Engineering stochasticity in gene expression

Stochastic fluctuations (noise) in gene expression can cause members of otherwise genetically identical populations to display drastically different phenotypes. An understanding of the sources of noise and the strategies cells employ to function reliably despite noise is proving to be increasingly important in describing the behavior of natural organisms and will be essential for the engineering of synthetic biological systems. Here we describe the design of synthetic constructs, termed ribosome competing RNAs (rcRNAs), as a means to rationally perturb noise in cellular gene expression. We find that noise in gene expression increases in a manner proportional to the ability of an rcRNA to compete for the cellular ribosome pool. We then demonstrate that operons significantly buffer noise between coexpressed genes in a natural cellular background and can even reduce the level of rcRNA enhanced noise. These results demonstrate that synthetic genetic constructs can significantly affect the noise profile of a living cell and, importantly, that operons are a facile genetic strategy for buffering against noise

Proximity ligation assays with peptide conjugate ‘burrs’ for the sensitive detection of spores

The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide–oligonucleotide–phycoerythrin conjugates (‘burrs’) that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins

Deoxyribozymes that recode sequence information

Allosteric nucleic acid ligases have been used previously to transform analyte-binding into the formation of oligonucleotide templates that can be amplified and detected. We have engineered binary deoxyribozyme ligases whose two components are brought together by bridging oligonucleotide effectors. The engineered ligases can ‘read’ one sequence and then ‘write’ (by ligation) a separate, distinct sequence, which can in turn be uniquely amplified. The binary deoxyribozymes show great specificity, can discriminate against a small number of mutations in the effector, and can read and recode DNA information with high fidelity even in the presence of excess obscuring genomic DNA. In addition, the binary deoxyribozymes can read non-natural nucleotides and write natural sequence information. The binary deoxyribozyme ligases could potentially be used in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of short nucleic acids such as microRNAs

Drop Test Release Mechanism

Many boxes for shipping undergo drop tests by the manufacturer to ensure their durability. Certain constraints are necessary to successfully carry out these tests such as not damaging the box prior to the drop and maintaining consistency throughout every drop. Our team has designed a Drop Test Release Mechanism that addresses these constraints. It provides repetitive drops for different objects that vary in shape and size such as small electronics or parts. This device utilizes a soft-clamping mechanism that can release an object with minimal force applied onto it prior to drop. A frame made of 80/20 was designed to provide rigidity to the soft-clamping mechanism. The soft clamping mechanism supports the object between it by utilizing foam and a friction pad to induce a high friction force. The clamp can be adjusted for multiple sized objects by use of sliding rails that allow it to widen or tighten. Our design focuses on just the release of the test object. A test stand to introduce varying heights must be designed for a fully functional drop test measurement process. The final prototype was tested to analyze the effectiveness of our design. The tests involved verifying the repeatability of the drop mechanism by testing the object to see if it fell in the same orientation and with the same impact each time. The drop mechanism passed these tests but failed at a usability test and an electronic test. The report goes into detail about the design and testing of the prototype

Sorting Nexin 1 Down-Regulation Promotes Colon Tumorigenesis

PURPOSE: Colon cancer is one of the most common human malignancies, yet studies have only begun to identify the multiple mechanisms that underlie the development of this tumor. In this study, we have identified a novel mechanism, dysregulation of endocytic sorting, which promotes colon cancer development. EXPERIMENTAL DESIGN: Immunohistochemical and microarray analyses were done on human colon cancer tissue specimens to determine the levels of one endocytic protein, sorting nexin 1 (SNX1). SW480 cells, a human colon cancer cell line that retains a relatively high level of SNX1 expression, were used to assess the effects of down-regulating this protein by small hairpin RNA. Activation of signal transduction cascades was evaluated in these cells using Western blotting, and multiple functional assays were done. RESULTS: We determined by immunohistochemistry that the level of SNX1 was significantly down-regulated in 75% of human colon cancers. In corroborative studies using microarray analysis, SNX1 message was significantly decreased (log(2) ratio less than -1) for 8 of 19 colon carcinomas. Cell lines with reduced SNX1 levels showed increased proliferation, decreased apoptosis, and decreased susceptibility to anoikis. They also showed increased activation of epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 in response to epidermal growth factor. This increased activation was abolished by inhibition of endocytosis. CONCLUSIONS: These data suggest that loss of SNX1 may play a significant role in the development and aggressiveness of human colon cancer, at least partially through the mechanism of increased signaling from endosomes. Further, these findings suggest that dysregulation of endocytic proteins may represent a new paradigm in the process of carcinogenesis.Fil: Nguyen, Lananh N.. University of Washington; Estados UnidosFil: Holdren, Matthew S.. University of Washington; Estados UnidosFil: Nguyen, Anthony P.. Baylor College of Medicine; Estados UnidosFil: Furuya, Momoko H.. University of Washington; Estados UnidosFil: Bianchini, Michele. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Levy, Estrella Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Mordoh, Jose. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Liu, Annie. University of Washington; Estados UnidosFil: Guncay, Gabriela D.. University of Washington; Estados UnidosFil: Campbell, Jean S.. University of Washington; Estados UnidosFil: Parks, W. Tony. University of Washington; Estados Unido

Tracking of TV and video gaming during childhood: Iowa Bone Development Study

<p>Abstract</p> <p>Background</p> <p>Tracking studies determine the stability and predictability of specific phenomena. This study examined tracking of TV viewing (TV) and video game use (VG) from middle childhood through early adolescence after adjusting for moderate and vigorous physical activity (MVPA), percentage of body fat (% BF), and maturity.</p> <p>Methods</p> <p>TV viewing and VG use were measured at ages 5, 8, 11, and 13 (n = 434) via parental- and self-report. MVPA was measured using the Actigraph, % BF using dual-energy x-ray absorptiometry, and maturity via Mirwald predictive equations. Generalized Estimating Equations (GEE) were used to assess stability and logistic regression was used to predict children "at risk" for maintaining sedentary behaviors. Additional models examined tracking only in overfat children (boys ≥ 25% BF; girls ≥ 32% BF). Data were collected from 1998 to 2007 and analyzed in 2010.</p> <p>Results</p> <p>The adjusted stability coefficients (GEE) for TV viewing were 0.35 (95% CI = 0.26, 0.44) for boys, 0.32 (0.23, 0.40) for girls, and 0.45 (0.27, 0.64) for overfat. For VG use, the adjusted stability coefficients were 0.14 (0.05, 0.24) for boys, 0.24 (0.10, 0.38) for girls, and 0.29 (0.08, 0.50) for overfat. The adjusted odds ratios (OR) for TV viewing were 3.2 (2.0, 5.2) for boys, 2.9 (1.9, 4.6) for girls, and 6.2 (2.2, 17.2) for overfat. For VG use, the OR were 1.8 (1.1, 3.1) for boys, 3.5 (2.1, 5.8) for girls, and 1.9 (0.6, 6.1) for overfat.</p> <p>Conclusions</p> <p>TV viewing and VG use are moderately stable throughout childhood and predictive of later behavior. TV viewing appears to be more stable in younger children than VG use and more predictive of later behavior. Since habitual patterns of sedentarism in young children tend to continue to adolescence, early intervention strategies, particularly to reduce TV viewing, are warranted.</p

Ionic and electronic properties of the topological insulator Bi2_2Te2_2Se investigated using β\beta-detected nuclear magnetic relaxation and resonance of 8^8Li

We report measurements on the high temperature ionic and low temperature electronic properties of the 3D topological insulator Bi$_2$Te$_2$Se using ion-implanted $^8$Li $\beta$-detected nuclear magnetic relaxation and resonance. With implantation energies in the range 5-28 keV, the probes penetrate beyond the expected range of the topological surface state, but are still within 250 nm of the surface. At temperatures above ~150 K, spin-lattice relaxation measurements reveal isolated $^8$Li$^{+}$ diffusion with an activation energy $E_{A} = 0.185(8)$ eV and attempt frequency $\tau_{0}^{-1} = 8(3) \times 10^{11}$ s$^{-1}$ for atomic site-to-site hopping. At lower temperature, we find a linear Korringa-like relaxation mechanism with a field dependent slope and intercept, which is accompanied by an anomalous field dependence to the resonance shift. We suggest that these may be related to a strong contribution from orbital currents or the magnetic freezeout of charge carriers in this heavily compensated semiconductor, but that conventional theories are unable to account for the extent of the field dependence. Conventional NMR of the stable host nuclei may help elucidate their origin.Comment: 17 pages, 12 figures, submitted to Phys. Rev.

Optimized pulses for the control of uncertain qubits

Constructing high-fidelity control fields that are robust to control, system, and/or surrounding environment uncertainties is a crucial objective for quantum information processing. Using the two-state Landau-Zener model for illustrative simulations of a controlled qubit, we generate optimal controls for \pi/2- and \pi-pulses, and investigate their inherent robustness to uncertainty in the magnitude of the drift Hamiltonian. Next, we construct a quantum-control protocol to improve system-drift robustness by combining environment-decoupling pulse criteria and optimal control theory for unitary operations. By perturbatively expanding the unitary time-evolution operator for an open quantum system, previous analysis of environment-decoupling control pulses has calculated explicit control-field criteria to suppress environment-induced errors up to (but not including) third order from \pi/2- and \pi-pulses. We systematically integrate this criteria with optimal control theory, incorporating an estimate of the uncertain parameter, to produce improvements in gate fidelity and robustness, demonstrated via a numerical example based on double quantum dot qubits. For the qubit model used in this work, post facto analysis of the resulting controls suggests that realistic control-field fluctuations and noise may contribute just as significantly to gate errors as system and environment fluctuations.Comment: 38 pages, 15 figures, RevTeX 4.1, minor modifications to the previous versio