Purification and characterization of Taq polymerase: A 9-week biochemistry laboratory project for undergraduate students

We have developed a 9-week undergraduate laboratory series focused on the purification and characterization of Thermus aquaticus DNA polymerase (Taq). Our aim was to provide undergraduate biochemistry students with a full-semester continuing project simulating a research-like experience, while having each week\u27s procedure focus on a single learning goal. The laboratory series has been taught for the past 7 years, and survey-based assessment of the effectiveness of the laboratory series was completed during the 2006 and 2007 fall semesters. Statistical analysis of the survey results demonstrate that the laboratory series is very effective in teaching students the theory and practice of protein purification and analysis while also demonstrating positive results in more broad areas of scientific skill and knowledge. Amongst the findings, the largest reported increases in knowledge were related to students\u27 understanding of how patent law relates to laboratory science, a topic of great importance to modern researchers that is readily discussed in relation to Taq polymerase. Overall, this laboratory series proves to be a very effective component in the curricula of undergraduate biology and chemistry majors and may be an appropriate laboratory experience for undergraduates. © 2010 by The International Union of Biochemistry and Molecular Biology

How Changes in Extracellular Matrix Mechanics and Gene Expression Variability Might Combine to Drive Cancer Progression

Changes in extracellular matrix (ECM) structure or mechanics can actively drive cancer progression; however, the underlying mechanism remains unknown. Here we explore whether this process could be mediated by changes in cell shape that lead to increases in genetic noise, given that both factors have been independently shown to alter gene expression and induce cell fate switching. We do this using a computer simulation model that explores the impact of physical changes in the tissue microenvironment under conditions in which physical deformation of cells increases gene expression variability among genetically identical cells. The model reveals that cancerous tissue growth can be driven by physical changes in the microenvironment: when increases in cell shape variability due to growth-dependent increases in cell packing density enhance gene expression variation, heterogeneous autonomous growth and further structural disorganization can result, thereby driving cancer progression via positive feedback. The model parameters that led to this prediction are consistent with experimental measurements of mammary tissues that spontaneously undergo cancer progression in transgenic C3(1)-SV40Tag female mice, which exhibit enhanced stiffness of mammary ducts, as well as progressive increases in variability of cell-cell relations and associated cell shape changes. These results demonstrate the potential for physical changes in the tissue microenvironment (e.g., altered ECM mechanics) to induce a cancerous phenotype or accelerate cancer progression in a clonal population through local changes in cell geometry and increased phenotypic variability, even in the absence of gene mutation

Intermediate filament protein synemin is transiently expressed in a subset of astrocytes during development

Synemin, a developmentally regulated protein first described in muscle cells, has recently been recognized as an intermediate filament (IF) protein. Because IF proteins are invaluable markers of cell origin within the nervous system, we were interested in determining the expression pattern of synemin in the brain. Our results show that, during development of the rat cortex, synemin is expressed only in a subpopulation of astrocytic cells expressing GFAP as well as vimentin and nestin. Unlike GFAP, however, synemin is not expressed in mature astrocytes and, unlike vimentin and nestin, synemin is not present in astrocytic precursors before GFAP expression. Taken together with morphological evidence, the time course of synemin expression, as determined by Western blotting, suggests that synemin is expressed in radial glial cells undergoing morphological transformation into astrocytes. Studies of synemin expression in vitro demonstrate that, early in primary culture, the majority of polygonal astrocytes are derived from synemin+ radial glial cells. With time in culture, however, polygonal astrocytes either stop expressing synemin or are overgrown by cells not expressing synemin. The unique pattern of synemin expression, both in vivo and in vitro, suggests that the use of synemin as a marker will add a new dimension to studies of astrocytic differentiation. (C) 2000 Wiley-Liss, Inc

Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

Human pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) promoted expression of the mesodermal marker PDGFRα upregulated characteristic (pro)epicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (pro)epicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (pro)epicardial-like cells displaying functional properties (adhesion and spreading over the myocardium) of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (pro)epicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration. The authors have shown that hPSCs can be instructed in vitro to differentiate into a specific cardiac embryonic progenitor cell population called the proepicardium. Proepicardial cells are required for normal formation of the heart during development and might contribute to the development of cell-based therapies for heart repair

Cell shape and mechanical changes accompany cancer progression in FVB C3(1)-SV40Tag transgenic mice.

<p>(<b>A</b>) Regional variations in mammary cancer progression observed in the same mammary gland isolated from a 16-week-old transgenic mouse. Note that normal ducts (<b>N</b>), hyperplastic ducts (<b>H</b>) and DCIS-resembling ducts (<b>D</b>) can be found in close proximity in the same gland (scale bar: 100 µm). (<b>B</b>) High magnification H&E stainings of normal, hyperplastic and DCIS ducts in 16-week-old transgenic females highlighting epithelial cell shape changes that accompany cancer progression when cells become increasingly pleiomorphic. (<b>C</b>) Histograms showing the Young’s moduli of epithelium and periductal stroma of different normal and DCIS ducts measured within the same 16-week-old transgenic mammary glands using AFM. (<b>D</b>) Average stiffnesses measured in the epithelial and stromal compartments of normal versus DCIS ducts within the same 16-week-old glands (*, <i>p</i><0.05).</p