October 22-27, 2008
Pathogenesis and Control of Emerging Infections and Drug-Resistant Organisms
Keystone Symposia PDF
Point-of-Care Detections of Multi-Drug Resistant Mycobacterium tuberculosis.
October 14-16, 2008
Centers of Excellence in Genomic Science
Sixth Annual Grantee Meeting, National Human Genome Research Institute
LATE-PCR Assays for Monitoring Transcriptional Responses to Hypoxia In Single Cells Recovered from Barrett's Esophagus.
Abstract: Barrett's esophagus is a model system established by the Brian Reid laboratory at the Fred Hutchinson Cancer Research Center (FHCRC) to study the impact of low oxygen tension (hypoxia) during neoplastic progression. We are using technologies developed at Brandeis University (LATE-PCR, PurAmp, PrimeSafeTM) to construct assays to measure the transcriptional responses to hypoxia of single Barrett's esophagus cells.
October 14-16, 2008
Open Session of the EuFMD Standing Technical Committee
New Detection technologies for pan-FMDV and serotype-specific assays in the field.
Kenneth E. Pierce, Rohit Mistry, Suki Bharya, Scott M. Reid, Katja Ebert, Donald P. King, and Lawrence J. Wangh
Introduction: Detection and serotype identification of foot-and-mouth disease virus (FMDV) RNA is complicated by high sequence variation between strains. Linear-After-The-Exponential (LATE)-PCR efficiently generates single-stranded DNA that is freely available to hybridize with fluorescently labeled, mismatch-tolerant probes over a wide temperature range, enabling the detection of numerous sequence variants. Two assays are being developed; a pan-FMDV assay intended for detection of all strain variants and a second to distinguish between FMDV serotypes.
Mitochondria and Chloroplasts Graduate Research Seminar
University of New England
Nucleoside Reverse Transcriptase Inhibitors: Impact on Mitochondrial Genomic Integrity
Adam Osborne and Lawrence Wangh, Brandeis University
Abstract: Recent studies have been directed toward the role of mitochondrion in diseases such as Alzheimer’s and diabetes. Interestingly, Nucleoside Reverse Transcriptase Inhibitors (NRTIs) used to treat HIV/AIDS (Figure 1), have also been shown to damage mitochondria. Such damage may lead to the onset of a host of mitochondrially related diseases such as diabetes. While mitochondrial dysfunction is widely accepted as playing a role in disease, the question still remains whether mitochondrial dysfunction is a cause or a symptom. Mitochondrial dysfunction and NRTI damage maybe manifested by genetic instability caused by mutations in mitochondrial DNA. Mitochondrial DNA (mtDNA) is being studied at the population as well as the single molecule level using Linear-After-The-Exponential (LATE)-PCR, a powerful form of asymmetric PCR that allows for the clean and efficient generation of single stranded DNA that can be easily sequenced. Thus we can determine mutational load, and its affect on disease by observing single mtDNA molecules over a time course. To this end the mitochondrial genes 12srRNA, ND1, ND2, ND4, CO1, CO2, CytB as well as HV1 and HV2 of the control region, are amplified in one multiplex LATE-PCR assay to study the mutational load. These studies will be conducted in a HepG2 cell line to study mitochondrial mutations and their long-term effects of NRTIs on mitochondrial DNA. Such work may, in the future, be able to track HIV/AIDS infected patients to see if, and when, NRTI treatment damages mtDNA before such damage leads to serious side effects.
29th World Veterinary Congress
Vancouver, British Columbia
Introduction: There is a growing need in veterinary and human medicine for diagnostic assays that can rapidly detect and analyse numerous species and strains of infectious organisms and viruses. RNA viruses are particularly challenging because they evolve rapidly. In the case of veterinary medicine, tests have to be doen at pen-side or in the field and the findings can have great economic impact, including slaughter of many animals. Currently, the use of RT-PCR is limited in scope and few, if any, assays provide results from multiplex data in the field. We have overcome these limitations by constructing assays based on LATE-PCR and (RT)-LATE-PCR. We are collaborating with Smiths Detection to implement these assays on an automated portable sample preparation and PCR system.
1st Workshop on LATE-PCR and its Diagnostic Applications
National Veterinary Institute
LATE-PCR and RT-LATE-PCR workshop
Speakers: Lawrence Wangh, Ken Pierce, Cristina Hartshorn, Bonnie Ronish
June 18-20, 2008
Cambridge Healthtech Institute
LATE-PCR and the BioSeeq: Maximum Information from a Field Based Instrument
Lawrence J. Wangh, Ph.D., Laboratory Director, Brandeis University Department of Biology, in collaboration with Smiths Detection, Inc., Watford, UK
June 4-6, 2008
European Union (EU) Network of Excellence for Epizootic Disease Diagnose and Control
Rapid Detection and Identification of Avian Influenza Subtypes in a Single Tube Using RT-LATE-PCR.
Cristina Hartshorn, A.H. Reis, Jr., K.E. Pierce, L.J. Wangh
Introduction: Pathogenic strains of avian influenza, particularly subtypes H5 and H7 continue to spread and evolve through out Eurasia and Africa, causing death and requiring culling of millions of birds. Several hundred people have died of avian influenza primarily following direct contact with infected poultry, but there is reason to fear that relatively few genetic changes could lead to efficient human-to-human transmission raising the specter of a devastating global pandemic. Rapid, accurate detection of the presence and type of influenza in people, birds, and other species is urgently needed, particularly in remote rural areas where people leave very close to their livestock. In collaboration with Smiths Detection, Inc. (Watford, U.K.) we are using two novel technologies developed in our laboratory at Brandeis University, LATE-PCR (U.S. Patent 7,198,897) and PrimeSafeTM (a mis-priming preventing compound), to construct a highly multiplexed assay that can detect and distinguish between several subtypes of avian influenza and their strains in a single tube. This assay is designed to work on a battery-powered point-of-care instrument, the BioSeeq, which is being designed and engineered by Smiths Detection, Inc.
April 8-13, 2008
Molecular Evolution as a Driving Force in Infectious Diseases
Beaver Run Resort - Breckenridge, Colorado
Infectious diseases have been a key driving force in evolution, both of the hosts and the pathogens. The era of genomics has made it possible to evaluate both infectious agents and their hosts at the genomic level. The evolutionary dynamics of viruses, bacteria and parasites, in their adaptive evolution to changes in host response or adaptation to new hosts is enormously complex. The burden of infectious diseases on people and domestic animals is staggering. We are only now beginning to achieve an understanding of how infectious organisms cause disease and how the complex patterns of immune response generated by their infections mitigate it. By bringing together experts in the evolution of viruses, bacteria and parasites, plus those studying host adaptation and evolution of the immune response, we hope that this meeting will stimulate further interaction between widely disparate lines of investigation.
LATE-PCR for Detection and Analysis of Infectious Diseases - Presentation
Lawrence Wangh, Cristina Hartshorn, Arthur Reis, John Rice, Kenneth Pierce. Department of Biology, Brandeis University, Waltham, MA, 02454.
LATE-PCR, invented in our laboratory, is an advanced form of asymmetric PCR that uses two primers of unequal concentration and differing melting temperatures to generate double-stranded DNA amplicons exponentially, followed by linear amplification of one strand of amplicon. One of the primers can also be used to synthesize cDNA from RNA prior to the start of amplification, thus allowing for RT-PCR.
Cristina Hartshorn, Odelya Hartung, Yanwei Jia, and Lawrence J. Wangh, Brandeis University
Abstract: We are using three novel technologies invented in our laboratory, LATE-PCR, PrimeSafe, and PurAmp to construct multiplexed assays for quantitative analysis of specific mRNAs in single cells and small groups of cells. LATE-PCR is an advanced form of asymmetric PCR in which efficient exponential amplification of double-stranded DNA is followed by cycles of linear amplification of single-stranded DNA. The resulting single strands are detected using fluorescent probes.
Using these technologies we have already constructed reactions for measurement of Xist RNA, Oct 4 mRNA, Cdx2 mRNA, and heatshock protein (hsp) 70i mRNA. Duplex assays in various combinations have been used to quantify the levels of these gene transcripts in single blastomeres recovered from mouse embryos at the 4 cell and 8 cell stages of early development, as wells as from intact embryos. These assays can also measure copy numbers of the respective genes when LATE-PCR is performed without RT. These experiments serve as controls with which to validate that our methods are sensitive down to single molecules of genomic DNA in single cells. They also allow us to compare several methods of reverse transcription in order to conveniently and reliably detect very low numbers of RNA molecules in single cells.
In collaboration with Brian Reid’s laboratory at the Fred Hutchinson Cancer Research Center we will use our optimized protocols to construction of duplex and multiplexed LATE-PCR assays for measurement of mRNAs levels in single normal and neoplastic esophageal cells. The following transcripts, COX-2, hsp27, p53, are of particular interest because they are known to change in response to stress stimuli like heat, low pH, and bile salts. In parallel with these studies we are also carrying out single-cell LATE-PCR assays to measure loss of heterozygosity (LOH) in the region of CDKN2A(p16) or TP53(p53) primers. These genetic changes are hallmarks of Barretts esophagus, the precursor to esophageal adenocarcinoma.