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CBT in Malaria and Cassava Research addresses 6/8 MDGs

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1 CBT in Malaria and Cassava Research addresses 6/8 MDGs
Thomas G. Egwang, PhD ED, African Academy of Sciences, Nairobi, Kenya DG/SD, Med Biotech Laboratories, Kampala, Uganda Science and Technology for Sustainable Development in Africa AAAS Boston 17th Feb 2008

2 Cassava Food to> 500 million Hardy and drought-resistant
Roots high in calories Leaves :Vitamins A and B Stalks : propagation Hardy and drought-resistant Poor soils, marginalized land No fertilizers/pesticides Animal feed Industrial applications Sugar, acetone, alcohol Food security to the poor

3 Cassava Family:Euphorbiaceae Genus: Manihot Genus: Manihot esculenta
Ploidy: Allotetraploid (n=9) Genome size: 770Mbp Cassava Genetic Resources: -5700 acc. (CIAT Gene Bank) -2,000 (IITA Gene bank) -2,500 (EMBRAPA) 98 wild Manihot relatives Monoecious Dicot

4 Cassava: A ‘Shining Example’ of
African Agriculture Production in Tons/Ha of the three principal producing regions Source: FAOSTAT

5 2004

6 Important issues Diseases Starch content
Cassava Mosaic Disease (CMD) Starch content Amylose versus amylopectin content Bitter taste: bitter strains, sweet strains Glycoside of HCN V. bitter forms > 100 pmm cyanide

7 S&T and cassava issues Molecular markers for breeders
Transgenic cassava modified starch content: low amylopectin

8 The Cassava Mosaic Disease (CMD)
A viral disease endemic in SS-Africa Vectored by the white fly (Bemisia tabaci) Est > $1 B/y Control: development of CMD resistant varieties: 6-10 years Molecular markers

9 Multiple Sources of Resistance to CMD
Multiple resistance to diseases and pests CMD-resistant CMD-susceptible Improved multiple disease and pest resistant clones showing high levels of resistance to CMD

10 Targeted training in Cassava Genomics
Elizabeth Balyejusa Kizito Yona Baguma Scientists at NARO (Uganda)-developing SLU CIAT (Colombia) -advanced

11 Cassava Biotechnology Research: CBT needs
Tissue Culture Genetic Transformation Embryo rescue Micro-propagation Conservation and exchange Molecular Markers BAC Libraries BAC Clone screening and fingerprinting Genetic Markers Positional Cloning Genomic/cDNA Libraries Genetic Mapping QTL Mapping Molecular Breeding

12 Applications of Molecular Markers in Cassava Breeding
Increase heritability in the identification of superior genotypes Follow specific chromosome segments from unadapted wild relatives in breeding schemes Reduce population sizes for more precise field evaluations Delineate heterotic pools or to choose parents that maximize additive variance Accelerate inbred line development

13 Molecular Marker Resources for Cassava at CIAT
5 small insert (1-2kb) genomic libraries 3000 low-copy RFLP marker probes 3 genomic SSR sequence enriched libraries 16 partial and full-length cDNA libraries 850SSR markers from genomic and cDNA libraries Two diversity array technology (DArT) chip with and 10,000 polymorphic clones respectively Unigene chip of 6,000 ESTs Web-based data base being prepared for easy access to these global public goods (GPG)

14 Cassava Map 2004: 202 SSRs, 307 RFLPs, 100 RAPDs, 3 isozymes
H Dist Marker cM Name GY85 13.1 OJ1 20.4 SSRY52 1.6 SSRY12 11.2 SSRY48 14.5 rGY199 12.6 rGY170 10.3 rGY104 6.4 GY127 19.7 rGY211 rSSRY91 24.3 SSRY178 16.9 SSRY63 SSRY164 rSSRY31 rSSRY52 7.2 rSSRY12 12.4 rQ11 19.0 rGY99 11.1 rGY8-1 12.0 GY85-1 9.1 nGY11 6.0 rSSRY178 37.4 rSSRY164 A ACP-1 9.2 rGY208 15.7 SSRY98 11.4 rGY75 9.3 GY12 8.2 GY28 6.8 GY213 5.1 GY209 rSSRY4 16.4 rGY22-1 19.5 rGY75-1 7.3 rSSRY98 8.8 rBEST-2 GY68 7.0 8.9 GY32 rGY155 3.9 rGY192 9.5 GY154 14.6 rSSRY111 15.3 SSRY59 1.8 SSRY10 rGY215 19.3 L7 28.1 AC-1 M C rGY144 2.5 rGY23 GY81 6.2 rGY89 14.0 rGY177 O11a 16.1 GY174 9.8 rSSRY183 rSSRY45 4.4 rSSRY95 17.8 rSSRY109 5.8 SSRY32 rK9a 20.1 rNI1.C2 7.6 GY23-1 13.4 rGY18 2.8 rGY26 rGY89-1 SSRY171 GY42 GY44 GY54 GY81-1 rA16 13.2 rGY191 17.5 rV17a 6.5 GY197 6.7 rGY147 5.6 rSSRY50 26.0 GY207 B rAi14a 17.2 GY72 10.9 rSSRY61 9.4 rGY65 5.0 rPASK1 17.1 SSRY113 SSRY21 rF19b 15.0 rGY118 12.7 GY217 11.0 rS2 rSSRY161 11.6 rGY176 20.3 rGY190 E SSRY84 rSSRY180 GY83 28.7 GY17 12.3 SSRY72 nGY162 15.6 rAE15 23.7 GY58 21.1 CDY123a 11.5 GY118 10.0 rI4a G13 D rGOT-2 rGY167 rGY180 1.2 GY222 GY181 3.7 GY219 GY50 GY125 GY179 3.8 rON1 SSRY3 0.0 SSRY120 3.6 rSSRY40 SSRY9 rSSRY169 10.6 GY4 14.8 SSRY108 rGY24 rGY59 GY78 rGY67H 15.8 rI18b rJ1a rGY57 20.5 rGY25 SSRY23 rSSRY55 10.7 rJ7 rG11 20.7 GY110 rSSRY100 7.9 SSRY88 19.2 Ai11b L 19.4 N15b 12.1 GY53 7.4 rSSRY88 8.0 rGY110 rSSRY172 22.0 rE14b 11.9 rJ9c 7.5 rCDY74 CDY76 AD1c 14.4 K2a 27.0 GY34 SSRY101 8.6 SSRY2 SSRY5 J SSRY47 O20 11.3 AE10a 11.8 rM8b GY7 GY34-1 25.9 K10 rGY93 GY97 SSRY135 rSSRY30 22.4 rSSRY38 7.8 rGY41 16.7 CYP79(152) G rSSRY135 23.4 rGA-131 10.4 rGY94 AM18 rK16d 13.3 20.8 rCPY79(152) rGY137 23.5 AB9a 24.8 SKDH-1 rAGPaseB 16.0 rSSRY106 rSSRY103 15.2 SSRY83 F GY196 GY203 GY218 GY122 CDY107 GY37 GY204 GY194 GY186 SSRY179 17.0 SSRY68 Z11b 12.5 GY21 rCDY107-1 10.1 CDY123b 4.6 GY80 21.0 rV20a 28.3 H14c GY16 23.0 rGY55 I rAC1 GY130 R1 13.6 rW3 rGY201-1a rD5a rB3a H14b GY128 17.4 GY143 CDY29 22.6 CDY68 CDY128 rGY19 rSSRY8 rS12c 23.8 rU2a rSSRY122 37.0 nrGY67 rGY36 3.0 rSSRY86 SSRY105 rGY87 W19b GY105 5.9 SSRY177 8.4 GY5 5.7 SSRY99 rK3 U K17 18.0 SSRY6 SSRY122 9.0 SSRY42 18.7 34.6 GY138 rGY164 GY223 4.5 SSRY170 CDY131-1 rGY113H 6.3 rGY92 SSRY185 2.3 SSRY20 21.5 U2c X rM5a CDY131 13.5 nCBB rSSRY20 rAF14a SSRY110 N rCDY99 rGBSSII SSRY13 rCDY44 rSSRY51 GY20 6.1 rGY10 GY108 GY52 5.3 GY13 Z18b GY88 17.3 rGY47 4.0 nGY143 rGY109 rGY148 rGY145 2.4 GY201-1b 18.8 AD4b SSRY35 rGY108 GY182 GY 124 rAF17 SSRY107 GY171 rGY210 5.5 ri4b rA18 GY131 13.9 rSSRY83 GY66 rSSRY34 9.9 SSRY102 2.6 SSRY182 5.4 GY39 28.6 AM6 rGY115 rGY9 rGY1 rSSRY28 Ai19 PO2 16.8 SSRY7 F19a AC11a GY71 GY183 12.8 i16b R V J13c 8.7 GY51 25.0 F15b 18.6 rSSRY19 rG4 rK11c rAC5 rR13b rP1a rGY156 21.2 GY134 rGY220 GY63 rAC9a GY100 31.4 rGY48 AE2 U1 rGY163 rGA-127 CDY106 27.8 rSSRY49 rGY119 K rGY117 rSSRY175 1.5 rSSRY85 6.9 GY82 8.3 W16b 14.1 AC9b GY119 rI2 SSRY174 rSSRY82

15 RFLP Marker Segregation in an F1 Cross of Non-inbred Parents

16 Gene Tagging of Multiple Sources of CMD Resistance Genes
Three different CMD Resistance genes have been mapped and Can now be pyramided using molecular markers D Y66 18.5 rI18b 20.5 rJ1a 20.0 rGY57 21.0 rGY25 21.2 SSRY9 23.9 SSRY3 16.2 SSRY23 CMD1 R Dist Marker cM Name rGY115 7.9 rGY9 15.6 CMD2 rGY1 16.1 rSSRY28 11.3 Akano et al. 2001 Ai19 M SSRY102 24.4 SSRY230 16.6 Ns905 9.1 GY39 11.2 SSRY299 SSRY102 SSRY102 Fregene et al. 2000 24.4 24.4 SSRY230 SSRY230 16.6 16.6 NS170 CMD3 Ns905 Ns905 9.1 9.1 GY39 GY39 11.2 11.2 SSRY182 SSRY182 Hurtado et al. 2007 SSRY299 SSRY299

17 Positional Cloning of CMD2
High resolution map around CMD2 Contigs of BAC clones around the CMD2 locus Candidate BAC Sequence

18 X Male Parent Female Parent TME3 TMS30555 1288 F1s Susceptible to CMD
High Starch Resistant to CMD (CMD2 gene) 1288 F1s

19 Cassava BAC Libraries Purification and cloning HindIII partial digestion 388kb 194kb 48.5kb Mega base pair-sized DNA in Agarose Plugs Size selection Cloning vector Ligation and Sizing of clones 3 BAC libraries with 5X, 10X, and 11X coverage of the genome respectively 97kb 6.9kb 48.5kb Clone picking by Robotics Sizing of clones

20 BAC Clone Fingerprinting
Digestion with Hind III

21 Construction of a Contig Around CMD2
Contig construction and genetic mapping of the ends of a BAC cone (BAC-33b) CMD2 BAC 33 SSRY-28 RME--1 NS158 RME--2 7cM 4cM

22 Bringing the Benefits of Modern Science to Farmer’s Fields
Molecular markers can help reduce breeding populations Proof of concept: Farmer participation demonstrate quickly superiority of new germplasm Social scientists/mol biologists

23 Two scientists Elizabeth Balyejusa: molecular markers
PhD Grant awards MBL and NARO Yona Baguma: modified starch content Heading Biotechnology Division, NARO

24 Achievement Award in Bean (Phaseolus) Improvement
granted by the Bean Improvement Cooperative (BIC) to Matthew Blair. CIAT-Outstanding Principal Staff Achievement Award (OPSA) granted to Segenet Kelemu CIAT-Outstanding Young Scientist-of-the-Year Award (OYSYA) granted to Marcela Quintero. CIAT-Outstanding Research Publication Award (ORPA) , granted to Elizabeth Balyejusa Kizito, Linley Chiwona-Karltun, Thomas Egwang, Martin Fregene, and Anna Westerbergh.

25 Hereditas Sep;144(4):129-36 Quantitative trait loci controlling cyanogenic glucoside and dry matter content in cassava (Manihot esculenta Crantz) roots Balyejusa Kizito E, Rönnberg-Wästljung AC, Egwang T, Gullberg U, Fregene M, Westerbergh A.

26 Genetica. 2007 Jul;130(3):301-18. Epub 2006 Nov 3.
Genetic diversity and variety composition of cassava on small-scale farms in Uganda: an interdisciplinary study using genetic markers and farmer interviews. Balyejusa Kizito E, Chiwona-Karltun L, Egwang T, Fregene M, Westerbergh A.

27 Plant Science 164: , 2003 Expression patterns of the gene encoding starch branching enzyme II in the storage roots of cassava (Manihot esculenta Crantz) Baguma Y, Sun C, Ahlandsberg S, Mutisya J, Palmqvist S, Rubaihayo PR, Magambo MJ, Egwang TG, Larsson H, Jansson C. .

28 Malaria

29 Malaria kills 110 Ugandan children <5 years old annually

30 Pregnant women (1st pregnancies) bear the brunt:
Poor pregnancy outcomes Maternal anemia Maternal deaths

31 Malaria control Drug treatment: RESISTANCE
Insecticide treated bed nets: RESISTANCE Vector control IRS: RESISTANCE; LOGISTICS Vaccines: future ray of hope

32 Targeted training Malaria drug resistance monitoring
Malaria vaccine studies

33 Malaria vaccines Development Clinical trials Total 13 4 27 37 9 46 2 1
Activity phase Vaccine Type Development Clinical trials Total PreErythrocytic 13 4 27 Asexual 37 9 46 Transmission- Blocking 2 1 3 Combination 6 8 58 16 74

34 Localization of SERA (serine repeat antigen)
in P. falciparum infected RBC by immuno-gold staining RBC membrane Merozoite SERA

35 Study Populations Apac, Uganda transmission : perennial
Longitudinal study School children Age 7-16 years Case-control study of severe & mild malaria Age 6-59 months To study this, we have used from low and high malaria transmission areas

36 Before processing After processing Recombinant proteins 図1
Serine repeats (35) 23 S H N 989 Before processing I III II After processing P47 P50 P6 P18 Recombinant proteins 17 382 SE47’ まず、実験室レベルの精製レコンビナント蛋白質、SE47’とSE50Aで疫学調査を行った。 P50A SE36 図1

37 Correlation between anti-SERA IgG
and malaria symptom or blood parasitemia Anti-SERA IgG and Fever Anti-SERA IgG and blood parasitemia Anti-SE47’ Anti-SE50A Anti-SE47’ Anti-SE50A 4 2 . 1 4 1 4 1 2 1 2 3 1 . 5 1 1 Parasitemia (1000/μl blood) Anti-E50A IgG1 ELISA OD 8 8 Anti-SE47' IgG3 ELISA OD 2 1 . 6 6 4 4 1 . 5 2 2 1 2 3 4 1 2 3 4 No Fever < 37.5 ℃ Fever > 37.5 ℃ No Fever < 37.5 ℃ Fever > 37.5 ℃ anti-SE47' IgG3 (OD at 405 nm) anti-SE50A IgG1 (OD at 405 nm)

38 Mean ODs for IgG subclass responses to SE47’ and SE50A
as a function of age in Apac District, Northern Uganda Anti-SE50A IgG Anti-SE47' IgG ELISA OD ELISA OD 一般にマラリア抗原分子に対して、宿主であるヒトはかなりよく反応するが、それが即座に防御免疫につながる保証は全くない。 次に、抗SERA抗体価とマラリア免疫の相関を見る。 0 - 1 2 - 5 6 - 8 9 - 10 > 40 0 - 1 2 - 5 6 - 8 > 40 11- 14 9 - 10 11- 14 Age group Age group I g G 1 2 3 4

39 Parasite growth inhibition by human serum
Hondurasー1 FCR3 K1 Patient Serum 75 50 Parasite culture Parasite Growth inhibition (%) Parasite Growth Inhibition Assay after 24 h. 25 102 103 104 105 IgG3 ELISA titer against SE47’

40 8 Recombinant 47’ neutralizes the inhibitory effect of patient serum on in vitro parasite growth 50 40 SE50A 30 Parasite Growth Inhibition (%) SE47’ 20 SE36 SE36の精製では、後々ヒトでのPhase I, Phase II の臨床試験に応用できるように、GMPに近い条件で精製した。 10 10 20 30 40 Concentration of recombinant protein for neutralization (μg/ml)

41 Median SE36 titre compared with level of pigmented leucocytes
MM SM SM IQR 7, ,500 1,500

42 Production of malaria vaccine SE36 under
GMP environments for clinical trials GMP production facilities for SE36 located in the Kanonji institute of The Research Foundation for Microbial Diseases of Osaka University (BIKEN)


44 P. falciparum: chloroquine resistance
Countries with at least one study indicating chloroquine total failure rate > 20% Countries with at least one study indicating chloroquine total failure rate > 10% No recent data available

45 P. falciparum: mefloquine treatment failure
Countries with at least one study indicating mefloquine total failure rate > 20% No failure reported Mefloquine total failure rate < 10% No recent data available Countries with at least one study indicating mefloquine total failure rate > 10%

46 P. falciparum: sulfadoxine-pyrimethamine resistance
Countries with at least one study indicating sulfadoxine-pyrimethamine total failure rate > 20% No failure reported Sulfadoxine-pyrimethamine total failure rate < 10% No recent data available Countries with at least one study indicating sulfadoxine-pyrimethamine total failure rate > 10%

47 P. vivax: chloroquine prophylactic or treatment failure
P. vivax prophylactic or treatment failure

48 Molecular monitoring of drug resistance
Finger prick blood spotted onto filter paper Sample was air-dried Storage at room temperature Shipment in luggage or by regular mail DNA extracted by simple methods for molecular analysis Sample ID In addition to the techniques we also developed very simple methods for sample collection: A few drops of blood from finger-prick are deposited on a small piece of filter paper cut into tiny rectangles at one end. The sample identification information are noted on the other end. The paper is then air dried, it can be stored and shipped at room temperature and can even be sent out by regular mail. Each blood-socked-tiny-rectangle can be cut out to extract DNA for molecular analysis.

49 Mutation Sites in the PfCRT Transmembrane Protein
NH2 COOH R371 H97 K76 Q271 I356 N75 M74 C72 N326 Tom Wellems’ group at the NIH, discovered pfcrt, the parasite gene that is responsible for chloroquine resistance. pfcrt has a few point mutations, the critical one for resistance being the K76T mutation. A220

50 Molecular Diagnosis of Chloroquine Resistance in the Field

51 PCR detection of pfcrt K76T polymorphisms
P4-w P4-m D1 D2 Apo1 DIAGNOSTIC PCR PCR1: P1+P2 PCR2: P3+P4-w or P4-m RESTRICTION PCR1: P1+P2 PCR2: D1+D2 Digestion with Apo1

52 Restriction digestion analysis of pfcrt K76T
M Dd2 3D7 C M 300bp This is a sample gel of the restriction analysis. You can see that samples #1 and 2 are uncut and therefore are mutated while samples #4 and 5 are cut and of the wild type. Sample 3 is a mixte infection of mutant and wild tupe alleles. Dd2 is the resistant ctrl, 3D7 is the sensitive ctrl and C represent a negative ctrl. M is a 100bp DNA ladder.

53 Distinguishing Recrudescent from reinfection with Microsatellite ta99
Dd2 Hb3 C

54 dihydrofolate reductase
Mode of action of antifolates GTP Sulfadoxine (SD) Pteridine pABA dihydropteorate synthase (DHPS) Pyrimethamine (PM) Dihydrofolate dihydrofolate reductase (DHFR) Tetrahydrofolate Thimidine, Methionine synthesis

55 Relevance of Malaria Studies to MDG
Change in CQ/SP drug policy Phase I-II trials of SERA5 IPTp assessment of Coartem-on-going Collaboration with Control Pgm

56 Targeted CBT to meet MDGs
Cassava: Molecular markers Elizabeth Bayejusa Kizito Cassava: Modified starch content Yona Baguma Malaria: Vaccine studies Brenda Okech Malaria Drug resistance Anne Nalunkuma Connie Agwang


58 Female scientists trained
Elizabeth Balyejusa Kizito PhD Ag biotech Best CIAT paper 2006 Woman science fellowship-USA 2007 Brenda Okech PhD Malaria vaccine studies Career development award TDR/GSK Management of malaria vaccine clinical trials UK and Japan Anne Kazibwe: MSc malaria drug resistance PhD drug resistance Connie Agwang: DipBSc drug resistance Best AMANET student paper 2006 Prossy Namuwulya Short term training Plasmodium transfection: LUMC

59 Gender equality/equity issues addressed?
Not entirely

60 Other training in malaria
Group workshops Bioinformatics HHMI Drug resistance genotyping MIM/DTR, IAEA Molecular biology primer WHO /TDR > 70 Africans from> 12 countries IAEA training for Latin America Panama, Colombia, Peru Malaria/HIV interactions Mentored clinical fellow under ILA EGPAF International Scholars Award EGPAF Media malaria advocacy and outreach Uganda Media for Health (UM4H) Malaria Consortium

61 CBT in Malaria and Cassava Research addresses 6/8 MDGs
Science and Technology for Sustainable Development in Africa

62 Acknowledgement HHMI Elizabeth Glaser Pediatric AIDS Fdn WHO/TDR
SIDA/SAREC IAEA MIM/TDR NIH EU Govt of Japan Malaria Consortium NARO CIAT MBL staff Martin Fregene Ann Westerbergh Urban Gullberg Christian Johannson LindleyChiwona-Karltun Eleanor Riley Lisa Ranford-Cartwright Cally Roper Toshihiro Horii Andy Waters Christine Clayton Alan Thomas

63 Thank you

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