Molecular, Cellular, and Tissue Engineering Commons^™

Efficient One‑Step Fusion Pcr Based On Dual‑Asymmetric Primers And Two‑Step Annealing, Yilan Liu, Jinjin Chen, Anders Thygesen

Department of Chemical and Biomolecular Engineering: Faculty Publications

Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized. In addition, the fusion products produced with this method were successfully applied in seamless genome editing. The …

Wild-Type P53 Enhances Endothelial Barrier Function By Mediating Rac1 Signalling And Rhoa Inhibition, Nektarios Barabutis, Christiana Dimitropoulou, Betsy Gregory, John D. Catravas

Bioelectrics Publications

Inflammation is the major cause of endothelial barrier hyper-permeability, associated with acute lung injury and acute respiratory distress syndrome. This study reports that p53 "orchestrates" the defence of vascular endothelium against LPS, by mediating the opposing actions of Rac1 and RhoA in pulmonary tissues. Human lung microvascular endothelial cells treated with HSP90 inhibitors activated both Rac1- and P21-activated kinase, which is an essential element of vascular barrier function. 17AAG increased the phosphorylation of both LIMK and cofilin, in contrast to LPS which counteracted those effects. Mouse lung microvascular endothelial cells exposed to LPS exhibited decreased expression of phospho-cofilin. 17AAG treatment …

Alteration Of Metabolite Profiling By Cold Atmospheric Plasma Treatment In Human Myeloma Cells, Dehui Xu, Yujing Xu, Ning Ning, Qingjie Cui, Zhijie Liu, Xiaohua Wang, Dingxin Liu, Hailan Chen, Michael G. Kong

Bioelectrics Publications

BACKGROUND: Despite new progress of chemotherapy in multiple myeloma (MM) clinical treatment, MM is still a refractory disease and new technology is needed to improve the outcomes and prolong the survival. Cold atmospheric plasma is a rapidly developed technology in recent years, which has been widely applied in biomedicine. Although plasma could efficiently inactivate various tumor cells, the effects of plasma on tumor cell metabolism have not been studied yet.

METHODS: In this study, we investigated the metabolite profiling of He plasma treatment on myeloma tumor cells by gas-chromatography time-of-flight (GC-TOF) mass-spectrometry. Meanwhile, by bioinformatic analysis such as GO and …

Exogenous Fniii 12-14 Regulates Tgf-Β1-Induced Markers, Hilmi M. Humeid

Theses and Dissertations

The extracellular matrix protein Fibronectin (FN) plays an important role in cell contractility, differentiation, growth, adhesion, and migration. The 12th -14th Type III repeats of FN (FNIII 12-14), also referred to as the Heparin-II domain, comprise a highly promiscuous growth factor (GF) binding region. This binding domain aids in cellular signaling initiated from the ECM. Additionally, FN has the ability to assemble into fibrils under certain conditions, mostly observed during cell contractile processes such as those that initiate due to upregulation of Transforming Growth Factor Beta 1 (TGF-β1) [1], [2]. Previous work from our lab has shown that self-assembly of …

Decellularized Matrices Effect On The Adaptive Immune Response, Kegan Sowers

Theses and Dissertations

Decellularized extracellular matrices have been a growing area of interest in the biomedical engineering fields of tissue engineering and regenerative medicine.As these materials move toward clinical applications, the immune response to these materials will be a driving force toward their success in clinical approaches. Fully digested decellularized matrix constructs derived from porcine liver, muscle and lung were created to test the adaptive immune response. Hydrogel characterization ensured that the materials had relatively similar stiffness levels to reduce variability, and in vitro studies were conducted. Each individual construct as well as a gelatin control were plated with a co-culture of macrophages …

Characterization Of Electroactive Nanofibers For Nerve Regeneration, Jean-Yves Azar

Research Opportunities for Engineering Undergraduates (ROEU) Program 2017-18

In the event of peripheral nerve injury, loss of function may occur as the result of a severed nerve. Hollow nerve gap conduits are used to provide a protected environment for nerves to reconnect and regain function, although therapies for nerve gaps larger than 3 cm lack a stable environment for nerves to reconnect. Development of a nerve guide conduit filler has been proposed to mimic the cell’s microenvironment. This would lead to increased functional regeneration. We are aiming to create a biomaterial to act as a filler for the hollow nerve gap conduits to promote functional regeneration of the …

Developing Vascular Graft From Adipose-Derived Stem Cells, Ashley C. Apil

Research Opportunities for Engineering Undergraduates (ROEU) Program 2017-18

This project aims to differentiate adipose-derived stem cells into fibroblasts through the addition of platelet-derived growth factor (PDGF) into the culture media. Differentiation protocol will be optimized, then successful differentiation will be verified through PCR analysis. The ASC-fibroblasts will then be seeded into a ring construct as outlined by the protocol by Lam et al. The ring constructs will be tensile tested and analyzed through histology to characterize their strength and cellularity.

Developing A 3d In Vitro Model By Microfluidics, Hung-Ta Chien

Dissertations and Theses

In vitro tissue models play an important role in providing a platform that mimics the realistic tissue microenvironment for stimulating and characterizing the cellular behavior. In particular, the hydrogel-based 3D in vitro models allow the cells to grow and interact with their surroundings in all directions, thus better mimicking in vivo than their 2D counterparts. The objective of this thesis is to establish a 3D in vitro model that mimics the anatomical and functional complexity of the realistic cancer microenvironment for conveniently studying the transport coupling in porous tissue structures. We pack uniform-sized PEGDA-GelMA microgels in a microfluidic chip to …

Using Crosslinked Hyaluronic Acid (Ha) And Collagen Scaffolds With Sustained Brain-Derived Neurotrophic Factor (Bdnf) Release For Post-Sci Nerve Regeneration, Panth Doshi

Undergraduate Research Posters

Traumatic events resulting in spinal cord injuries (SCIs) often leave people paralyzed or with partial loss of motor function. The physical disabilities arising from traumatic events prevent people from functioning at the same level as pre-injury. My work aims to identify a plausible method to overcome the inhibitory post-SCI environment and to regenerate nervous tissue in order to restore neural function and, subsequently, motor function. I identified components of a new, hypothetical nerve scaffold based on the immune response after SCIs and the efficacy of currently used scaffolds for nerve regeneration. Hyaluronic acid (HA) polymer scaffolds and collagen-based scaffolds are …

Targeting Ovarian Cancer And Endothelium With An Allosteric Ptp4a3 Phosphatase Inhibitor, Kelley E. Mcqueeney, Joseph M. Salamoun, James C. Burnett, Nektarios Barabutis, Paula Pekic, Sophie L. Lewandowski, Danielle C. Llaneza, Robert Cornelison, Yunpeng Bai, Zhong-Yin Zhang, John D. Catravas

Bioelectrics Publications

Overexpression of protein tyrosine phosphatase PTP4A oncoproteins is common in many human cancers and is associated with poor patient prognosis and survival. We observed elevated levels of PTP4A3 phosphatase in 79% of human ovarian tumor samples, with significant overexpression in tumor endothelium and pericytes. Furthermore, PTP4A phosphatases appear to regulate several key malignant processes, such as invasion, migration, and angiogenesis, suggesting a pivotal regulatory role in cancer and endothelial signaling pathways. While phosphatases are attractive therapeutic targets, they have been poorly investigated because of a lack of potent and selective chemical probes. In this study, we disclose that a potent, …

A Purification And In Vitro Activity Assay For A (P)Ppgpp Synthetase From Clostridium Difficile, Astha Pokhrel, Asia Poudel, Erin B. Purcell

Chemistry & Biochemistry Faculty Publications

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of γ-³²-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for …

Restoring Cholesterol Homeostasis In Familial Hypercholesterolemia Cell Line Model, Lubna Hindi

Electronic Theses and Dissertations

Familial hypercholesterolemia (FH) is an autosomal dominant disorder that results in elevated levels of low-density lipoprotein cholesterol (LDL-C). This increase in serum cholesterol level has been shown to result in premature coronary artery disease (CAD) with devastating symptoms from a young age. Today, the prevalence of heterozygous FH (HeFH) and homozygous FH (HoFH) is estimated to be 1 in 320 and 1 in 160,000 people, respectively. FH is referred to as an underdiagnosed disease due to the large number of mutations that continues to grow. These mutations often exist in one of or a combination of three genes: low-density lipoprotein …

Molded Features In Pdms For Fabricating Bacterial Cellulose For Various Geometries, Mitchell Habegger

Williams Honors College, Honors Research Projects

The purpose of producing features on bacterial cellulose (BC) is to facilitate the elongation and alignment for cells, in this case Normal Human Dermal Fibroblast (NHDF) cells. The elongated cells have applications in wound healing, tissue engineering, disease diagnostics, and many other fields. Experiments were run to test the effectiveness of transferring features to BC sheets from features induced by fracturing on polydimethylsiloxane (PDMS) and those duplicated from molds with existing features. The features were duplicated to BC sheets by either air drying or Guided Assembly-Based Biolithography (GAB). The research results showed that fracture inducing on PDMS produced very small …

Modification Of Bacterial Cellulose Using Organosilanes To Enhance Cell Adhesion And Growth, Kristi Ferrato

Williams Honors College, Honors Research Projects

In this project, drying bacterial cellulose (BC) to maintain its structural integrity, and treating BC with organosilanes 3-aminopropyltriethoxysilane (APTES) and (3-Mercaptopropyl)trimethoxysilane (MPTMS) as well as with various concentration of gelatin to increase the cells per area was studied. Drying BC with hexamethyldisilazane (HMDS) by soaking the BC in ascending concentrations of ethanol and HMDS did not protect the structural integrity of the BC grown in lab but did for the BC grown in Dr. Siriporn Taokaew’s lab. Freeze drying also did not fully protect the structural integrity as the BC was placed in a freezer for several hours at -20oC …

Multiple Factors Drive Variation Of Forest Root Biomass In Southwestern China, Hao Zhang, Kelin Wang, Zhaoxia Zeng, Zhigang Zou, Yanfang Xu, Fuping Zeng

Nebraska Center for Biotechnology: Faculty and Staff Publications

The roots linking the above-ground organs and soil are key components for estimating net primary productivity and carbon sequestration of forests. The patterns and drivers of root biomass in forest have not been examined well at the regional scale, especially for the widely distributed forest ecosystems in southwestern China. We attempted to determine the spatial patterns of root biomass (RB, Mg/ha), annual increment root biomass (AIRB, Mg/ha/year), ratio of root and above-ground (RRA), and the relative contributions of abiotic and biotic factors that drive the variation of root biomass. Forest biomass and multiple factors (climate, soil, forest types, and stand …

Diversity And Functional Evolution Of Terpene Synthases In Dictyostelid Social Amoebae, Xinlu Chen, Tobias G. Kollner, Gad Shaulsky, Qidong Jia, Jeroen S. Dickschat, Jonathan Gershenzon, Feng Chen

Nebraska Center for Biotechnology: Faculty and Staff Publications

Dictyostelids, or social amoebae, have a unique life style in forming multicellular fruiting bodies from unicellular amoeboids upon starvation. Recently, dictyostelids were found to contain terpene synthase (TPS) genes, a gene type of secondary metabolism previously known to occur only in plants, fungi and bacteria. Here we report an evolutionary functional study of dictyostelid TPS genes. The number of TPS genes in six species of dictyostelids examined ranges from 1 to 19; and the model species Dictyostelium purpureum contains 12 genes. Using in vitro enzyme assays, the 12 TPS genes from D. purpureum were shown to encode functional enzymes with …

Non-Targeted Colonization By The Endomycorrhizal Fungus, Serendipita Vermifera, In Three Weeds Typically Co-Occurring With Switchgrass, Prasun Ray, Yingqing Guo, Jaydeep Kolape

Nebraska Center for Biotechnology: Faculty and Staff Publications

Serendipita vermifera (=Sebacina vermifera; isolate MAFF305830) is a mycorrhizal fungus originally isolated from the roots of an Australian orchid that we have previously shown to be beneficial in enhancing biomass yield and drought tolerance in switchgrass, an important bioenergy crop for cellulosic ethanol production in the United States. However, almost nothing is known about how this root-associated fungus proliferates and grows through the soil matrix. Such information is critical to evaluate the possibility of non-target effects, such as unintended spread to weedy plants growing near a colonized switchgrass plant in a field environment. A microcosm experiment was conducted …

The Effects Of Emerging Technology On Healthcare And The Difficulties Of Integration, Skyler J. Pavlish-Carpenter

Honors Undergraduate Theses

Background: Disruptive technology describes technology that is significantly more advanced than previous iterations, such as: 3D printing, genetic manipulation, stem cell research, innovative surgical procedures, and computer-based charting software. These technologies often require extensive overhauls to implement into older systems and must overcome many difficult financial and societal complications before they can be widely used. In a field like healthcare that makes frequent advancements, these difficulties can mean that the technology will not be utilized to its full potential or implemented at all.

Objective: To determine the inhibiting factors that prevent disruptive technology from being implemented in conventional healthcare.

Methods: …

Mechanical And Material Characterization Of Advanced Materials For Tissue Regeneration, Bailey Kylara Ricketts Jackson

Theses and Dissertations

In natural disasters and military deployment operations, injury to long bone can cause extensive damage and be difficult to treat and heal quickly. Explosive devices and trauma often produce long-bone (>2-cm length) wounds that are more complicated than common fractures. Due to the size and complexity of these injuries, they can be difficult to treat in a battlefield environment and take a long time to heal completely and properly. Even after the long healing period, these injuries often require rehabilitation to strengthen the muscles that have weakened in the wounded limb. For military applications especially, these effects can put …

Surface Functionalization Via Photoinitiated Radical Polymerization For Rare Cell Isolation And Mechanical Protection, Calvin Frank Cahall

Theses and Dissertations--Chemical and Materials Engineering

Surface functionalization of living cells for cell therapeutics has gained substantial momentum in the last two decades. From encapsulating islets of Langerhans, to cell laden gels for tissue scaffolds, to individual cell encapsulation in thin hydrogels, to surface adhesives and inert surface camouflage, modification of living cell surfaces has a wide array of important applications. Here we use hydrogel encapsulation of individual cells as a mode of protection from mechanical forces for high throughput cell printing, and chemical stimuli for the isolation of rare cells in blood.

In the first study, we review methods of surface functionalization and establish a …

Fibronectin Mechanics And Signaling In Tgf-Β1-Induced Epithelial To Mesenchymal Transition, Lauren Griggs

Theses and Dissertations

Epithelial to Mesenchymal Transition (EMT) is a dynamic process by which a distinct change in the phenotype and function of epithelial cells render them as mesenchymal cells. Characteristics of mesenchymal cells include the ability to invade, increased migratory kinetics and heightened resistance to apoptosis. Therefore, there is a strong need to fully understand the mechanism for the induction of EMT in pathological conditions such as carcinoma progression. Recent advances highlight two pivotal contributors, soluble growth factor (gf) signals, and mechanical signals, in the process. However, to date, no clear mechanism exists linking the two in epithelial transdifferentiation. Transforming Growth Factor-β1 …

Nuclear Rupture In Progeria Expressing Cells, Kranthidhar Bathula

Theses and Dissertations

Cells regularly take on various types of force in the body. They have structures that are able to mediate, transfer and respond to the forces. A mutation in force regulating proteins such as lamin in the nucleus or the KASH domain which connects the nucleus to the cytoskeleton of the cell can cause catastrophic events to occur. The aims of this study were to better understand the response of the nucleus when structural proteins are mutated or are not present while under force. Progeria, a rare disease where an additional farnesyl group is attached to lamin was used in this …

Engineering Surface Properties To Modulate Inflammation And Stem Cell Recruitment Through Macrophage Activation, Kelly M. Hotchkiss

Theses and Dissertations

Biomaterials are becoming the most commonly used therapeutic method for treatment of lost or damaged tissue in the body. Metallic materials are chosen for high strength orthopaedic and dental applications. Titanium (Ti) implants are highly successful in young, healthy patients with the ability to fully integrate to surrounding tissue. However the main population requiring these corrective treatments will not be healthy or young, therefore further research into material modifications have been started to improve outcomes in compromised patients. The body’s immune system will generate a response to any implanted material, and control the final outcome. Among the first and most …