Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter.

Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors

Surface μ Heavy Chain Signals Down-Regulation of the V(D)J-Recombinase Machinery in the Absence of Surrogate Light Chain Components

Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre–B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible μHC transgene in Rag2−/− pro–B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-α. Furthermore, in wild-type mice and in mice lacking either λ5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in μHC+LC− pre–B cells. Surprisingly, μHC without LC is expressed on the surface of pro–/pre–B cells from λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− mice. Thus, SLC or LC is not required for μHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components

Metric tensor as the dynamical variable for variable cell-shape molecular dynamics

We propose a new variable cell-shape molecular dynamics algorithm where the dynamical variables associated with the cell are the six independent dot products between the vectors defining the cell instead of the nine cartesian components of those vectors. Our choice of the metric tensor as the dynamical variable automatically eliminates the cell orientation from the dynamics. Furthermore, choosing for the cell kinetic energy a simple scalar that is quadratic in the time derivatives of the metric tensor, makes the dynamics invariant with respect to the choice of the simulation cell edges. Choosing the densitary character of that scalar allows us to have a dynamics that obeys the virial theorem. We derive the equations of motion for the two conditions of constant external pressure and constant thermodynamic tension. We also show that using the metric as variable is convenient for structural optimization under those two conditions. We use simulations for Ar with Lennard-Jones parameters and for Si with forces and stresses calculated from first-principles of density functional theory to illustrate the applications of the method.Comment: 10 pages + 6 figures, Latex, to be published in Physical Review

Additive fiber-cerclages in proximal humeral fractures stabilized by locking plates: No effect on fracture stabilization and rotator cuff function in human shoulder specimens

Background and purpose The effect of additive fiber-cerclages in proximal humeral fractures stabilized by locking plates on fracture stabilization and rotator cuff function is unclear. Here it was assessed in a human cadaver study

Boundary Conditions on Internal Three-Body Wave Functions

For a three-body system, a quantum wave function $\Psi^\ell_m$ with definite $\ell$ and $m$ quantum numbers may be expressed in terms of an internal wave function $\chi^\ell_k$ which is a function of three internal coordinates. This article provides necessary and sufficient constraints on $\chi^\ell_k$ to ensure that the external wave function $\Psi^\ell_m$ is analytic. These constraints effectively amount to boundary conditions on $\chi^\ell_k$ and its derivatives at the boundary of the internal space. Such conditions find similarities in the (planar) two-body problem where the wave function (to lowest order) has the form $r^{|m|}$ at the origin. We expect the boundary conditions to prove useful for constructing singularity free three-body basis sets for the case of nonvanishing angular momentum.Comment: 41 pages, submitted to Phys. Rev.

Large scale quantum simulations: C_60 impacts on a semiconducting surface

We present tight binding molecular dynamics simulations of C_60 collisions on the reconstructed diamond(111) surface, carried out with an O(N) method and with cells containing 1140 atoms. The results of our simulations are in very good agreement with experiments performed under the same impact conditions. Furthermore our calculations provide a detailed characterization of the microscopic processes occuring during the collision, and allow the identification of three impact regimes, as a function of the fullerene incident energy. Finally, the study of the reactivity between the cluster and the surface gives insight into the deposition mechanisms of C_60 on semiconducting substrates

Linac Coherent Light Source Undulator RF BPM System

The Linac Coherent Light Source (LCLS) will be the world's first x-ray free-electron laser (FEL) when it becomes operational in 2009. The LCLS is currently in the construction phase. The beam position monitor (BPM) system planned for the LCLS undulator will incorporate a high-resolution X-band cavity BPM system described in this paper. The BPM system will provide high-resolution measurements of the electron beam trajectory on a pulse-to-pulse basis and over many shots. The X-band cavity BPM size, simple fabrication, and high resolution make it an ideal choice for LCLS beam position detection. We will discuss the system specifications, design, and prototype test results

Mechanically Induced Amorphization of Diaqua-bis(Omeprazolate)-Magnesium Dihydrate

The influence of milling diaqua-bis(omeprazolate)-magnesium dihydrate (DABOMD), an active pharmaceutical ingredient (API), was investigated. DABOMD was processed in a planetary ball mill at different milling times, from 1 to 300 min. The milling process resulted in a prominent comminution (size reduction) and amorphization of the API. DABOMD amorphization was identified with various characterization techniques including thermogravimetric analysis, differential scanning calorimetry, powder X-ray diffraction, and attenuated total reflection-Fourier transform infrared spectroscopy. The solid–solid crystalline to amorphous phase transformation is driven by compression, shear stresses, and heat generated in the planetary ball mill. This leads to distortion and breakage of hydrogen bonds, release of water molecules from the crystalline lattice of DABOMD and the accumulation of defects, and eventually a collapse of the crystalline order. Model fitting of the kinetics of comminution and the amorphization of DABOMD revealed a series of events: a rapid comminution at the start of milling driven by crystal cleavage of DABOMD, followed by partial amorphization, which is driven by rapid water diffusion, and subsequently, a slow steady comminution and amorphization

Design and Performance of the LCLS Cavity BPM System

In this paper we present the design of the beam position monitor (BPM) system for the LCLS undulator, which features a high-resolution X-band cavity BPM. Each BPM has a TM{sub 010} monopole reference cavity and a TM{sub 110} dipole cavity designed to operate at a center frequency of 11.384 GHz. The signal processing electronics features a low noise single-stage three-channel heterodyne receiver that has selectable gain and a phase locking local oscillator. We will discuss the system specifications, design, and prototype test results

Scarring Effects on Tunneling in Chaotic Double-Well Potentials

The connection between scarring and tunneling in chaotic double-well potentials is studied in detail through the distribution of level splittings. The mean level splitting is found to have oscillations as a function of energy, as expected if scarring plays a role in determining the size of the splittings, and the spacing between peaks is observed to be periodic of period {$2\pi\hbar$} in action. Moreover, the size of the oscillations is directly correlated with the strength of scarring. These results are interpreted within the theoretical framework of Creagh and Whelan. The semiclassical limit and finite-{$\hbar$} effects are discussed, and connections are made with reaction rates and resonance widths in metastable wells.Comment: 22 pages, including 11 figure