Accomplishments V Shyamala

V. Shyamala 1

Accomplishments (Selected)

Venkatakrishna Shyamala, PhD

•Consultant: Research Diagnostics, Inc.

•Consultant: Novartis Dx- Scientific Affairs

•CLSI Document writer - Molecular Diagnostics

•Innovative Biosensors, Inc.•Senior VP, Research and Development

• Digene Corporation•Director, Research and Development

• Chiron Corporation

•Associate Director, Research

• Univ of California, Berkeley•Visiting Scientist

V. Shyamala 2











V. Shyamala 3



• Research Diagnostics, Inc. Bengaluru, India

V. Shyamala (2014) Nucleic acid technology (NAT) testing for blood

screening: Impact of Individual donation and minipool NAT testing on

Analytical sensitivity, Screening sensitivity and Clinical sensitivity. ISBT

Science Series. In press.

V. Shyamala (2014) Transfusion transmitted infections in Thalassemics:

Need for reappraisal of blood screening strategy in India. Transfusion

Medicine. 2,79-88.

V. Shyamala (2014) Factors in Enhancing blood safety by Nucleic Acid

Technology Testing for Human Immunodeficiency virus, Hepatitis C virus

and Hepatitis B virus. Asian J. Trans. Sci. 8, 13-18.

V. Shyamala, T. Sandison and J. A. Holmberg (2014) Individual Donation

Nucleic Acid Technology testing to minimize Human Immunodeficiency

virus-1, Hepatitis C virus and Hepatitis B virus Transfusion Transmitted

Infections. Asian J. Trans. Sci. 8, 68.

•

V. Shyamala 4

Nucleic acid technology (NAT) testing for blood screening: Impact of

Individual donation and minipool NAT testing on Analytical sensitivity,

Screening sensitivity and Clinical sensitivity

V. Shyamala

ISBT Science Series, in press (2014)

•Blood screening requires the most sensitive assays

•Globally the donated blood is screend for HIV, HCV and HBV

•The impact of NAT testing formats and its effect on limit of detection (LOD) is elaborated

•In individual donation NAT testing, the Analytical sensitivity is equivalent to LOD

•The LOD in pooled testing decreases depending on the pool size, and is called the detection limit is called as Screening sensitivity

•The pool size dependent increase in risk days, demonstrated experimentally, and projected by calculation is presented

•Reported cases of minimum infectious units for causing infection and the compromise with pooled testing is discussed

•The results of regional variations in serology prevalence in India, the NAT detected units, the NAT testing formats are analyzed

V. Shyamala 5

Transfusion transmitted infections in Thalassemics: Need for

reappraisal of blood screening strategy in India.

V. Shyamala

Transfusion Medicine. 2,79-88 (2014)

•The donated blood in India is tested by serology assays for human immunodeficiency viruses (HIV)1/2, Hepatitis C virus (HCV), and Hepatitis B virus (HBV).

•The markers are anti-HIV-1/2, anti-HCV and HBsAg, and a prevalence of 0.3%, 0.4% and 1.2% in the Indian donors is reported.

•The development of these markers in an infected donor requires time. In the interim the unit though infectious is negative by the serology assays and released for use

•Using repeat recipient thalassemics as the model a prevalence of up to 26% of HCV, 6% of HBV, and 4% of HIV is reported

•Individual donation blood screening in India by nucleic acid technology assays shows serology marker negative NAT yields in the range of 1/350-1/2200.

• Though NAT testing is not mandated because of cost, the enormity of transfusion transmitted infections requires a reappraisal

V. Shyamala 6

Factors in Enhancing blood safety by Nucleic Acid Technology

Testing for Human Immunodeficiency virus, Hepatitis C virus and

Hepatitis B virus

V. Shyamala

Asian J Transfusion Science 8,13-18 (2014)

•Nucleic acid technology (NAT) testing is being increasingly adopted to detect HIV-1/2, HCV and HBV in the donated blood.

• The testing can be done either in the individual donation (ID) format or in the pooled format

•The pooled format compromises sensitivity of detection

•Globally with pooled testing several cases of transfusion transmitted infections (TTI) of HIV-1, HCV and HBV have been reported.

•Since very low copies of the viruses can be infectious there have been two cases of TTI by ID format testing also

•In nearly all of the cases infectious units are realized through a repeat donor turning positive.

In the absence of repeat donor base ID-NAT testing offers safer blood versus pooled NAT testing.

V. Shyamala 7



• Consultant: Scientific Affairs

Novartis Vaccines & Diagnostics

• Monitoring Blood screening by Procleix ® Nucleic acid technology (NAT) assays at blood banks in India

•Assist in product trials, monitoring trials, data analysis

• Provide support with NAT awareness Seminars, Workshops, Conference Presentations and Publications

V. Shyamala 8



• Document writer - Molecular Diagnostics

Clinical & Laboratory Standards Institute

Reviewer , Consensus Committee on Molecular Methods

Contributor:

MM06: Quantitative Molecular Methods for Infectious Diseases

MM19: Establishing Molecular Testing in Clinical Laboratories

MM01-A3: Diagnostic Molecular Methods for Genetic Diseases

MM09: Nucleic acid sequencing methods in Diagnostic Laboratory Medicine

MM22: Microarrays for Diagnosis and Monitoring of Infectious Diseases

POCT14: Point-of-Care Testing for Infectious Diseases

V. Shyamala 9

Molecular Methods and Platforms for Infectious Diseases Testing

A review of FDA Approved and Cleared Assays*

R. Emmadi, J. B. Boonyaratanakornkit, R. Selvarangan, V. Shyamala,B. L. Zimmer, L. Williams, B. Bryant, T. Schutzbank, M. M.

Schoonmaker, J. A. AmosWilson, L. Hall, P. Pancholi and K. Bernard

J. Mol. Diagnostics 13, 583-604 (2011)

•The performance of various assays as described in public forums, Product Inserts and publications are reviewed

•The challenges, limitations, testing and indications related to implementation of various assays in a clinical laboratory setting is summarized

•The categories of diseases include Sexually Transmitted Diseases -HPV, CT-GC, HIV-1, and HSV; Hospital Acquired Infections - MRSA, VRE, C. difficile, Respiratory tract and CNS infections, MTB, and CNS viral infections, other infections such as HCV, HBV, GBS, Culture tests for fungal and bacterial identification.

*A by-product of CLSI MM19 document

V. Shyamala 10











V. Shyamala 11

Innovative Biosensors, Inc.,

http://www.innovativebiosensors.com/VP%20R&D_July2007.pdf

Management Team

IBI's management has extensive experience in the

commercialization of innovative technologies in the

pharmaceutical, research and diagnostic markets.

V. Shyamala, Ph.D. — Senior Vice President of Research

and Development

Dr. Venkatakrishna Shyamala has extensive experience in

commercializing NAT technologies, primer/probe designs and assay

development, cDNA library screening, molecular cloning, sequencing

and expression. She has also participated in IND and BLA

preparations and submissions with broad project and people

management experience. Dr. Shyamala has been cited as inventor on

a dozen patents and published nearly fifty peer reviewed articles in

leading industry journals. She has a PhD in Biochemistry and

Molecular Biology.

V. Shyamala 12

V. Shyamala

Senior Vice President Research and Development

Accomplishments

http://www.innovativebiosensors.

com/pressreleases_pathogen_detection.html

•Innovative Biosensors Inc. Expands Scientific Advisory Board —

August 5, 2008

•Innovative Biosensors Inc. Raises $11.5 Million — Series B

Financing Combines Equity and Debt Capital — May 19, 2008

•Innovative Biosensors Inc. and ATCC® Partner to Develop High-

speed Test for Avian Flu — April 9, 2008

•Innovative Biosensors, Inc. and the University of Maryland

Receive Funding for Handheld Biosensor Development —

February 25, 2008

V. Shyamala 13

V. Shyamala

Senior Vice President Research and Development

Accomplishments

•NSF Grant Reviewer – Ohio State Interdisciplinary Grant —

October 2007

•AACC Oakridge Conference poster — A Rapid, Sensitive,

Specific Assay for the Detection of Chlamydia trachomatis By F.

Benahmed, J. Simpson, N. Chakraborty, I. Mielzynska, K. Modarress,

T. Hazel and V. Shyamala — April 2008

•Completion of MRSA CANARYTM Assay Feasibility — Feb 2008

•Pre-IDE Presentation — April 2008

•Alpha Clinical trials — Univ MD Shock Trauma Center; Indiana

University — July 2008

•Three Provisional Patents — Feb - April 2008

V. Shyamala 14











V. Shyamala 15

V. Shyamala

Director, Research and Development

Accomplishments

•Evaluate comparator assays for Chlamydia trachomatis of Roche

Amplicor, Gen-Probe Aptima, Abbott m2000, with in-house HC2,

helicase-isothermal, whole genome amplification and real-time

PCR technologies

•Optimize nucleic acid extraction with silica from large sample

volume for increased Diagnostic Sensitivity

•Five Invention Disclosures — Dec 2007 - July 2008

V. Shyamala 16











V. Shyamala 17

Alternative NAT HCV assay- Development, Validation , Implementation

Screen for Procleix ® HIV-HCV Clinical positives

S. Nguyen, P. Arcangel, D. Madriaga, David Chien, V. Shyamala, B. Phelps, BSRI members

Chiron Corporation, Blood Systems Research Institute

•Development of an Alternative HCV Assay. June 2000

•Alternate Technology: Target amplification by PCR

•Nucleic acid isolation: Organon-Teknika NucliSens reagent

•Amp-Det: Roche COBAS Amplicor

•Validation July-2000

•BSRI- reference lab Aug-Sep 2000

•Timeline BLA filing Oct 2000

•FDA Approval Feb 2002

V. Shyamala 18

Conference presentations: Hepatitis C virus RNA assay

VIIth European congress of the ISBT meeting, 2001,15th-18th July, Paris, France

1. V. Shyamala, D. Chien, S. Nguyen, N. Lagwinski, D. Madriaga, P. Carmichael,

B. Phelps. J. Heitman, D. Hirschkorn, L. Tobler, and M. Busch. Development

and Evaluation of Alternative NAT assay: a highly sensitive RT-PCR based

diagnostic assay for HCV RNA.

54th Annual AABB meeting, 2001, 13th-17th Oct. San Antonio, TX.

2. L. H. Tobler, J. M. Vargo, K. M. Smith, D. Hirschkorn, J. Heitman, C. Degula, V.

Shyamala, D. Chien, B. Phelps, L. Mimms, M.P.Busch. Sensitivity and Specificity

of an HCV supplemental NAT assay. Transfusion, 41, 83.

AACC meeting, 2000, 16th-18th Nov, Anaheim, CA.

3. Shyamala, D. Madriaga, D. Chien and B. Phelps. A highly sensitive,

Transcription mediated amplification (TMA) and Polymerase chain reaction (PCR)

dual amplification assay for the detection of Hepatitis C viral RNA molecules.

V. Shyamala 19

Alternative NAT WNV assay- Development, Validation , Implementation

Screen for Procleix ® WNV Clinical positives

D. Madriaga, J. Cottrell, P. Arcangel, David Chien, V. Shyamala, B. Phelps, BRTL members

Chiron Corporation, Bayer Research and Testing Labs

•Development of an Alternative WNV Assay Nov 2002 – Feb 2003

•Alternate Technology: Target amplification by PCR

•Nucleic acid isolation: Specific target capture technology

•Amp-Det: PCR- TaqMan technology

•Validation March 2003

•BRTL- Implement April 2003

•Timeline IND filing May 2003

•Blood Screening 2003-2004 WNV season

V. Shyamala 20

Analytical and Clinical sensitivity of West Nile virus RNA screening

and Confirmatory assays

M Busch, L Tobler, J Saldahna, S Caglioti, V Shyamala, J Linnen, J Gallarda, B Phelps, R Smith, S Kleinman

BSRI , the Canadian blood Services, Blood Systems Laboratory, Chiron Corporation, Gen-Probe, Inc., Roche Molecular Systems, the National Genetics Institute, the National Microbiology Lab-Canada, Univ of BC

Transfusion 45, 492 - 499 (2005)

•Coded samples, the First generation Chiron WNV assay implemented at Bayer Reference and Testing Labs had a 95% LoD of 33 Cps/mL and the improved Chiron assay had a 95% LoD of 6.4 Cps/mL

LoD Copies/mL Neat Minipools

Assay

Study Code

G-P

A

Roche

B

NGI

C

Bayer

(Chiron)

D

G-P

E

Chiron

F

G-P

1:4

G

G-P

1:16

H

Roche

1:6

I

95% LoD 15 125 26 33 6.4 6.4 55 184 1336

50% LoD 3.4 29 6.1 7.7 1.5 1.5 13 43 309

V. Shyamala 21

Detection of West Nile Virus RNA and antibody in frozen plasma

components from a voluntary market withdrawal during the 2002 peak

epidemicL Tobler, C Bianco, S A Glynn, G B Schriber, B J Dille, H E Prince, R S

Lanciotti, J M Linnen, J Gallarda, V Shyamala, D Smith, S H Kleinman, and M P Busch for the REDS study Group

BSRI, America’s Blood Centers, Westat, Abbott Laboratories, Focus Technologies, The CDC and Prevention, Blood, Gen-Probe, Inc., Roche

Molecular Systems, Chiron Corporation, UCSF

Transfusion 45, 480 - 486 (2005) F

o

•During the year 2002 in the WNV peak epidemic regions 60,000 plasma units were voluntarily withdrawn from the market

• Of this 1468 were retrospectively screened by immunoassays (Focus Technologies, Abbott Labs) and RNA detection assays (Gen-Probe Inc., and Roche Molecular Systems)

•RNA screening yielded one positive sample that was negative by the immunoassays

•RNA quantitation by Target-capture RT PCR (Chiron Corporation) suggested 440 cps/mL in the positive unit

V. Shyamala 22

Screening the Blood supply for West Nile virus RNA by Nucleic Acid

Amplification testing

M Busch, S Caglioti, G Robertson, J McAuley, L Tobler, H Kamel, J Linnen, V Shyamala, P Tomasulo, S Kleinman

Blood Systems Research Institute, Dept. Of Laboratory Medicine, Blood Systems Laboratory, Blood Systems, Gen-Probe, Inc., Chiron Corporation,

Univ of British Columbia

New Eng J Med 353, 460-467 (2005)

ms Foundation

•For the early part of 2003 WNV RNA screening assay was performed by minipool (16 samples) testing

•For the regions with highest reactivity in minipool screening, retrospective individual screening was performed

•Individual unit testing yielded additional positives that was IgM-negative

•Recommend Staged Minipool to Individual testing during the year

V. Shyamala 23

Conference presentations: West Nile virus RNA assay

58th Annual AABB meeting, 2005, Seattle, WA

1. J. Cline, C. Deza, R. Cory, A. Garcia, M. Lewis, A. Broulik, M. Deras, V. Shyamala, S. Pichuantes,

C. Giachetti, J. M. Linnen. Stability of WNV Viral RNA from tissue culture and blood donor samples in

stored blood. Transfusion 45, 149.

2. L. H. Tobler, V. Shyamala, J. Saldhana, C. Cameron, R. Lanciotti, R. Smith, I. Walsh, B. Munneke,

B. H. Phelps, D. Chien, M. P. Busch. West Nile virus (WNV) viral load comparison study. Transfusion

45, 151.

3. S. Nguyen, V. Shyamala, H. Huang, D. Madriaga, M. Badgett, J. Hedges, C. WalkerPeach, D.

Chien, B. Phelps, S. Pichuantes. Cloning of West Nile virus internal control and nucleotide fragments

spanning the full-length viral genome for production of stable RNA standards. Transfusion 45, 151.

2005 National conference on West Nile Virus, San Jose, CA

4. . S. Cagliotti, G. F. Robertson, J. McAuley, S. H. Kleinman, L. H. Tobler, H. Kamel, J. M. Linnen, V.

Shyamala, P. A. Tomasulo, M. P. Busch. Screening the blood supply for West Nile Virus RNA by

nucleic acid amplification testing.

57th Annual AABB meeting, 2004, Baltimore, MD

5. V. Shyamala, D. Madriaga, S. Pichuantes, B. Jaitner, D. Chien and B. Phelps. Performance

characteristics of the Validated and Improved qualitative and quantitative Target-Capture PCR WNV

NAT assays. Transfusion 44, 140.

6. M. Busch, L. Tobler, J. McAuley, J. Linnen, V. Shyamala, G. Robertson, D. Wright, S. Kleinman, S.

Cagliotti. West Nile Virus RNA dynamics and antibody evolution based on follow-up of viremic blood

donors. Transfusion 44, 2.

7. J. Cline, M. Lewis, W. Wu, S. Miller, A. Broulik, J. Savage, V. Shyamala, M. Cass, C. Giachetti, J.M.

Linnen. Gen-Probe Alternative WNV assay: A TMA-based confirmatory assay for West Nile Virus.

Transfusion 44, 138.

V. Shyamala 24

Conference presentations: West Nile virus RNA assay (Cont.)

28th congress of the ISBT meeting, 2004, Edinburgh, UK.

8. V. Shyamala, S. Pichunates, B. Jaitner, D. Madriaga, P. Arcangel, J. Cottrell, S. Nguyen, H.

Huang, A. Medina-Selby, D. Coit, D. Chien B. Phelps. Performance characteristics of the qualitative

and quantitative Target-Capture PCR WNV NAT assay. Vox Sanguinis, 87, 26.

9. L. H. Tobler, H. Prince, G. Hafner, B. Dille, R. A. Gutierrez, W. Andrews, C. Harrington, V.

Shyamala, J. McAuley, V. Winkelman, S. Cagliotti, M. P. Busch. Relative performance of four West

Nile Virus antibody assays in viremic blood donor specimens. Vox Sanguinis, 87, 65.

56th Annual AABB meeting, 2003, San Diego, CA

10. V. Shyamala, S. Pichuantes, B. Jaitner, D. Madriaga, P. Arcangel, J. Cottrell, S. Nguyen, H.

Huang, A. Medina-Selby, D. Coit, C. McCoin, D. Chien, B. Phelps. Detection and Quantitation of West

Nile Virus RNA by the Alternative NAT WNV Assay. Transfusion. 43, 128.

11. B. Jaitner, V. Shyamala, S. Nguyen, H. Huang, Y-L Fong, D. Chien, B. Phelps, S. Pichuantes.

Propagation, quantitation, and inactivation of West Nile Virus to support nucleic acid and IgM assay

development. Transfusion. 43, 128.

12. V. Shyamala, P. Arcangel, D. Madriaga, J. Cottrell, J. Linnen, D. Chien, B. Phelps. Compatibility

of ProcleixR- West Nile Virus (WNV) assay in various anticoagulants. Transfusion. 43, 129.

10th EPFA/NIBSC workshop & SOGAT meeting, 2003, Langen, Germany

13. V. Shyamala, S. Pichuantes, B. Jaitner, D. Madriaga, P. Arcangel, J. Cottrell, S. Nguyen, H.

Huang, A. Medina-Selby, D. Coit, C. McCoin, D. Chien, B. Phelps. Use of quantitative NAT assay to

correlate West Nile Virus titration bioassay (pfu/ml) with genomic copy numbers (geq/mL).

V. Shyamala 25

Alternative NAT HBV assay- Development, Validation , Implementation

Screen for Procleix ® Ultrio Clinical positives

J. Cottrell, P. Arcangel, D. Madriaga, David Chien, V. Shyamala, B. Phelps, BRTL members

Chiron Corporation, Bayer Research and Testing Labs

Development of Target-Capture PCR HBV DNA Alternative

Assay for ProcleixR Ultrio Clinical Trials 2001

Technology: Target-Capture PCR

Nucleic acid isolation: Specific Target-Capture

Amplification-Detection: TaqMan assay

Agreement with BRTL as the Reference lab Aug-Sep 2002

BLA filing Oct 2004

FDA Approval 2006-2008

Used to Support TUV submission for Ultrio, Tigris-Ultrio,

RAS, FEP

V. Shyamala 26

Assessment of the Target-Capture PCR Hepatitis B Virus (HBV) DNA

Quantitative Assay and Comparison with Commercial HBV DNA

Quantitative Assays

V. Shyamala, P. Arcangel, J. Cottrell, D. Coit, A. Medina-Selby, C. McCoin, D. Madriaga, D. Chien and B. Phelps

Chiron Corporation

J. Clin. Microbiol. 42, 5199 - 5204 (2004)

•The performance of the Target-Capture HBV assay was demonstrated with Reference panels and Standards

•The range of Quantitation was 10-50 IU/mL

•The accuracy of quantitation of several concentrations of serially diluted WHO standard was between 100-142% and of the QCMD 2003 six member panel was in the 74-140% range

•The comparative commercial assays included Roche Amplicor, National Genetics Institute SuperQuant, Bayer Quantiplex version 2.0, and Digene Hybrid Capture assay

•The Target-Capture HBV assay was more sensitive, accurate, high-throughput, rapid, and reproducible.

ProcleixTM Ultrio Registration and Market Trials

V. Shyamala

V. Shyamala 28

External quality assessment for the detection of blood-borne viruses

in plasma by nucleic acid amplification technology: the first human

immunodeficiency virus and hepatitis B virus studies (HIV EQA/1 and

HBV EQA/1) and the fifth hepatitis C virus study (HCV EQA/5).

G. Pisani, K. Cristiano, J. Saldanha, M. Wirz, G. M. Bisso, C. Mele, G. Gentili and the EQA Participants.

Department of Infectious, parasitic and immune-mediated Diseases, Rome, Italy, Canadian Blood services, Ottawa, Canada

Vox Sanguinis 87, 91- 95 (2004)

• Sixteen laboratories received HBV EQA/1 coded panel of two HBV concentrations

• All qualitative assays detected both members

•The Chiron Target-Capture PCR assay (Lab 46) assigned a mean titer of 1506 IU/mL and 104 IU/mL to the 1000 IU/mL and 100 IU/mL samples respectively.

V. Shyamala 29

The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana

J.-P. Allain, D. Candotti, K. Soldan, F. Sarkodie, B. Phelps, C. Giachetti, V. Shyamala, F. Yeboah, M. Anokwa, S. Owusu-Ofori, and

O. Opare-Sem

Division of Transfusion Medicine, Cambridge, UK, Departments of Medicine and Biochemistry, Komfo Anokye Teaching Hospital, Kumasi, Ghana, Chiron

Corporation, Gen-Probe, Inc.

Blood 101, 2419-2425 (2003)

• In Africa more than 50% of blood donors and recipients are HBV positive through natural exposure

•There are currently no HBV screening programs

•Relative merits of various antigen screening methods such as Particle agglutination, dipstick, and EIA assays were compared

•The risk of HBV transmission was predicted by screening HBsAg negative donors and a group of potential blood recipients for HBV DNA (0.05% DNA positivity)

•The risk of transmission for <10 years old ranged between 1:11 and 1:326 for unscreened vs.EIA screened. This risk decreased four fold in adults due to natural exposure to HBV.

V. Shyamala 30

Conference presentations: Hepatitis B virus DNA assay

59th Annual AABB meeting, 2006, Miami Beach, FL

1. Y.-L. Fong, D. Madriaga, V. Shyamala, J. Cottrell, R. Lewis, L. Eudey, G. Crutcher, N. Lelie, A.

Heaton, B. Phelps, D. Chien. Evaluation of Analytical Sensitivity of Chiron Target Capture HBV DNA

Assay for HBV Detection, and Comparison with NGI SuperQuantTM HBV DNA Assay. Transfusion 46,

96.

57th Annual AABB meeting, 2004, Baltimore, MD

2. V. Shyamala, P. Arcangel, J. Cottrell, D. Coit, A. Medina-Selby, C. McCoin, D. Chien, B.Phelps.

Performance characteristics of the qualitative and quantitative Target-Capture PCR HBV NAT assay.

Transfusion 44, 85.

56th Annual AABB meeting, 2003, San Diego, CA

3. V. Shyamala, J.Cottrell, P. Archangel, D. Coit, A. Medina-Selby, C. McCoin, J. Turczyn, D. Chien and

B. Phelps. Validation Of Alternative NAT HBV Assay: A Highly Sensitive PCR Based Assay For HBV

DNA. Transfusion. 43, 125.

27th congress of the ISBT meeting, 2002, Vancouver, Canada.

4. V. Shyamala, P. Arcangel, J. Cottrell, J. Linnen, C. Giachetti, D. Chien, B. Phelps. Performance

characteristics of hepatitis B virus DNA confirmatory assay for ProcleixR triplex assay. Vox Sanguinis,

83, 183.

5. J. Linnen, A. Umali, A. Broulik, D. Kolk, J. Dockter, S. McDonough, V. Shyamala, J. Cottrell, P.

Arcangel, L. Mimms and C. Giachetti. Effect of donor mini-pool size on closure of the HBV detection

window: A comparison of Triplex TMA to surface antigen detection. Vox Sanguinis, 83, 42.

54th Annual AABB meeting, 2001, San Antonio, TX.

6. J. Linnen, M. Ho-Sing-Lloy, M. Miyano, D. Kolk, A. Menez, A. Vaughn, E. Peterson, V. Shyamala, P.

Arcangel, D. Chien, B. Phelps. Performance of the TMA Triplex Assay which simultaneously detects

HIV-1, HCV and HBV nucleic acid. Transfusion, 41, 82.

V. Shyamala 31

Prevalence and quantitation of Parvovirus B19 DNA levels in blood

donors using a sensitive PCR screening assay

S. Kleinman, S. Glynn, T.-H. Lee, L. Tobler, L. Montalvo, D. Todd, J. Kiss, V. Shyamala, M. Busch

Westat, Blood Systems Research Institute, The Institute for Transfusion Medicine, Chiron Corporation; National Heart, Lung, and Blood Institute

Retrovirus Epidemiology Donor Study (REDS-II)

Transfusion 47, 1756- 1764 (2007)

•A retrospective study was conducted with 5020 plasma samples for Parvo B19 DNA detection by Target Capture-PCR assay

•The Assay performance was 50% LoD at 1.6 IU/mL and 95% LoD at 16.5 IU/mL

•B19 DNA prevalence was 0.88 percent with 40 positives

•IgM positivity was associated with high DNA levels (median concn 105 IU/mL) indicating acute resolving infection

•IgG but not IgM positivity is indicative of chronic and persistent phase of B19 infection

V. Shyamala 32

Characterization of the terminal regions of hepatitis C viral RNA:

Identification of conserved sequences in the 5' untranslated region

and poly(A) tails at the 3' end

J. H. Han, V. Shyamala, K. H. Richman, M. J. Brauer, B. Irvine, M. S. Urdea, P. Tekamp-Olson, G. Kuo, Q.-L. Choo, and M.

Houghton

Chiron Corporation

Proc. Natl. Acad. Sci. USA 88, 1711-1715 (1991)

• The nucleotide sequence at the 5' and 3' termini of the hepatitis C

virus (HCV) genome has been determined, with the sequence in the 5'

untranslated region highly conserved among geographical isolates

•There are several features indicating relatedness of HCV to pestivirus

but not to other flavi viruses such as, (a) blocks of 5’ nucleotide

sequence and position of short open reading frames, (b) poly(A) tails

present on 3' subgenomic RNAs, (c) RNAs truncated at the 5’ and 3’

end.

•However, HCV also appears to be substantially different from pesti

virus with assignment to a separate viral genus.

V. Shyamala 33

Receptor recognition and specificity of interleukin-8 is determined by

residues that cluster near a surface-accessible hydrophobic pocket

M. E. Wernette-Hammond, V. Shyamala, M. A. Siani, C. A. Gallegos,, P. H. Feucht, J. Abbott, G. Reza-Lapointe, M. Moghadam, H. Khoja, J.

Zakel, and P. Tekamp-Olson

Chiron Corporation

J. Biol. Chem. 271, 8228-8235 (1996)

•Chemokine IL8 (C-C) binds both R1 and R2 receptors and gro gamma (CXC) binds only R2 receptor. Chimeric C-C and CXC ligands were used to determine the specificity and affinity of binding to recombinant R1 and R2 cell lines

•Substitution into C-C of CXC aa at the 1st beta sheet reduced binding to both R1 and R2, and of 3rd beta sheet reduced binding to R1 but not R2. Substitution into CXC of C-C aa at the second beta sheet conferred high affinity binding to both R1 and R2.

•Individual aa substitutions were made and the results explained through a homology model suggests that a hydrophobic pocket is essential for both R1 and R2 binding, while surrounding residues play an additional role for R1 binding.

V. Shyamala 34

Interleukin-8 receptors R1 and R2 activate mitogen-activated protein

kinases and induce c-fos, independent of Ras and Raf-1 in Chinese

hamster ovary cells

V. Shyamala, and H. Khoja

Chiron Corporation

Biochemistry 37, 15918-15924 (1998)

• Biological effects of interleukin-8 (IL-8) are realized by binding to the two seven-transmembrane receptors IL-8 R1 and IL-8 R2.

• IL-8 R1 and R2 have been shown to interact with Galphai2 and Galpha16, with activation of several mitogen-activated protein kinases

•In CHO cells stably expressing either IL-8 R1 or R2 receptors results demonstrate that: (a) IL-8 activates ERK and ERK kinases (MEK) through R1. Both IL-8 and GROalpha activate ERK and MEK through R2, whereas MIP-1alpha, a beta chemokine, does not activate these kinases through either of these receptors. (b) ERK activation is inhibited by pertussis toxin and MEK1 inhibitor. (c) ERK activation is independent of the upstream mediators Ras and Raf-1. (d) The downstream effects of ERK activation result in increase of c-fos mRNA through both R1 and R2 receptors.

V. Shyamala 35

High-throughput screening for ligand-induced c-fos mRNA

expression by branched DNA assay in chinese hamster ovary cells

V. Shyamala, H. Khoja, M. L. Anderson, J.-X. Wang, H. Cen, and W. M. Kavanaugh

Chiron Corporation

Anal. Biochem. 266, 140-147 (1999)

• Cell based High-throughput screening requires use of standardized cell lines for universal signal read-outs for use with a variety of targets.

• The screening assay for receptor agonists and antagonists is in Chinese Hamster Ovary (CHO) cells overexpressing the relevant receptors.

•A universal signal readout is of endogenous c-fos mRNA which responds to a wide spectrum of stimuli.

•The signal readout was amplified with branched chain DNA (bDNA) assay which is highly sensitive, quantifiable, amenable to high-throughput analysis, and easy to execute.

•The combined benefit of the above three features was proven with CHO cells overexpressing insulin receptor to compare conventional signaling assays with the high-throughput c-fos branched DNA assay.

V. Shyamala 36

Tumor Necrosis factor alpha induced activation of c-jun N-terminal

kinase is mediated by TRAF2

C. Reinhard, B. Shamoon, V. Shyamala, and L. T. Williams

Chiron Corporation

EMBO J. 16,1080-1092 (1997)

• Tumor necrosis factor alpha (TNF alpha) a pro-inflammatory cytokine is

an endogenous mediator of septic shock, inflammation, anti-viral

responses and apoptotic cell death through 55 kDa (TNF-RI) and 75 kDa

(TNF-RII) receptors.

•TNF-RII specific signaling was examined by chimeric receptor with

extracellular domain mouse CD4 antigen and intracellular domain TNF-

RII, and activated it through anti-CD4 antibodies

•Results show that: (i) TNF-RII activates ERK and JNK; (ii)

Overexpression of TRAF2, a molecule that binds TNF-RII activates JNK ;

(iii) dominant-negative TRAF2 blocks JNK activation ; (iv) TRAF2 signals

activation of JNK and NF-kappaB through different pathways.

•TNF alpha-mediated JNK activation in fibroblasts is independent of the

cell death pathway.

.

V. Shyamala 37

Cloning of CCRL1, an orphan seven transmembrane receptor related

to chemokine receptors, expressed abundantly in the heart

H. Khoja, G. Wang, C. T. Ng, J.Tucker, T. Brown, V. Shyamala

Chiron Corporation

Gene 246, 229-238 (2000)B

Involved in stroke

• To identify novel chemokine receptor genes, cDNA expressed sequence tags (EST) were analyzed for a significant homology with mammalian chemokine receptors.

•One EST clones sequence was used to generate a full-length cDNA encoding a putative seven transmembrane receptor, CCRL1-CC chemokine receptor like 1, encoding a polypeptide of 350 amino acids with 35% homology to the chemokine receptors CCR6 and CCR7. Coupled transcription-translation of CCRL1 cDNA yielded a glycosylated polypeptide of about 45kDa.

•Northern blot analysis indicates predominant expression of about 5.0, 2.0 and 1.3kb mRNA forms in human heart tissue. In-situ hybridization confirmed the presence of CCRL1 mRNA in cardiac muscle cells.

•CCRL1 maps to chromosome 6 and has one intron in the 5' untranslated region.

V. Shyamala 38

Semirational design of a potent, artificial agonist of fibroblast growth

factor receptors

M.D. Ballinger, V. Shyamala, L. D. Forrest, M. Deuter-Reinhard, L. V. Doyle, J. X.Wang, L. Panganiban-Lustan, J. R. Stratton, G. Apell, J. A.

Winter, M. V. Doyle, S. Rosenberg, W.M. Kavanaugh

Chiron Corporation

Nature Biotechnology 17, 1199 - 1204 (1999)B

Involved in stroke

• A 26 amino acid polypepetide with FGF receptor binding activity unrelated to any known FGF was identified through phage display.

•A heparin binding, and dimerizaion enabling domain of c-jun leucine zipper was tailored onto this peptide

•Such a synthetic polypeptide reproduced the intracellular kinase cascade, and mitogenic and morphogenic properties of bFGF with similar potency

•The synthetic peptide also reproduced corneal vascularization properties of FGF

•Artificial FGFR peptide and non-peptide agonists may be useful alternatives to FGF in the treatment of ischemic vascular disease.

V. Shyamala 39











V. Shyamala 40

Amplification of bacterial genomic DNA by the polymerase chain

reaction and direct sequencing after asymmetric amplification:

application to the study of periplasmic permeases

V. Shyamala, and G. F.-L. Ames

Dept. of Biochemistry, UC Berkeley, CA

J. Bacteriol. 171, 1602-1608, (1989)

•The polymerase chain reaction (PCR) was used to amplify bacterial genomic DNA and PCR amplicons of 4,400 base pairs obtained

•We discuss problems inherent in the direct sequencing of the amplified product, and solved the problems by developing an "asymmetric amplification" method in which one of the oligonucleotide primers is used in limiting amounts, thus allowing the accumulation of single-stranded copies of only one of the DNA strands.

•As an illustration of the use of PCR in bacteria, we have amplified, sequenced, and subcloned several DNA fragments carrying mutations in genes of the histidine permease operon. These mutations are part of a preliminary approach to studying protein-protein interactions in transport, and their nature is discussed.

V. Shyamala 41

Genome walking by single-specific-primer polymerase chain

reaction: SSP-PCR

V. Shyamala, and G. F. L. Ames

Division of Biochemistry and Molecular Biology, UC Berkeley, CA

Gene 84, 1-8, (1989)

•We have extended the use of the polymerase chain reaction (PCR) to

amplify double-stranded DNA when sequence information is available

only at one extremity sufficient to design a gene-specific primer.

• The unknown end is ligated to a vector and the gene-specific primer

is used in combination with a second generic vector primer.

•Restriction, ligation, amplification and sequencing of the products can

be achieved within three days.

•This method eliminates the laborious steps of shotgun cloning, colony

screening and culturing of cells.

•We demonstrate the usefulness of this technique for chromosome

walking in the absence of any restriction data.

V. Shyamala 42

Tandem chromosomal duplications: role of REP sequences in the

recombination event at the join-point

V. Shyamala, E. Schneider and G. F. L. Ames


EMBO J. 9, 939 - 946 (1990)

•A family of prokaryotic repetitive sequences, called REP (repetitive

extragenic palindromic) is involved in the formation of chromosomal

rearrangements such as duplications.

•Here through SSP-PCR we have characterized the join-points of

seven RecA+ tandem duplications in Salmonella typhimurium, that

fuse the hisD gene to distant foreign promoters

• Such a recombination takes place even in a RecA- background.

Thus, REPs can recombine with each other by a RecA(-)-independent

mechanism

•Some RecA- duplications occurred outside of REP sequences by

recombination within a 7 bp homology.

•Possible roles for the known interaction between DNA gyrase and

REP in chromosomal rearrangements are discussed.

Blood

V. Shyamala 43

Genome walking by Single-Specific Primer Polymerase Chain Reaction



Methods in Enzymology 217, Part H, 436 – 446 (1993) Ed. R. Wu B

ood

V. Shyamala 44

Use of exonuclease for rapid polymerase-chain-reaction-based

in vitro mutagenesis



Gene 97, 1-6, (1991)

• In a previous publication we proposed Asymmetric Amplification as a

method to preferentially amplify one of the two PCR strands to

facilitate direct sequencing

•Here the extended application of Asymmetric amplification for in vitro

mutagenesis has been demonstrated

•We also demonstrate a second method for in vitro mutagenesis

following treatment of PCR fragments with lambda exonuclease. This

requires kinasing one of the primers.

•The entire procedure of kinasing the primer, amplification by PCR,

Exo lambda digestion and second step of PCR can be performed in

less than 6 h. to generate a number of mutations in the S. typhimurium

hisP gene of the histidine transport operon.

V. Shyamala 45

Antimalarial activity of optical isomers of quinacrine dihydrochloride

against chloroquine-sensitive and -resistant plasmodium falciparum

in vitro

R. V. Webster, J. C. Craig, V. Shyamala, G. C. Kirby, and D. C. Warhurst

Dept. of Pharmacology, UCSF, Pacific Presbyterian Hospital

Biochem. Pharmacol. 42, S225-S227, (1991)

• Both enantiomers of quinacrine and the racemic form of the drug

showed equal activity in vitro against chloroquine-sensitive and -

resistant strains of Plasmodium falciparum, without detectable

stereoselectivity.

•This contrasts with observations on chloroquine, where a similar lack

of stereoselectivity in vitro is accompanied by a 10-fold loss of activity

against the resistant strain.

•The reported in vivo differences for the enantiomers of chloroquine

and the observations on the optically active metabolites of chloroquine

and quinacrine may be ascribed to a difference in the

pharmacokinetics of their enantiomers.

Documents

Accomplishments V Shyamala