The Role of MMP9 in Satellite Cell Activation After Increased Activity

The unique adaptive ability of skeletal muscle to meet functional demands is exemplified in its response to exercise. Though little is known about the molecular mechanisms that regulate this plasticity, the extracellular matrix (ECM) is believed to play a large role. The basal lamina is a specialized layer of ECM that lies in direct contact with the cell membrane of muscle fibers and facilitates environment-to-cell interactions. Matrix metalloproteinase-9 (MMP-9) is an enzyme in the basal lamina that regulates much of these adaptive processes. During exercise, the regenerative process of damaged tissue requires the activation of muscle-specific stem cells known as satellite cells. Satellite cell activity has been proposed to be activated by MMP-9; However, there are no studies that look at this interaction. Thus the overall goal of the present proposal is to 1) determine the effects of MMP-9 on muscle hypertrophy via satellite cell activation and 2) assess whether hypertrophy of the plantaris muscle observed after functional overload (FO) is due to the increased size of existing muscle fibers or the addition of new muscle fibers. FO of the plantaris muscle, a calf muscle responsible for ankle extension, was performed in WT and MMP-9 knockout (MMP-9 KO) mice (~4 mos of age) by removing the soleus and gastrocnemius muscles and randomly placed into the following groups: 1) 2-day FO (n=5/group) and 14-day FO (n=5/group). A 0-day time point for each group was added as a baseline control (n=5/group). Fluorescence immunohistochemistry was performed using anti-Pax7 and anti-laminin antibodies to label satellite cells and the basal lamina, respectively. There was a general increase in satellite cells after 14-days FO compared to 0- and 2-days in both the WT and MMP-9 KO mice. There also was no observable trend or pattern in fiber count between time points for both WT and MMP-9 KO mice. However as the sample size is based on n=1 for each group at each time point no inference to statistical significance can be made. At this juncture, more samples are being analyzed to determine these relationships

Unlocking biomarker discovery: Large scale application of aptamer proteomic technology for early detection of lung cancer

Lung cancer is the leading cause of cancer deaths, because ~84% of cases are diagnosed at an advanced stage. Worldwide in 2008, ~1.5 million people were diagnosed and ~1.3 million died – a survival rate unchanged since 1960. However, patients diagnosed at an early stage and have surgery experience an 86% overall 5-year survival. New diagnostics are therefore needed to identify lung cancer at this stage. Here we present the first large scale clinical use of aptamers to discover blood protein biomarkers in disease with our breakthrough proteomic technology. This multi-center case-control study was conducted in archived samples from 1,326 subjects from four independent studies of non-small cell lung cancer (NSCLC) in long-term tobacco-exposed populations. We measured >800 proteins in 15uL of serum, identified 44 candidate biomarkers, and developed a 12-protein panel that distinguished NSCLC from controls with 91% sensitivity and 84% specificity in a training set and 89% sensitivity and 83% specificity in a blinded, independent verification set. Performance was similar for early and late stage NSCLC. This is a significant advance in proteomics in an area of high clinical need

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

A Single Molecule Scaffold for the Maize Genome

About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/∼23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5% (2,103.93 Mb/∼2,300 Mb) of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66 (2,032.42 Mb) optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars

Clinical characteristics of NSCLC case and control sets for training and verification.

§<p>For continuous data the differences were tested using t-tests. For categorical data significant differences were tested using the Pearson Chi-Squared Test for independence.</p>‡<p>Pack-years: product of the self reported number of packs of cigarettes smoked per day and the number of years of smoking.</p

Clinical characteristics of NSCLC cases in the training and verification sets.

§<p>Clinical staging for 17 Stage I, 5 Stage II and 29 Stage III cases, NOS not otherwise specified.</p