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Modifying Peptide/Lipid-Associated Nucleic Acids (PLANAs) for CRISPR/Cas9 Ribonucleoprotein Delivery
Get PDFWith the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies
Panel 1: Federal Right of Publicity?
No full textBrandon Anand, Managing Attorney, Anand Law PC
Kevin J. Greene, Professor of Law and John J. Schumacher Chair, Southwestern Law School
John Tehranian, Professor of Law and Paul W. Wildman Chair, Southwestern Law School
Moderator: Nate Camuti, Professor of Law, Chapman University, Fowler School of La
Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector \u3cem\u3eIn Situ\u3c/em\u3e
No full textcAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 µM forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells
Regular Functions on the Scaled Hypercomplex Numbers
Get PDFIn this paper, we study the regularity of R-differentiable functions on open connected subsets of the scaled hypercomplex numbers {Ht}t∈R by studying the kernels of suitable differential operators {∇t}t∈R, up to scales in the real field R
Stabilizing Two-Qubit Entanglement with Dynamically Decoupled Active Feedback
Get PDFWe propose and analyze a protocol for stabilizing a maximally entangled state of two noninteracting qubits using active state-dependent feedback from a continuous two-qubit half-parity measurement in coordination with a concurrent, noncommuting dynamical decoupling drive. We demonstrate that such a drive can be simultaneous with the measurement and feedback, while also playing a key part in the feedback protocol itself. We show that robust stabilization with near-unit fidelity can be achieved even in the presence of realistic nonidealities, such as time delay in the feedback loop, imperfect state-tracking, inefficient measurements, dephasing from 1/f-distributed qubit-frequency noise, and relaxation. We mitigate feedback-delay error by introducing a forward-state-estimation strategy in the feedback controller that tracks the effects of control signals already in transit. More generally, the steady state is globally attractive without the need for ancillas, regardless of the error state, in contrast to most known feedback and error-correction schemes
Cinema\u27s Poetic Function: Creating an Amorous Distance
Get PDFThe aim of this thesis is to examine how cinema can embrace its poetic function to avoid its assimilation into preexisting hermeneutic structures, which would leave it vulnerable to myth as defined by Roland Barthes, and instead be a generative force, encouraging its viewer to engage with the full potential of the text. This mode of spectatorship is termed the “amorous distance,” which Barthes describes as his simultaneous fascination with the film and that which exceeds it. The amorous distance finds further articulation through the work of Roman Jakobson and Julia Kristeva. Jakobson’s schema of six language functions describes the poetic function as that which orients a message toward itself, highlighting its own signifying status to bring attention to the process through which it acquires meaning. Kristeva’s distinction between the Semiotic and Symbolic modes illuminates the scope of that process, the former mode referring to the preverbal bringing together of nonsignifying materials into meaning connection, and the latter to the solidification of those materials into an exclusive unity. By synthesizing Barthes, Jakobson, and Kristeva, this thesis argues that the poetic function of cinema puts the viewer at an amorous distance whereby they actively engage with the signifying process in its full involvement of the Semiotic and Symbolic modes. This argument is supported through a close reading of several films: the experimental short film “Psalm II: Walking Distance” (1999), the silent test film “Monkeyshines No. 1” (1890), and the narrative feature films 71 Fragments of a Chronology of Chance (1994) and Code Unknown (2000)
From Film Sets to Front Lines and Back Again: Reinventing Star Image in Post-World War II Hollywood
Get PDFWhen World War II broke out in 1939, and as the United States entered the conflict after Pearl Harbor was bombed in 1941, men and women all across America left their homes to serve the war effort, some of which included Hollywood stars. There were famous actors in the 1950s and 60s who served as unknown young men, but this thesis focuses on established movie stars who voluntarily left their lives of luxury to serve and returned to a changed postwar film industry, specifically James Stewart, Robert Montgomery, Marlene Dietrich, and Myrna Loy. By looking at each of these actors’ prewar star images and their individual acts of war service, this thesis analyzes how their postwar star images were altered by their war experiences and absence from Hollywood to argue that not only could their youthful, light-hearted star images no longer stand, but that their service ultimately did their film careers a disservice. Stewart’s journey with trauma after the war changed him from an endearing everyman to a rugged, battle-tested everyman. The various types of silence thrust upon Montgomery took him away from Hollywood and pushed him into the periphery of the political sphere. The effects of ageism on Dietrich hindered her glamour girl image and forced her out of Hollywood, while ageism turned Loy into an agreeable wife and mother, rather than an exotic, clever young woman. These actors’ extremely personal choices to leave the film industry to serve were at odds with Hollywood’s machine-like nature that saw them as forms of currency, thereby requiring them to reinvent their star images to survive in a hardened, postwar world
Breaking Boundaries: Women in Mesopotamia
Get PDFWe must examine women\u27s contributions to society to comprehend women\u27s roles and significance in ancient Mesopotamia. Women\u27s roles in society and civilization are still frequently overlooked, both then and now. We must not ignore the importance of women\u27s performance throughout history in these societies. Instead, we need to recognize these achievements made by women in the fields of politics, economic standpoints, and family life. We can distinguish the experiences of women in ancient Mesopotamia and contrast them with those of women in modern society by using textual sources and comparative analysis and research. Ancient writings, artwork, and literature can be helpful in determining how women were viewed in this society, as well as in gaining a deeper comprehension of the daily lives that they led and the required duties. I hope that a more comprehensive knowledge of gender dynamics in ancient societies will emerge from careful examination of a variety of sources and circumstances. This allows for the revelation of trends in women\u27s positions and duties throughout Mesopotamia\u27s various eras and time periods. This will allow for critical new perspectives on the general patterns of gender relations in Mesopotamia and the modern-day age
Effects of Chemical and Physical Properties of Water on Hagfish Slime Structure
No full textHagfishes are elongate saltwater fishes that defend themselves using thick slime composed of skein threads and mucus vesicles that clogs the gills of predators. The 3D structure of hagfish slime is currently unknown. Flash freezing then freeze-drying (lyophilizing) slime could help determine the 3D structure of the slime. Instead of seawater which would show salt crystals, deionized (DI) water was used to produce slime from pacific hagfish (Eptatretus stoutii). It is hypothesized that slime in DI water forms too quickly causing the threads to tangle. Varying ethanol (EtOH) and water temperatures might help slow down unraveling and produce a slime structure that is more representative of natural slime. Although there were no obvious directional effects of EtOH on mucus fiber diameter, there was a significantly different mucus fiber diameter between the 5% EtOH (0.339 μm) samples and the 10% EtOH (0.524 μm) samples (p \u3c 0.05). Furthermore, mucus threads at 35°C were significantly thinner than the 1°C and 20°C samples (p \u3c 0.05). Despite no obvious directional effects between the EtOH and temperature treatments, higher EtOH concentrations and higher temperatures seem to have an effect on hagfish slime structure