Life Sciences Commons^™

Mechanical Test Methods For Assessing Porcine Carotid And Uterine Artery Burst Pressure Following Ex Vivo Ultrasonic Ligature Seal And Transection, Carissa M. Krane, Margaret Pinnell, Courtney Gardner, Mercedes Thompson, James Coleman, Robert Wilkens

Robert J. Wilkens

A test method was developed to identify those variables important for assessing the performance of ultrasonic surgical devices in ex vivo ligature sealing of porcine carotid and uterine arteries. Ruggedness testing using a small sample size in pilot experiments was conducted using a newly developed test method in an effort to assess the usefulness of this methodology and to identify test variables that might warrant further testing. The development of this test method included the use of a custom-designed prototypic tension device for load-controlled ex vivo vessel stretching during saline perfusion and subsequent seal and transection of porcine arteries with …

Jennifer Maurer Phd Thesis.Pdf, Jennifer Maurer

Jennifer Maurer

Intracellular Signaling And Trafficking In Cancer: Role Of Rab5-Gtpase In Migration And Invasion Of Breast Cells, Nicole Porther

Nicole Porther

Metastasis is characterized pathologically by uncontrolled cell invasion, proliferation, migration and angiogenesis. Steroid hormones, such as estrogen, and growth factors, which include insulin growth factor I/II (IGF-1/IGF-2) therapy has been associated with most if not all of the features of metastasis. It has been determined that IGF-1 increases cell survival of cancer cells and potentiate the effect of E2 and other ligand growth factors on breast cancer cells. However not much information is available that comprehensively expounds on the roles of insulin growth factor receptor (IGFR) and Rab GTPases may play in breast cancer. The latter, Rab GTPases, are small …

Use Of Imaging Biomarkers To Assess Perfusion And Glucose Metabolism In The Skeletal Muscle Of Dystrophic Mice, Nabeel Ahmad, Ian Welch, Robert Grange, Jennifer Hadway, Savita Dhanvantari, David Hill, Ting-Yim Lee, Lisa M Hoffman

Lisa Hoffman

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease that affects 1 in 3500 boys. The disease is characterized by progressive muscle degeneration that results from mutations in or loss of the cytoskeletal protein, dystrophin, from the glycoprotein membrane complex, thus increasing the susceptibility of contractile muscle to injury. To date, disease progression is typically assessed using invasive techniques such as muscle biopsies, and while there are recent reports of the use of magnetic resonance, ultrasound and optical imaging technologies to address the issue of disease progression and monitoring therapeutic intervention in dystrophic mice, our study aims to validate …

The Fossil Record Of Phenotypic Integration And Modularity: A Deep-Time Perspective On Developmental And Evolutionary Dynamics, Anjali Goswami, Wendy J. Binder, Julie Meachen, Robin O'Keefe

F. Robin O’Keefe

Variation is the raw material for natural selection, but the factors shaping variation are still poorly understood. Genetic and developmental interactions can direct variation, but there has been little synthesis of these effects with the extrinsic factors that can shape biodiversity over large scales. The study of phenotypic integration and modularity has the capacity to unify these aspects of evolutionary study by estimating genetic and developmental interactions through the quantitative analysis of morphology, allowing for combined assessment of intrinsic and extrinsic effects. Data from the fossil record in particular are central to our understanding of phenotypic integration and modularity because …

Automated Requirements Analysis For A Molecular Watchdog Timer, Samuel J. Ellis, Eric R. Henderson, Titus H. Klinge, James I. Lathrop, Jack H. Lutz, Robyn R. Lutz, Divita Mathur, Andrew S. Miner

Robyn Lutz

Dynamic systems in DNA nanotechnology are often programmed using a chemical reaction network (CRN) model as an intermediate level of abstraction. In this paper, we design and analyze a CRN model of a watchdog timer, a device commonly used to monitor the health of a safety critical system. Our process uses incremental design practices with goal-oriented requirements engineering, software verification tools, and custom software to help automate the software engineering process. The watchdog timer is comprised of three components: an absence detector, a threshold filter, and a signal amplifier. These components are separately designed and verified, and only then composed …

Requirements Analysis For A Product Family Of Dna Nanodevices, Robyn R. Lutz, Jack H. Lutz, James I. Lathrop, Titus H. Klinge, Divita Mathur, D. M. Stull, Taylor G. Bergquist, Eric R. Henderson

Robyn Lutz

DNA nanotechnology uses the information processing capabilities of nucleic acids to design self-assembling, programmable structures and devices at the nanoscale. Devices developed to date have been programmed to implement logic circuits and neural networks, capture or release specific molecules, and traverse molecular tracks and mazes. Here we investigate the use of requirements engineering methods to make DNA nanotechnology more productive, predictable, and safe. We use goal-oriented requirements modeling to identify, specify, and analyze a product family of DNA nanodevices, and we use PRISM model checking to verify both common properties across the family and properties that are specific to individual …

Engineering And Verifying Requirements For Programmable Self-Assembling Nanomachines, Robyn Lutz, Jack Lutz, James Lathrop, Titus Klinge, Eric Henderson, Davita Mathur, Dalia Abo Sheasha

Robyn Lutz

We propose an extension of van Lamsweerde's goal-oriented requirements engineering to the domain of programmable DNA nanotechnology. This is a domain in which individual devices (agents) are at most a few dozen nanometers in diameter. These devices are programmed to assemble themselves from molecular components and perform their assigned tasks. The devices carry out their tasks in the probabilistic world of chemical kinetics, so they are individually error-prone. However, the number of devices deployed is roughly on the order of a nanomole (a 6 followed by fourteen 0s), and some goals are achieved when enough of these agents achieve their …

Impact Of The C-Mybe308g Mutation On Mouse Myelopoiesis And Dendritic Cell Development, Peter Papathanasiou, Sawang Petvises, Ying-Ying Hey, Andrew C Perkins, Helen C O'Neill

Helen O'Neill

Booreana mice carrying the c-Myb308G point mutation were analyzed to determine changes in early hematopoiesis in the bone marrow and among mature cells in the periphery. This point mutation led to increased numbers of early hematopoietic stem and progenitor cells (HSPCs), with a subsequent reduction in the development of B cells, erythroid cells, and neutrophils, and increased numbers of myeloid cells and granulocytes. Myelopoiesis was further investigated by way of particular subsets affected. A specific question addressed whether booreana mice contained increased numbers of dendritic-like cells (L-DC subset) recently identified in the spleen, since L-DCs arise in vitro by direct …

The Glia Response After Peripheral Nerve Injury: A Comparison Between Schwann Cells And Olfactory Ensheathing Cells And Their Uses For Neural Regenerative Therapies, Matthew J Barton, James St John, Alison Wright, Jenny Ekberg

Jenny Ekberg

The peripheral nervous system (PNS) exhibits a much larger capacity for regeneration than the central nervous system (CNS). One reason for this difference is the difference in glial cell types between the two systems. PNS glia respond rapidly to nerve injury by clearing debris from the injury site, supplying essential growth factors and providing structural support; all of which enhances neuronal regeneration. Thus, transplantation of glial cells from the PNS is a very promising therapy for injuries to both the PNS and the CNS. There are two key types of PNS glia: olfactory ensheathing cells (OECs), which populate the olfactory …

Epitaxially Grown Collagen Fibrils Reveal Diversity In Contact Guidance Behavior Among Cancer Cells, Juan Wang, Joseph W. Petefish, Andrew C. Hillier, Ian C. Schneider

Andrew C. Hillier

Invasion of cancer cells into the surrounding tissue is an important step during cancer progression and is driven by cell migration. Cell migration can be random, but often it is directed by various cues such as aligned fibers composed of extracellular matrix (ECM), a process called contact guidance. During contact guidance, aligned fibers bias migration along the long axis of the fibers. These aligned fibers of ECM are commonly composed of type I collagen, an abundant structural protein around tumors. In this paper, we epitaxially grew several different patterns of organized type I collagen on mica and compared the morphology …

Crispr-Cas9 Nuclear Dynamics And Target Recognition In Living Cells, Hanhui Ma, Li-Chun Tu, Ardalan Naseri, Maximiliaan Huisman, Shaojie Zhang, David Grünwald, Thoru Pederson

David Grünwald

The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence …

Genetic Diversity Among Eight Dendrolimus Species In Eurasia (Lepidoptera: Lasiocampidae) Inferred From Mitochondrial Coi And Coii, And Nuclear Its2 Markers, Alexander Kononov, Kirill Ustyantsev, Baode Wang, Victor C. Mastro, Victor Fet, Alexander Blinov, Yuri Baranchikov

Victor Fet

Background: Moths of genus Dendrolimus (Lepidoptera: Lasiocampidae) are among the major pests of coniferous forests worldwide. Taxonomy and nomenclature of this genus are not entirely established, and there are many species with a controversial taxonomic position. We present a comparative evolutionary analysis of the most economically important Dendrolimus species in Eurasia. Results: Our analysis was based on the nucleotide sequences of COI and COII mitochondrial genes and ITS2 spacer of nuclear ribosomal genes. All known sequences were extracted from GenBank. Additional 112 new sequences were identified for 28 specimens of D. sibiricus, D. pini, and D. superans from five regions …

Central Role Of Il-23 And Il-17 Producing Eosinophils As Immunomodulatory Effector Cells In Acute Pulmonary Aspergillosis And Allergic Asthma, Evelyn V. Santos Guerra, Chrono K. Lee, Charles A. Specht, Bhawna Yadav, Haibin Huang, Ali Akalin, Jun R. Huh, Christian Mueller, Stuart M. Levitz

Christian Mueller

Aspergillus fumigatus causes invasive pulmonary disease in immunocompromised hosts and allergic asthma in atopic individuals. We studied the contribution of lung eosinophils to these fungal diseases. By in vivo intracellular cytokine staining and confocal microscopy, we observed that eosinophils act as local sources of IL-23 and IL-17. Remarkably, mice lacking eosinophils had a >95% reduction in the percentage of lung IL-23p19+ cells as well as markedly reduced IL-23 heterodimer in lung lavage fluid. Eosinophils killed A. fumigatus conidia in vivo. Eosinopenic mice had higher mortality rates, decreased recruitment of inflammatory monocytes, and decreased expansion of lung macrophages after challenge with …

Hepatitis C Virus-Induced Monocyte Differentiation Into Polarized M2 Macrophages Promotes Stellate Cell Activation Via Tgf-Beta, Banishree Saha, Karen Kodys, Gyongyi Szabo

Gyongyi Szabo

BACKGROUND and AIMS: Monocyte and macrophage (MPhi) activation contributes to the pathogenesis of chronic hepatitis C virus (HCV) infection. Disease pathogenesis is regulated by both liver-resident MPhis and monocytes recruited as precursors of MPhis into the damaged liver. Monocytes differentiate into M1 (classic/proinflammatory) or M2 (alternative/anti-inflammatory) polarized MPhis in response to tissue microenvironment. We hypothesized that HCV-infected hepatoma cells (infected with Japanese fulminant hepatitis-1 [Huh7.5/JFH-1]) induce monocyte differentiation into polarized MPhis. METHODS: Healthy human monocytes were co-cultured with Huh7.5/JFH-1 cells or cell-free virus for 7 days and analyzed for MPhi markers and cytokine levels. A similar analysis was performed on …

A Sumo-Targeted Ubiquitin Ligase Is Involved In The Degradation Of The Nuclear Pool Of The Sumo E3 Ligase Siz1, Oliver Kerscher, Jason W. Westerbeck, Nagesh Pasupala, Mark Guillotte, Eva Szymanski, Brooke C. Matson, Cecilia Esteban

Oliver Kerscher

The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a …

Budding Yeast Protein Extraction And Purification For The Study Of Function, Interactions, And Post-Translational Modifications, Eva P. Szymanski, Oliver Kerscher

Oliver Kerscher

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. …

Tim18p Is A New Component Of The Tim54p-Tim22p Translocon In The Mitochondrial Inner Membrane, Oliver Kerscher, Naresh B. Sepuri, Robert E. Jensen

Oliver Kerscher

The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex). To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitivetim54-1 mutant. We identified a new gene,TIM18, that encodes an integral protein of the inner membrane. The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress thetim54-1 growth defect; thetim18::HIS3 disruption is synthetically lethal …

Recognizing Chromosomes In Trouble: Association Of The Spindle Checkpoint Protein Bub3p With Altered Kinetochores And A Unique Defective Centromere, Oliver Kerscher, Luciana B. Crotti, Munira A. Basrai

Oliver Kerscher

Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). We show for the first time that green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G₂/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, we tested …

Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Flup, Josh La, Wei-Chun Au, Nagesh Pasupala, Ruben Levy-Meyers, Jack Warren, Alexander Strunnikov, Richard E. Baker, Oliver Kerscher, Kerry Bloom, Munira A. Basrai

Oliver Kerscher

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and …

The Yeast Hex3·Slx8 Heterodimer Is A Ubiquitin Ligase Stimulated By Substrate Sumoylation, Yang Xie, Oliver Kerscher, Mary B. Kroetz, Heather F. Mcconchie, Patrick Sung, Mark Hochstrasser

Oliver Kerscher

Hex3 and Slx8 are Saccharomyces cerevisiae proteins with important functions in DNA damage control and maintenance of genomic stability. Both proteins have RING domains at their C termini. Such domains are common in ubiquitin and ubiquitin-like protein ligases (E3s), but little was known about the molecular functions of either protein. In this study we identified HEX3 as a high-copy suppressor of a temperature-sensitive small ubiquitin-related modifier (SUMO) protease mutant, ulp1ts, suggesting that it may affect cellular SUMO dynamics. Remarkably, even a complete deletion of ULP1 is strongly suppressed. Hex3 forms a heterodimer with Slx8. We found that the Hex3·Slx8 …

The Yeast Nuclear Pore Complex Functionally Interacts With Components Of The Spindle Assembly Checkpoint, Tatiana Louk, Oliver Kerscher, Robert J. Scott, Munira A. Basrai, Richard W. Wozniak

Oliver Kerscher

Aphysical and functional link between the nuclear pore complex (NPC) and the spindle checkpoint machinery has been established in the yeast Saccharomyces cerevisiae. We show that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle. There they are associated with a subcomplex of nucleoporins containing Nup53p, Nup170p, and Nup157p. The association of the Mad1p–Mad2p complex with the NPC requires Mad1p and is mediated in part by Nup53p. On activation of the spindle checkpoint, we detect changes in the interactions between these proteins, including the release of Mad2p …