I have been working in Malawi for about six months now, and though I miss home (Oregon), I am beginning to feel comfortable and increasingly productive here. Malawi is a wonderful place to work. I have met many good people and seen much of the land. I have yet to explore so much more, but I have been very busy and very content with work.
Last Thursday, February 4th, I had the fortunate opportunity to present at a National Data Standards Meeting, convened at the Cresta Hotel in Lilongwe by Chris Moyo of the Central Monitoring and Evaluation Division of Malawi’s Ministry of Health. These slides offer a brief overview of FrontlineSMS:Medic’s evolution in Malawi, the current work that has kept me busy for the last six months, a few lessons learned, and some of our vision and strategy for the future.
If you have questions, ideas, critiques, or general feedback, I’d love to see it in the comments or on Twitter.
Note: If you click through to this presentation on slideshare.net, you can select the Notes tab underneath the presentation to see more or less the full text of this talk.
A dam near St. Gabriel’s Mission Hospital in Namitete, Malawi
A few weeks ago I packed my bags and moved to Malawi; I’ll be living here at least a year. In case you were wondering where I am, why I left my beloved Cascadia, or what I’m doing, I’m now the Field Director for FrontlineSMS:Medic. I co-founded this nonprofit with a few friends to help clinics use mobile phones to provide better care in under-served communities. I’m currently the only member of our team who will be working full-time for the coming year based out of East Africa. My work in Malawi is supported by a Compton Mentor Fellowship, a program which exists:
to promote the creativity and support the commitment of graduating seniors from participating schools as they move beyond academic preparation to focus on continuing “real world” application and contribution.
This fellowship has a few basic ingredients:
1. Select a cause you care about, and explain how serving this cause will enrich your life and the lives of others.
The Compton Foundation gets bright young people to work full-time on service projects right after they finish undergrad, that critical juncture where we are making career choices that will affect how happy and productive we are well into the future. A big part of my application was explaining why mHealth is right for me – why I find it so invigorating that a year in the field is sure to get me hooked on service work for the rest of my life. Medicine has made sense for a long time, however, until recently I thought dabbling in technology was just a hobby.
Then I decided I wanted to work in East Africa before starting medical school… what skills did I have to offer? I was honestly considering everything from teaching English to mopping floors when I realized that medicine needs geeks too! In a countless number of ways, having access to the information you need, when you need it, is essential to helping people be healthy. Somehow that was the difficult realization for me. Once I began looking at medical information problems in East Africa, cell phones seemed an obvious game changer and it was only a matter of time before I would discover FrontlineSMS and OpenMRS.
2. Find a mentor who you admire, who serves the same cause as you, and who would like to help you grow.
I’m very fortunate to be working with Ken Banks, founder and champion of the FrontlineSMS platform. Ken writes extensively about technology (especially mobile phones) and development from an anthropological perspective. My ethnographic work in Havana convinced me that participant observation reveals a certain subtlety that is essential to service outside of one’s homeland. If an estimate is an educated guess, I might say that ethnography is educated empathy. More important still, Ken is a great guy and an excellent communicator; he’s already been very helpful with my project.
3. Develop a plan of action to make full use of your intellectual resources and more than $35K from the Compton foundation over the course of one year.
My first plan of action was called MobilizeMRS. A far cry from what FrontlineSMS:Medic is doing today, but the basics were there – I wanted to integrate OpenMRS (the medical records system) with FrontlineSMS (for managing large groups of text messages). The plan had just enough spunk to become a finalist in the USAID sponsored Mobile Development Challenge. That small nod of encouragement helped put me in closer touch and eventually a working relationship with Josh Nesbit, and later Lucky Gunesekara and Nadim Mahmud, and the re-launch as FrontlineSMS:Medic is history (which you can read about at MobileHealthNews.com).
Fast-forward a few months – I now live in room #2 of a guest house at St. Gabriel’s Hospital in Namitete, Malawi. This rural mission hospital serves about 250,000 patients spread 100 miles in every direction. A year ago Josh Nesbit helped local staff train about 75 volunteer health workers to use FrontlineSMS to coordinate and provide better home-based care. I’ll be helping the same staff double the number of community health workers using phones in the field, train the community volunteers to provide more structured descriptions of their activities, and use PatientView to sort the data being sent to the hospital. At the same time, I will be helping the hospital transition from paper forms to a computer-based system for some of their medical records keeping. I’m also responsible for developing our Field Guide or Do It Yourself guide explaining our implementation model, and helping (via skype and email) clinics all over the place put this field guide to good use.
The eventual goal is to integrate the mobile phone program with some components of the electronic medical records system, probably beginning with the treatment program for persons with HIV/AIDS. After working in the clinic for a couple weeks, I couldn’t be more excited about the people I’m working with, and the project’s potential here.
I presented these slides at the Scotland Malawi Partnership Conference at City Chambers in Edinburgh, Scotland, 2009. If you are interested in the slides, particularly if you were able to attend the conference, I encourage you to contact me with follow up questions.
The short one-day conference was great. A few interesting tid bits I learned: More than half of all international health and development aid from the Scottish government goes to Malawi. Until Malawi began to build its first medical school just a few decades ago, most Malawian doctors were trained in St. Andrews. Blantyre (Malawi’s largest city) was named after Blantyre, Scotland, the birth-place of Dr. David Livingston. Dr. Livingston (of the oft’ quoted question “Dr. Livingston, I presume?”) was a Scottish Doctor who was the first European to explore much of East Africa, and his story is at the root of much of the long relationship between Scotland and Malawi. It was great being at a conference with so many people with extensive experience working in Malawi, and I particularly enjoyed meeting Maeghan Ray and the other conference organizers and speakers.
Since the conference I’ve been as busy as ever, meeting up with Ken Banks in Cambridge, visiting Dutch friends and host family that I hadn’t seen since I was an exchange student in the Netherlands (4 years ago!), and most recently re-launching the FrontlineSMS:Medic website.
Isaac Holeman (Me!), giving FrontlineSMS:Medic’s final pitch at the Netsquared conference, 2009.
I was fortunate enough to attend the Netsquared conference in 2008 with the featured project Squarepeg, and it was a wonderful experience. We didn’t take home a top cash prize that year, but I learned so much and met so many great people. So when I helped get FrontlineSMS:Medic started and we were in the process of meeting new people, continuing to explore our field, and looking for funding, I knew the opportunity to attend N2Y4 would be great. The community certainly didn’t disappoint. I feel like I am a slightly more thoughtful person for having had the pleasure of brain-storming, competing, laughing, eating, and drinking with all of you for this short whirlwind conference.
Our team also walked away from the conference with our first major organizational funding: the $25k top prize based on the Netsquared conference vote, the top $15K prize from the Microsoft Mobile Development Challenge, and a $5K social justice award from the French American Charitable Trust (FACT). Thank you, thank you, thank you, and THANK YOU!
For all of our friends who could not attend the conference and for those of you who attended and are (like us) still trying to piece together a flurry of ideas and experiences into a few memorable lessons, I’d like to share a few observations about why I think we were successful.
APT stands for Accesible Para Todos (Accessible For All in Spanish): Sasha from VozMob coined (I think) this tech acronym during his closing pitch, and I picked it up just minutes later during our closing pitch, using it to describe FrontlineSMS:Medic. What does APT mean for us? We’re working with a platform that is optimized for low-end and prevalent phones, that supports many roman and non-roman languages, and we’re trying to bring these tools to people who might not be able to access them without a little support.
Often the very DNA of an innovation (for example, look to our friends The Extraordinaries, also winners at this conference) requires a tool like a smart phone that just isn’t going to be accessible to everyone. That’s okay. But all of us can brainstorm about how to become more APT, and it was wonderful to have the Netsquared community affirm all the thought we’ve put into this topic.
We’re young and scrappy: I’m 23, and no one on our team is much older. On one hand, this means we know how to gird ourselves in caffeine and put the pedal to the metal 24/7 when we have a deadline. On the other hand, our project doesn’t have an MD, PhD, or M.A. at the helm. Many people would see that as a risk for such an ambitious project, and perhaps they would be right. The Netsquared voters, Microsoft, and FACT all decided to trust the core platform we work with, our successful pilot, and the passion with which we speak about our work rather than some of the traditional credentials of experience and expertise (initials like MD). Thank you for taking that risk, for being committed to meritocracy. We’re going to work tirelessly over the next year to prove your investment worthwhile. To that end… why don’t you help us succeed!
Here are a few ways you can help:
1) Go to hopephones.org, print out a pre-paid shipping label, and send us your old mobile phone so that we can repurpose it for global health. Consider contacting us to get a donation box at your work, school, or Church. If you blog or tweet, why not let the world know about our recycling campaign?
2) If you are a developer, designer, global health activist, philanthropist, or experienced entrepreneurial type, and are interested in contributing, we’d love to see how you can help. If you gave us a card or email at the conference, don’t worry, we’ll be in touch ;-)
Thank you once more friends and funders for an incredible conference.
- Isaac
For more than three years I worked in Deborah Lycan’s molecular biology lab at Lewis & Clark, where I studied ribosome biogenesis in a laboratory variant of bakers yeast. In the spring of 2009 I completed a 40 page research thesis, summarized below. Ultimately I was awarded departmental honors for successfully completing the written thesis, giving a seminar to faculty, students in my program, and a few daring members of the general public (slides above), and orally defending the thesis before the BCMB faculty.
Deborah was an extraordinary mentor, and over the course of several years became a dear friend. Greg Hermann provided critical feedback as my thesis reader, and I also appreciate the support of department chair Janis Lochner, and the rest of BCMB faculty for cultivating my understanding of science during those 4 wonderful years at Lewis & Clark.
Background
The yeast S. cerevisiae can complete its cell cycle in as little as ninety minutes. Reproducing all of the protein that underlies cell structure and function requires the constant activity of over 200,000 ribosomes per cell. Ribosome production reaches rates as high as 33 new ribosomal subunits every second, and is the primary energetic cost for rapidly growing yeast cells and the primary limit on the rate at which cells can divide (Warner 1999).
Ribosome biogenesis is a highly conseved process in eukaryotic organisms, beginning in the nucleolus where three major rRNAs, the 18S, 5.8S, and 25S, are transcribed as a single 35S pre-rRNA precursor. Ribosomal proteins, non-ribosomal proteins, and small nucleolar ribonucleoprotein particles bind to the nascent rRNA, ultimately forming a large 90S pre-ribosome. Cleavage of the 35S rRNA splits the 90s pre-ribosome into a pre- 40S pre-ribosomal subunit and a larger pre-66S subunit. The 40S subunit sheds most of the 30+ processing factors associated with the 90S particle and is exported from the nucleus into the cytoplasm shortly after the 90S cleavage (Schafer et al., 2003).
Small Subunit Export
Nuclear export of macromolecules such as the ribosomal subunits is regulated by export and import receptors called karyopherins. Crm1 is the major export karyopherin in yeast; it binds and exports proteins that have a conserved, leucine-rich nuclear export signal [NES]. Neither ribosomal subunit is thought to contain any NES elements in cis; instead, Crm1 mediates large subunit export by binding an export adapter, Nmd3, which contains an NES. Nmd3 in turn binds the 60S to enable export (Ho et al., 2000). Although small subunit export also depends on Crm1 (Moy and Silver 1999), no adapter analogous to Nmd3 has yet been identified. The Lycan lab has proposed that Ltv1 is a non-essential export adapter for the 40S subunit (Seiser et al., 2006). Ltv1 is coded by a nonessential gene, and it shuttles in and out of the nucleus in a Crm1-dependent manner. Disruption of Ltv1 affects the cellular localization of components of the 40S subunit (Seiser et al. 2006, Loar et al. 2004). If Ltv1 is a Crm1-dependent export adapter, it should bind Crm1 directly and also bind the 40S subunit. Ltv1 has been independently reported to interact with Crm1 in two hybrid assays, and the Lycan lab recently found that deletion of an inhibitory 43aa N terminus of Ltv1 is necessary for the two-hybrid interaction between Ltv1 and Crm1 (unpublished observations). In addition, Several lines of evidence indicate that Ltv1 binds the 40S subunit. Ltv1 co-sediments in sucrose gradients with the 43S/40S subunit (Loar et al., 2004), and co-precipitates with late pre-40S subunits when affinity purified with TAP-tagged Ltv1 or Enp1 (Schafer et al., 2006).
Ribosomal protein Rps3 May Anchor the Adapter Complex
The Lycan lab began investigating the small subunit protein Rps3 as a potential site at which Ltv1 may anchor the Crm1 export complex to the small subunit after confirming a reported two-hybrid interaction between Ltv1 and RpS3 (Ito et al, and unpublished observations). RpS3 is a conserved 40S sub-unit protein with proposed roles in translation initiation (Westermann et al. 1981) and decoding accuracy (Hendrick et al. 2001). A role for Rps3 in 40S export is supported by experiments that show that cells depleted of Rps3 specifically accumulate 20S rRNA in the nucleoplasm, a phenotype associated with a specific defect in export rather than processing (Ferreira-Cerca et. al. 2005).
Two additional proteins that interact with both Rps3 and Ltv1 are of interest because they may also function in 40S export. For example, In vivo, a complex containing Rps3, Ltv1 and 40S, and the biogenesis factor Enp1 can be salt extracted from 43S pre-ribosomes, but not mature cytoplasmic 40S ribosomes (Schafer et al., 2006). Phosphorylation of Rps3 by the kinase Hrr25 also leads to dissociation of the complex in vitro, and Schafer et al. suggest that this phosphorylation may mediate conformational flexibility that enables the small subunit to fit through the nuclear pore (Schafer et al., 2006). In addition to Enp1, the small ankyrin-repeat protein Yar1 is of interest because it physically interacts with Rps3 as well as with Ltv1. Mutants lacking Yar1 have aberrant polysome profiles, with a reduced number of 40S subunits and excess of free 60S subunits. Over expression of RPS3 in the Δyar1 mutants suppresses this defect, suggesting that Yar1, in connection with Rps3, has a non-essential role in 40S biogenesis (Loar et al., 2004).
Generation and Analysis of RPS3 Mutants
To explore Rps3’s role in small ribosomal subunit assembly and function, previous thesis student Kelsey Rogers generated a library of random point mutations in the RPS3 gene, and inserted the mutagenized fragment in a vector that contained Green Fluorescent Protein and the native RPS3 promoter. Rogers screened for mutants using a yeast strain in which the Gal1 promoter has been integrated upstream of the chromosomal RPS3 gene. This means that the endogenous RPS3 gene is expressed when cells grow on galactose and repressed when cells are grown on glucose. The result is that yeast containing a plasmid which expresses non-functional RPS3 should grow on galactose but not glucose, whereas expression of wild type RPS3 from the plasmid should produce viable yeast on either medium. Screening for a glucose-dependent mutant phenotype helps distinguish between mutations in RPS3 (the only gene whose expression is sugar-dependent) and random mutations in the genome that may have occured during transformation of the plasmid into yeast.
In this thesis, I characterized the collection of RPS3 mutants that Kelsey Rogers had generated. I recovered 19 plasmids from yeast, purified the DNA, and transformed them back into yeast to test if the plasmids could reconfer the original slow-growth phenotype. I used a more sensitive assay for growth this time and screened for nonconditional mutants that exhibited growth defects. I used GFP microscopy to test if any of these mutants were specifically defective in 40S export as predicted by our model for 40S export. I determined the sequence of confirmed mutant plasmids and mapped the mutations onto the proposed structure of yeast Rps3. Interestingly, all 6 mutations clustered to the solvent exposed surface of RpS3, as opposed to surfaces likely to interact with either the rRNA or with other small subunit ribosomal proteins. Finally I am testing whether these mutations affect RpS3’s interaction with either Ltv1 or Yar1 by testing whether over expression of either suppresses the slow growth phenotype of any of these Rps3 mutants.
If you just have time for one background reference, check out this recent paper by our lab.
Seiser, R. M., Sundberg, A. E., Wollam, B. J., Zobel-Thropp, P., Baldwin, K., Spector, M. D., and Lycan D. E. (2006). Ltv1 Is Required for Efficient Nuclear Export of the Ribosomal Small Subunit in Saccharomyces cerevisiae. Genetics 174, 679-691.
Complete List of References
De Boulle, K., Verkerk, A.J., Reyniers, E., Vits, L., Hendrickx, J., Van Roy, B., Ban den Bos, F., de Graaff, E., Oostra, B.A. and Willems, P.J. (1993) A point mutation in the FMR-1 gene associated with fragile X mental retardation. Nature Genet., 3, 31-35.
Fatica, A., and Tollervey, D. (2002). Making Ribosomes. Curr. Opin. Cell Biol. 14, 313-318.
Gibson, T.J., Thompson, J.D. and Heringa, J. (1993) KH domains within the FMR1 sequence suggest that fragile X syndrome stems from a defect in RNA metabolism. Trends Biochem. Sci., 18, 331-333.
Grishin, N.V. 2001. KH domain: One motif, two folds. Nucleic Acids Res. 29: 638–643.
Ho, J. H., Kallstrom, G. and Johnson, A.W. (2000). Nmd3 is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. J. Cell Biol. 151, 1057-1066.
Fatica, A., and Tollervey, D. (2002). Making Ribosomes. Curr. Opin. Cell Biol. 14, 313-318.
Ferreira-Cerca, S., G. Poll, P. Gleizes, H. Tschochner, and P. Milkereit. (2005). Roles of eukaryotic ribosomal proteins in maturation and transport of pre-18S rRNA and ribosome function. Mol. Cell 20: 263-275.
Ito, T., T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, 2001 A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. USA 98: 4569-4574.
Jensen, T. H., Neville, M., Rain, J.C., and Rosbash, M. (2000). Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor Ace2p shows cell cycle-independent nucleocytoplasmic shuttling. Mol. Cell. Biol. 20, 8047-8058.
Johnson, A. W., E. Lund, and J. Dahlberg, 2002 Nuclear export of ribosomal subunits. TIBS 27: 580-585.
Loar, J. W., R. M. Seiser, A. E. Sunderberg, H. J. Sagerson, N. Ilias et al., 2004 Genetic and biochemical interactions among Yar1, Ltv1, and RPS3 define novel links between environmental stress and ribosome biogenesis in Saccharomyces cerevisiae. Genetics 168: 1877-1889.
Moy, T. I., and P. A. Silver, 1999 Nuclear export of the small ribosomal subunit requires the ran-GTPase cycle and certain nucleoporins. Genes Dev 13: 2118-2133.
Moy, T. I., and P. A. Silver, 2002 Requirements for the nuclear export of the small ribosomal subunit. J Cell Sci 115: 2985-2995.
Neuber, A., Franke, J., Wittstruck, A., Schlenstedt, G. Sommer, T., Stade, K. (2008). Nuclear Export Receptor Xpo1/Crm1 Is Physically and Functionally Linked to the Spindle Pole Body in Budding Yeast. Mol. Cell. Biol. No. 20, 0270-7306
Oldenburg, K. R., K. T. Vo, S. Michaelis, and C. Paddon. (1997). Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res. 25: 451-452.
Passmore, L. A., T. M. Schmeing, D. Maag, D. J. Applefield, M. G. Acker, M. A. Algire, J. R. Lorsch, and V. Ramakrishnan, 2007 The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome. Molecular Cell 26: 41-50.
Schafer, T., B. Maco, E. Petfalski, D. Tollervey, B. Bottcher, U. Aebi, and E. Hurt. (2006). Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit. Nature 441: 651-655.
Schafer, T., D. Strauss, E. Petfalski, D. Tollervey, and E. Hurt, 2003 The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes. EMBO J. 22: 1370-1380.
Seiser, R. M., A. E. Sundberg, B. J. Wollam, P. Zobel-Thropp, K. Baldwin, M. D. Spector, and D. E. Lycan, 2006 Ltv1 is required for efficient nuclear export of the ribosomal small subunit in S. cerevisiae. Genetics 174(2): 679-691.
Siomi H, Matunis MJ, Michael WM, Dreyfuss G. 1993a. The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. Nucleic Acids Res 21:1193–1198.
Siomi, H., Choi, M., Siomi, M.C., Nussbaum, R.L. and Dreyfuss, G. (1994) Essential role for the KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell, 77, 33-39.
Spahn, C. M., R. Beckmann, N. Eswar, P. A. Penczek, A. Sali, G. Blobel, and J. Frank. (2001). Structure of the 80S ribosome from Saccharomyces Cerevisiae tRNA-ribosome and subunit-subunit interactions. Cell 107: 373-386.
Spahn, C. M., M. G. Gomez-Lorenzo, R. A. Grassucci, R. Jorgensen, G. R. Andersen, R. Beckmann, P. A. Penczek, J. P. Ballesta, and J. Frank, 2004 Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO Journal 23: 1008-1019.
Tindall K. R., and T. A. Kunkel. (1988). Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27: 6008-6013.
Warner, J. R., 1999 The economics of ribosome biosynthesis in yeast. Trends Biochem Sci 24: 437-440.
I work with 