Many Blood Tests May Soon Be Replaced By Spit Tests

One day soon patients may spit in a cup, instead of bracing for a needle prick, when being tested for cancer, heart disease or diabetes. A major step in that direction is the cataloguing of the "complete" salivary proteome, a set of proteins in human ductal saliva, identified by a consortium of three research teams, according to an article published in the Journal of Proteome Research. Replacing blood draws with saliva tests promises to make disease diagnosis, as well as the tracking of treatment efficacy, less invasive and costly.



Saliva proteomics and diagnostics is part of a nationwide effort to create the first map of every human protein and every protein interaction, as they contribute to health and disease and as they act as markers for disease states. Following instructions encoded by genes, protein "machines" make up the body's organs and regulate its cellular processes. Defining exact protein pathways on a comprehensive scale enables the development of early diagnostic testing and precise drug design. In the current study, researchers sought to determine the "complete" set of proteins secreted by the major salivary glands (parotid, submandibular (SM) and sublingual (SL)). Recent, parallel efforts that mapped the blood (plasma) and tear proteomes allows for useful comparisons of how proteins and potential disease markers are common or unique to different body fluids.



"Past studies established that salivary proteins heal the mouth, amplify the voice, develop the taste buds and kill bacteria and viruses," said James E. Melvin, D.D.S., Ph.D., director of the Center for Oral Biology at the University of Rochester Medical Center, and an author on the paper. "Our work, and the work of our partners, has shown that salivary proteins may represent new tools for tracking disease throughout the body - tools that are potentially easier to monitor in saliva than in blood," said Melvin, who conducts his research at the Eastman Dental Center, in collaboration with the research labs of Mark Sullivan, Ph.D., and Fred K. Hagen, Ph.D.



The National Institute of Dental and Craniofacial Research (NIDCR), part of the National Institutes of Health, funded the current study. The saliva proteome study represents a consortium effort with research teams at The Scripps Research Institute (John R. Yates III), University of Rochester, University of Southern California (Paul Denny), The University of California at San Francisco (Susan J. Fisher) and UC Los Angeles (David T. Wong, Joseph A. Loo).



Not Your Parent's Saliva



To describe the results of the current study, it is important to note that the definition of saliva is evolving. Saliva once referred to everything in oral fluid, including: bacterial waste products, dead cells that had shed from mucous membranes and substances oozing from gum crevices. Among researchers today, however, the term saliva is increasingly reserved for just the salivary gland secretions (ductal saliva). The new definition is significant because of the emerging theory that the mix of proteins in ductal saliva tracks closely with that of blood, making saliva a potential diagnostic stand-in for blood.
















To construct a credible protein list for saliva, the teams used competing techniques both to capture the greatest number of protein candidates for the list and to lend extra credibility to those found using different methodologies. Each team subjected saliva collected from patients to some form of mass spectrometry, which determines the identity of proteins based on measurements of their mass and charge. Saliva was collected from 23 adults of several races and both sexes. Although small, the set of study subjects was large enough to serve as a baseline list for near-future comparisons between healthy people and individuals with major diseases, researchers said.



Using mass spectrometry techniques, three teams at five institutions identified 1,166 proteins in parotid and submandibular/sublingual saliva. The results indicated that more than a third of saliva proteins were found in the blood proteome, as well. Comparison of these proteins against known protein pathways and other proteomes provided a first glimpse of the function of the core proteins. In addition, a number of the salivary proteins were found to match proteins with known roles in Alzheimer's, Huntington's and Parkinson's diseases; breast, colorectal and pancreatic cancer; and type I and II diabetes. Specifically, a majority of the proteins were found to be part of signaling pathways, which is central to the body's response to (and thus diagnosic of) system-wide diseases, researchers said.



Determining the salivary proteome is only the first step toward salivary-based diagnosis and treatment. These findings provide crucial protein information that is already being incorporated into microarray technology, a high-speed test that can determine the levels of multiple proteins, during disease progression. Related work is underway under within the NIH-funded Bioengineering Nanotechnology Initiative to design biochips, nano-scale computer chips packed with salivary protein chains. Protein probes on the chip react with proteins in a saliva sample, say from the mouth of someone with oral cancer, and inform a computer about which proteins are present.



"We believe these projects will dramatically accelerate diagnosis and improve prognosis by treating diseases at the earliest stages," said Mireya GonzГЎlez BegnГ©, D.D.S., Ph.D., research assistant professor of Dentistry in the Center for Oral Biology at the Medical Center. "Researchers have already shown that saliva proteins can be used to detect oral cancer and HIV infection. We think this list will soon expand to include leading causes of death like cancer and heart disease, which, if caught early, are much more likely to be successfully treated."







Source: Greg Williams


University of Rochester Medical Center

Unselfish Molecules May Have Helped Give Birth To The Genetic Material Of Life

One of the biggest questions facing scientists today is how life began. How did non-living molecules come together in that primordial ooze to form the polymers of life? Scientists at the Georgia Institute of Technology have discovered that small molecules could have acted as "molecular midwives" in helping the building blocks of life's genetic material form long chains and may have assisted in selecting the base pairs of the DNA double helix. The research appears in the online early edition of the Proceedings of the National Academy of Sciences beginning March 8, 2010.



"Our hypothesis is that before there were protein enzymes to make DNA and RNA, there were small molecules present on the pre-biotic Earth that helped make these polymers by promoting molecular self-assembly," said Nicholas V. Hud, professor in the School of Chemistry and Biochemistry at the Georgia Institute of Technology. "We've found that the molecule ethidium can assist short oligonucleotides in forming long polymers and can also select the structure of the base pairs that hold together two strands of DNA."



One of the biggest problems in getting a polymer to form is that, as it grows, its two ends often react with each other instead of forming longer chains. The problem is known as strand cyclization, but Hud and his team discovered that using a molecule that binds between neighboring base pairs of DNA, known as an intercalator, can bring short pieces of DNA and RNA together in a manner that helps them create much longer molecules.



"If you have the intercalator present, you can get polymers. With no intercalator, it doesn't work, it's that simple," said Hud.



Hud and his team also tested how much influence a midwife molecule might have had on creating DNA's Watson-Crick base pairs (A pairs with T, and G pairs with C). They found that the midwife used could determine the base pairing structure of the polymers that formed. Ethidium was most helpful for forming polymers with Watson-Crick base pairs. Another molecule that they call aza3 made polymers in which each A base is paired with another A.



"In our experiment, we found that the midwife molecules present had a direct effect on the kind of base pairs that formed. We're not saying that ethidium was the original midwife, but we've shown that the principle of a small molecule working as a midwife is sound. In our lab, we're now searching for the identity of a molecule that could have helped make the first genetic polymers, a sort of 'unselfish' molecule that was not part of the first genetic polymers, but was critical to their formation," said Hud.



The work was supported by the National Aeronautics and Space Administration and the National Science Foundation.



Source:

David Terraso

Georgia Institute of Technology

Entire Yeast Genome Mapped By University Of Toronto Scientists

University of Toronto scientists have devised a tool to help understand and predict the state of a cell by successfully mapping all 70,000 nucleosomes in yeast. Nucleosomes wrap DNA before it is transformed into proteins and are critical indicators and regulators of a cell's state.



Led by Corey Nislow, a U of T Assistant Professor with the Banting and Best Department of Medical Research and Department of Molecular Genetics, the team created a complete, three-dimensional map of the yeast genome. This information was fed into a computer to build a software program that can predict where nucleosomes should be. The program worked remarkably well, and its accuracy will only improve with more data.



"When control is lost, cells make inappropriate proteins or divide inappropriately, which is what happens in diseases like cancer," says Nislow, whose team worked closely with U of T Professor Timothy Hughes on the project. "Knowing where nucleosomes are is the first step in identifying what is going on in a cell and what the cell plans to do next, so this initial research could have big implications down the road for early detection of certain diseases."



Scientists can tell by the presence of nucleosomes which genes are actively being converted into protein, and this information can function as an important first clue to disease detection.







The research appeared in the scientific journal Nature Genetics.



Corey Nislow, Lead Author



Source: April Kemick


University of Toronto

While Infecting Humans Tiny Fungi May Reproduce Sexually

A fungus called microsporidia that causes chronic diarrhea in AIDS patients, organ transplant recipients and travelers has been identified as a member of the family of fungi that have been discovered to reproduce sexually. A team at Duke University Medical Center has proven that microsporidia are true fungi and that this species most likely undergoes a form of sexual reproduction during infection of humans and other host animals.



The findings could help develop effective treatments against these common global pathogens and may help explain their most virulent attacks.



"Microsporidian infections are hard to treat because until now we haven't known a lot about this common pathogen," says Soo Chan Lee, Ph.D., lead author and a postdoctoral researcher in the Duke Department of Molecular Genetics and Microbiology. "Up to 50 percent of AIDS patients have microsporidial infections and develop chronic diarrhea. These infections are also detected in patients with traveler's diarrhea, and also in children, organ transplant recipients and the elderly."



Of the 1200 species of microsporidia, more than a dozen infect humans. Their identity had been obscured because these tiny fungi cannot live outside of an infected host cell and they have a small number of genes which are rapidly evolving.



The Duke scientists used two genetic studies to show that microsporidia apparently evolved from sexual fungi and are closely related to the zygomycete fungus in particular.



They found that microsporidia share 33 genes out of 2,000 with zygomycetes. which the microsporidia did not share with other fungi. This genomic signature also shows that microsporidia and zygomycetes likely shared a common ancestor and are more distantly related to other known fungal lineages.



In addition, these two types of fungi have the same sex-locus genes - and in the same order - in their DNA. Other genes involved in sexual reproduction are also present. The findings suggest that microsporidia may have a genetically controlled sexual cycle, and may be undergoing sexual reproduction while they infect the host, Lee said.



Lee said the next step is to explore the sexual reproduction of these species, which may cause more severe (more virulent) infections because they use the host's cellular environment and machinery as a safe haven in which to reproduce.



"These studies resolve the enigma of the evolutionary origins and proper placement of this highly successful group of pathogens, and provide better approaches to their experimental study," said senior author Joseph Heitman, M.D., Ph.D., director of the Center for Microbial Pathogenesis and director of the Duke University Program in Genetics and Genomics.



The team will pursue further studies with Duke genetic researchers Raphael Valdivia, Ph.D., and Alejandro Aballay, Ph.D., using cultured cells and C. elegans, a worm that researchers recently found is a natural host for microsporidia. "Using this roundworm may prove to be a useful way to study microsporidia genetics in a living creature," Heitman said.







This work was published online in the Oct. 30 edition of Current Biology, and was supported by grants from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, and by the Canadian Institutes for Health Research.



Other authors on this study include Nicolas Corradi and Patrick J. Keeling of the Canadian Institute for Advanced Research, Department of Botany, University of British Columbia - Vancouver; Edmond J. Byrnes III of the Duke Department of Molecular Genetics and Microbiology; Santiago Torres-Martinez of the Departamento de Genetica y Microbiologia, Facultad de Biologia, Universidad de Murcia, Spain; and Fred S. Dietrich of Duke Molecular Genetics and Microbiology and the Duke Institute for Genome Sciences and Policy.



Source: Mary Jane Gore


Duke University Medical Center

Models Begin To Unravel How Single DNA Strands Combine

Using computer simulations, a team of University of
Wisconsin-Madison researchers has identified some of the pathways through
which single complementary strands of DNA interact and combine to form the
double helix.



Present in the cells of all living organisms, DNA is composed of two
intertwined strands and contains the genetic "blueprint" through which all
living organisms develop and function. Individual strands consist of
nucleotides, which include a base, a sugar and a phosphate moiety.



Understanding hybridization, the process through which single DNA strands
combine to form a double helix is fundamental to biology and central to
technologies such as DNA microchips or DNA-based nanoscale assembly. The
research by the Wisconsin group begins to unravel how DNA strands come
together and bind to each other, says Juan J. de Pablo, UW-Madison Howard
Curler Distinguished Professor of Chemical and Biological Engineering.



The team published its findings in the Proceedings of the
National Academy of Sciences. In addition to senior author de Pablo, the
group included David C. Schwartz, a UW-Madison professor of chemistry and
genetics, and former postdoctoral research fellow Edward J. Sambriski, now
an assistant professor of chemistry at Delaware Valley College in
Pennsylvania.



The three drew on detailed molecular DNA models developed by de Pablo's
research group to study the reaction pathways through which
double-stranded DNA undergo denaturation, where the molecule uncoils and
separates into single strands, and hybridization, through which
complementary DNA strands bind, or "hybridize." In Watson-Crick base
pairing, A (adenine) pairs with T (thymine), while G (guanine) pairs with
C (cytosine). Reaction pathways are the trajectories single DNA strands
follow to find each other and connect via such complementary pairs.



The researchers studied both random and repetitive base sequences. Random
sequences of the four bases - A, T, G and C - contained little or no
regular repetition. To the researchers' surprise, a couple of bases
located toward the center of the strand associate early in the
hybridization process. The moment they find each other, they bind and the
entire molecule hybridizes rapidly and in a highly organized manner.


Conversely, in repetitive sequences, the bases alternated regularly, and
the group found that these sequences bind through a so-called diffusive
process. "The two strands of DNA somehow find each other, they connect to
each other in no particular order, and then they slide past each other for
a long time until the exact complements find one another in the right
order, and then they hybridize," says de Pablo.



Results of the team's study show that DNA hybridization is very sensitive
to DNA composition, or sequence. "Contrary to what was thought previously,
we found that the actual process by which complementary DNA strands
hybridize is very sensitive to the sequence of the molecules," he says.



Knowledge of how the process occurs could enable researchers to more
strategically design technologies such as gene chips. For example, says de
Pablo, if a researcher wanted to design sequences that bind very rapidly
or with high efficiency, he or she could place certain bases in specific
locations, so that the hybridization reaction could occur faster or more
reliably.



Ultimately, the research could help biologists understand why some
hybridization reactions are faster or more robust than others. "One of the
really exciting things about this work is that the hybridization reaction
between two strands of DNA is really fundamental to life itself," says de
Pablo. "It is the basis for much of biology. And it is amazing to me that,
until now, we knew little of how this reaction actually proceeds."



The National Science Foundation-funded Nanoscale Science and Engineering
Center on Templated Synthesis and Assembly at the Nanoscale at UW-Madison
sponsored the research.




Source
University of Wisconsin-Madison

Newly Discovered Epidermal Growth Factor Receptor Active In Human Pancreatic Cancers

Finally some promising news about pancreatic cancer, one of the most fatal cancers, due to the difficulties of early detection and the lack of effective therapies: Johns Hopkins University pathologist Akhilesh Pandey has identified an epidermal growth factor receptor aberrantly active in approximately a third of the 250 human pancreatic cancers studied.



In a presentation April 18, at Experimental Biology 2009 in New Orleans, Dr. Pandey explained why this finding and related work in his Hopkins laboratory is promising in terms of both a new treatment for a large subset of pancreatic cancers and a potential blood or urine screening tool that might eventually do for pancreatic cancer detection what biomarkers like prostate-specific antigen levels have done for prostate cancer. His presentation was part of the scientific program of the American Society for Investigative Pathology.



Personalized treatment. Phosophorylated epidermal growth factor receptor (pEGFR), the receptor identified by Dr. Pandey, is closely related to HER-2, a growth factor receptor found and used as a drug target in a subset of breast cancers. After he found and profiled the pEGFR activated in the pancreatic cancers, Dr. Pandey realized the same receptor had been found by other researchers to be activated in a subset of lung cancers. And, most promising, an EGFR inhibitor named erlotinib already has been through the long and complex Food and Drug Administration approval process and is in use for treatment of these specific lung cancers.



But would the drug work in pancreatic cancers? Dr. Pandey's group moved from studies of human cell lines to studies in mice in which human pancreatic tumor cells with activated EGFT had been placed. The tumors began growing. But when treated with erlotinib, they began to shrink. Other tumors without activated pECFR showed no response.



The promise - and the challenge - of using pEGFR is that of personalized medicine, says Dr. Pandey. Obviously a growth factor receptor that is activated only in a subset of all pancreatic cancers cannot be a one-size-fits-all target for treatment. Earlier studies in other laboratories and clinical trials already had tried EGF inhibitors as a treatment for pancreatic cancer and concluded that they did not work. When Dr. Pandey's collaborators allowed them to re-examine their samples, they found that the only case in 12 cases that had responded to the EGF inhibitor was the only case with an activated EGF receptor. Dr. Pandey would like to see other researchers go back and re-analyze their data, separating patients with and without the activated receptor, and then determining the success rate. He believes it would tell a different, more hopeful story.



Screening for pancreatic cancer. Dr. Pandey's other goal in his research is to use mass spectrometry to find additional markers of pancreatic cancer in the tumors themselves but also in blood and urine, which would avoid the problems of invasive biopsies. As a first step, his team has gone through the scientific literature to create a compendium of several hundred proteins and genes reported to be overexpressed in pancreatic cancers, making them excellent candidates for further study. The compendium already is being used by a consortium of investigators who are developing antibodies against the 60 most promising targets.



Co-authors of the Experimental Biology study are Hopkins faculty Dr. Antonio Jimeno, Dr. Henrik Molina, Dr. Ralph Hruban, Dr. Anirban Maitra, and Dr. Manuel Hidalgo; and H. C. Harsha, a graduate student in Dr. Pandey's laboratory who is also a member of the Institute of Bioinformatics in Bangalore, India. The research was supported by the Sol Goldman Trust for Pancreatic Cancer Research.



Source:
Sylvia Wrobel


Federation of American Societies for Experimental Biology

Microbial Analysis, Micropatterning Methods Featured In Cold Spring Harbor Protocols

Microbial populations have traditionally been studied in carefully controlled, laboratory-grown cultures. New metagenomic approaches are being developed to study these organisms in environmental or medical samples. The July issue of Cold Spring Harbor Protocols presents a method developed by Holger Daims from the University of Vienna for quantifying populations of microorganisms in a variety of naturally occurring conditions such as plankton samples or biofilms. Use of Fluorescence In Situ Hybridization and the daime Image Analysis Program for the Cultivation-Independent Quantification of Microorganisms in Environmental and Medical Samples combines fluorescent in situ hybridization using rRNA-targeted probes with digital image analysis. The results show an organism's "biovolume fraction" in a given sample; this indicates the share of biochemical reaction space occupied by the quantified population and can be more relevant ecologically than absolute cell numbers. The article is freely available on the website for Cold Spring Harbor Protocols.



Micropatterning methods are rapidly becoming standard approaches for investigating cellular behaviors such as growth and migration. Adhesive Micropatterns for Cells: A Microcontact Printing Protocol from Matthieu Piel and colleagues at the Institut Curie offers a simple, fast, and efficient method for generating micropatterns for cellular studies. Employing an elastomeric stamp to print proteins on the substrate of choice, this technique does not require much of the expensive equipment and technical expertise needed for most micropatterning methods, making it easier to implement in biology laboratories. The authors have provided a movie that illustrates the technique step-by-step as part of the protocol. The article is freely accessible on the website for Cold Spring Harbor Protocols.


Source

Cold Spring Harbor Protocols