mapping where the unit of analysis is breed. They're looking for linked loci or alleles that are major determinants of phenotypes (for example, dog size, see Sutter et al. 2007, Science 316). They perform regression on body size, again at a breed level analysis, and model avg breed body size (using stepwise regression). They then look at predicted size against the observed, for validation and to determine outliers.
They claim that the distribution of phenotypes is not independent of the distribution of how the breeds are mapped into "genetic space" (in other words, how closely related are they). They look for PCA (but he didn't exactly describe what the high-dimensional feature vector is), assuming that the axes you come up with would relate to the phenotypic differences among the breeds.
bottom line: PCA reconstructs aspects of known breed history.
What's the punchline?
In the same way, they do a spatial prediction of ancestry... in HUMANS, and come up with a remarkable geographically associated demographic history. So we can have some insight into human demographic history migration and admixture, by looking at ourselves at the 'breed' level...

The next talk was by Andersson from Uppsala University about pigmentation mutation in horses ("grey") identified by three phenotypes (these are in fact 'white horses' in whom 'greying with age' appears at a very early age):
1. loss of hair pigmentation
2. melanoma (not because of the UV affect- their skin is actually very dark), but because of intrinsic aspects of the mutation.
3. vitiligo
4. speckling and 'blood marks' (pointing to somatic instability? <- this assumption was questioned by the audience)
They looked for the mutation responsible for this phenotype and found the locus to be a duplication in an INTRON, using hi resolution IBD mapping (a duplication that showed correlation with the phenotype). This "greying with age" is a cis-acting regulatory mutation, but its effect on expression isn't exactly clear yet.
With respect to the black spots appearing on many white horses: says that with a duplication you can recruit silencing. Others questioned whether it could be an epigenetic silencing of the alleles (without implying DNA instability).

Also of note was the grand "unveiling" of the Platypus genome sequence. This week in Nature and accompanying pubs in Genome Research.

There were many posters last night that are of direct interest to Plasmodium genomes (notably the work of Dan Jeffares, Jeff Chang, and one more). The abstracts will be available in the abstract book. There was also a talk that pertained directly to Lucia's work in building phylogenies and hypothesizing when/where lateral gene transfer occured-- although this was on Prokaryote genomes (work by Tal Dagan).

I'm checking out. Better go line up for lunch (the meeting was oversubscribed, and there are even overflow flat panel screens out on the rainy foyer, for those not fitting in the warm and musty auditorium...)

Greetings from CSHL

Greetings from Cold Spring Harbor.

So far the sessions I've attended have comprised of a variety of focus-applications of large sequencing projects.
These have been:
Functional and Cancer Genomics
Genetics of Complex Traits
and
High Throughput Genomics & Genetics

The latter session was really all about new sequencing technologies and large genome sequencing projects. The most news-worthy talk was Chad Nusbaum's presentation of preliminary findings from their research using the SOLiD(TM) System. There were a number of science publications that covered this this morning (for instance here's the CNBC piece), so I won't go into it here.

One of the interesting ideas promoted by EA Grice from NISC comparative sequencing program was to look for a core microbiome. First, is there a core microbiome? In her talk titled "core diversity profile of human skin microbiome in health and in disease," the center is asking whether we should consider the dominant and rare bacterial populations in normal individuals as a taxonomic
unit when we're predicting disease or drug response (given that our drug digestion or response is sometimes dependent on the response of our microbiome). Their approach was all about the "Phylogenetic architecture" of the microbiome. While they were looking at skin microbiome, they said that the results (and ability to differentiate healthy and diseased tissue) could be generalized. However, they did recognize that skin is special because they have terminal differentiation.

I also presented my poster last night. The set-up is a bit weird because they make you put your poster up and down within a 4 hour period. We got some interest from the usual suspects, but I think there was greater interest in J.D. Watson's wine and cheese birthday party taking place on the lawn. So I got to see J. Watson in a cowboy hat. Personal opinion aside, everyone questioned CSHL's love-hate relationship with Watson...
J.D. Watson's wine and cheese birthday party






It was hard for me to see other posters last night since I was attending my own, but a few interesting things I saw:
Adam Siepel's lab is looking at some interesting ways of finding already characterized regulatory motifs under selection in multiple genomes with a phyloHMM approach (fabsCons derivative method)
Manuel Garber, Michelle Clamp and Xiaohui Xie looking for selection signatures. The idea is to identify sites with unlikely substitution patterns using diversion from stationary distribution that is in background. They tried this over the encode regions.

The evening session went until 11:30pm (!) finishing with R. Durbin's talk on the "1000 genome project." Since the meeting is oversubscribed, many of us are staying several miles away (and I didn't bring my car) so I'm dependent on the shuttle. So I didn't get to leave the labs until 11:45, and it wasn't because I hung out at the bar...

This morning's session is on Computational Genomics.
From JGI: Microbial genomes are a rich source of biological info. Doing comparative prokaryote genomics, they identified antimicrobial peptides, small RNAs and new restriction enzymes that may act as barriers to horizontal gene transfer (see Sorek, Science 2007).

From E birney's lab:
Herrero presented Enredo (which we heard about from Birney on his April 22nd visit)
Also introdued Ortheus
addressing inference of insertion deletion histories and substitution events
Uses a multiple alignment as guiding input
subs are using tamura nei nucleotide subs model
ancestral sequences are represented using weighted sequence graphs, but works progressively.
so check out
enredo, pecan and ortheus.
at their respective websites (which went past on the screen way too fast for me to catch).

Manolis Kellis talking about comparative genomics of mammalian phylogeny and drosophila.

In mammals: want to discover regions of increased selection, detect func elements by increased conservation, and add more genomes in which we can detect smaller elements, and subtle selection
also: discover diff classes of fun elements, patters of change that distinguish diff types of func element.
(I'm not sure what is new here)
They were promoting the "evolutionary signatures" of protein coding genes respecting frame shifting gaps and synonymous substitution signature (again, not new)
built a model for expected substitution counts (diff codons have diff distribution for synonymous subs counts) and score windows for depletion of syn subs-- but this only looks for purifying selection.
Also looking for signatures for microRNA genes (in Drosophila) (see Stark et al, Genome Research 2007)

BioMalPar mtg

Notes from Monday BioMalPar mtg

New edition of Methods in Molecular Biology ... should post on PlasmoDB.

Peter Preiser: antibodies to two STEVOR proteins show stain RBC surface differently in different isolates. Haven't done colocalization or selections.

Tony Holder: Nice talk on new GPI-anchored MN protein ... PF8_008 (filtered based on proteomics data, SP+, hypothetical, expressed in merozoites and sporozoites only)

Freddy Frischknecht: Holding pathogen imaging workshop in Heidelberg ... of possible interest to Ina?

M Prudencio (Maria Mota's lab): nice talk on siRNA kinome screen for slatered spz invasion of hepatocytes. PKCzeta best bet, although probably nonspecific?

Rowena Martin (Kiaran Kirk's lab): Beautiful talk on PfCRT expressed in Xenopus oocytes. Shows Xport of labelled CQ in K76T mutant if external pH=6. Block with 76 K or S163R. Reverse with verapamil (IC50-30uM). Suggestive data indicates possible transport of polypeptide fragments of Hb with two aromatic AAs.

Jake Baum (WEHI): Beautiful talk on formin nucleation of actin (see recent publication)

Welcome to the Roos Lab