Spatiotemporal Control of Vascular Endothelial Growth Factor Expression Using a Heat-Shock-Activated, Rapamycin-Dependent Gene Switch

A major challenge in regenerative medicine is to develop methods for delivering growth and differentiation factors in specific spatial and temporal patterns, thereby mimicking the natural processes of development and tissue repair. Heat shock (HS)-inducible gene expression systems can respond to spatial information provided by localized heating, but are by themselves incapable of sustained expression. Conversely, gene switches activated by small molecules provide tight temporal control and sustained expression, but lack mechanisms for spatial targeting. Here we combine the advantages of HS and ligand-activated systems by developing a novel rapamycin-regulated, HS-inducible gene switch that provides spatial and temporal control and sustained expression of transgenes such as firefly luciferase and vascular endothelial growth factor (VEGF). This gene circuit exhibits very low background in the uninduced state and can be repeatedly activated up to 1 month. Furthermore, dual regulation of VEGF induction in vivo is shown to stimulate localized vascularization, thereby providing a route for temporal and spatial control of angiogenesis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140102/1/hgtb.2013.026.pd

Spectra of Doubly Heavy Quark Baryons

Baryons containing two heavy quarks are treated in the Born-Oppenheimer approximation. Schr\"odinger equation for two center Coulomb plus harmonic oscillator potential is solved by the method of ethalon equation at large intercenter separations. Asymptotical expansions for energy term and wave function are obtained in the analytical form. Using those formulas, the energy spectra of doubly heavy baryons with various quark compositions are calculated analytically.Comment: 19 pages, latex2e, published at PRC61(2000)04520

High Hepatitis E Seroprevalence Among Displaced Persons in South Sudan.

AbstractLarge protracted outbreaks of hepatitis E virus (HEV) have been documented in displaced populations in Africa over the past decade though data are limited outside these exceptional settings. Serological studies can provide insights useful for improving surveillance and disease control. We conducted an age-stratified serological survey using samples previously collected for another research study from 206 residents of an internally displaced person camp in Juba, South Sudan. We tested serum for anti-HEV antibodies (IgM and IgG) and estimated the prevalence of recent and historical exposure to the virus. Using data on individuals' serostatus, camp arrival date, and state of origin, we used catalytic transmission models to estimate the relative risk of HEV infection in the camp compared with that in the participants' home states. The age-adjusted seroprevalence of anti-HEV IgG was 71% (95% confidence interval = 63-78), and 4% had evidence of recent exposure (IgM). We estimated HEV exposure rates to be more than 2-fold (hazard ratio = 2.3, 95% credible interval = 0.3-5.8) higher in the camp than in the participants' home states, although this difference was not statistically significant. HEV transmission may be higher than previously appreciated, even in the absence of reported cases. Improved surveillance in similar settings is needed to understand the burden of disease and minimize epidemic impact through early detection and response

Connections Between Connexins, Calcium, and Cataracts in the Lens

There is a good deal of evidence that the lens generates an internal micro circulatory system, which brings metabolites, like glucose, and antioxidants, like ascorbate, into the lens along the extracellular spaces between cells. Calcium also ought to be carried into the lens by this system. If so, the only path for Ca2+ to get out of the lens is to move down its electrochemical gradient into fiber cells, and then move by electrodiffusion from cell to cell through gap junctions to surface cells, where Ca-ATPase activity and Na/Ca exchange can transport it back into the aqueous or vitreous humors. The purpose of the present study was to test this calcium circulation hypothesis by studying calcium homeostasis in connexin (Cx46) knockout and (Cx46 for Cx50) knockin mouse lenses, which have different degrees of gap junction coupling. To measure intracellular calcium, FURA2 was injected into fiber cells, and the gradient in calcium concentration from center to surface was mapped in each type of lens. In wild-type lenses the coupling conductance of the mature fibers was ∼0.5 S/cm2 of cell to cell contact, and the best fit to the calcium concentration data varied from 700 nM in the center to 300 nM at the surface. In the knockin lenses, the coupling conductance was ∼1.0 S/cm2 and calcium varied from ∼500 nM at the center to 300 nM at the surface. Thus, when the coupling conductance doubled, the concentration gradient halved, as predicted by the model. In knockout lenses, the coupling conductance was zero, hence the efflux path was knocked out and calcium accumulated to ∼2 μM in central fibers. Knockout lenses also had a dense central cataract that extended from the center to about half the radius. Others have previously shown that this cataract involves activation of a calcium-dependent protease, Lp82. We can now expand on this finding to provide a hypothesis on each step that leads to cataract formation: knockout of Cx46 causes loss of coupling of mature fiber cells; the efflux path for calcium is therefore blocked; calcium accumulates in the central cells; at concentrations above ∼1 μM (from the center to about half way out of a 3-wk-old lens) Lp82 is activated; Lp82 cleaves cytoplasmic proteins (crystallins) in central cells; and the cleaved proteins aggregate and scatter light

Molecular mechanism of Gαi activation by non-GPCR proteins with a Gα-Binding and Activating motif

Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Gα. Although activation is predominantly carried out by G-protein-coupled receptors (GPCRs), non-receptor guanine-nucleotide exchange factors (GEFs) have emerged as critical signalling molecules and therapeutic targets. Here we characterize the molecular mechanism of G-protein activation by a family of non-receptor GEFs containing a Gα-binding and -activating (GBA) motif. We combine NMR spectroscopy, computational modelling and biochemistry to map changes in Gα caused by binding of GBA proteins with residue-level resolution. We find that the GBA motif binds to the SwitchII/α3 cleft of Gα and induces changes in the G-1/P-loop and G-2 boxes (involved in phosphate binding), but not in the G-4/G-5 boxes (guanine binding). Our findings reveal that G-protein-binding and activation mechanisms are fundamentally different between GBA proteins and GPCRs, and that GEF-mediated perturbation of nucleotide phosphate binding is sufficient for Gα activation

Over-expression of Thioredoxin-1 mediates growth, survival, and chemoresistance and is a druggable target in diffuse large B-cell lymphoma

Diffuse Large B cell lymphomas (DLBCL) are the most prevalent of the non-Hodgkin lymphomas and are currently initially treated fairly successfully, but frequently relapse as refractory disease, resulting in poor salvage therapy options and short survival. The greatest challenge in improving survival of DLBCL patients is overcoming chemo-resistance, whose basis is poorly understood. Among the potential mediators of DLBCL chemo-resistance is the thioredxoin (Trx) family, primarily because Trx family members play critical roles in the regulation of cellular redox homeostasis, and recent studies have indicated that dysregulated redox homeostasis also plays a key role in chemoresistance. In this study, we showed that most of the DLBCL-derived cell lines and primary DLBCL cells express higher basal levels of Trx-1 than normal B cells and that Trx-1 expression level is associated with decreased patients survival. Our functional studies showed that inhibition of Trx-1 by small interfering RNA or a Trx-1 inhibitor (PX-12) inhibited DLBCL cell growth, clonogenicity, and also sensitized DLBCL cells to doxorubicin-induced cell growth inhibition in vitro. These results indicate that Trx-1 plays a key role in cell growth and survival, as well as chemoresistance, and is a potential target to overcome drug resistance in relapsed/refractory DLBCL

Dominant cataracts result from incongruous mixing of wild-type lens connexins

Gap junctions are composed of proteins called connexins (Cx) and facilitate both ionic and biochemical modes of intercellular communication. In the lens, Cx46 and Cx50 provide the gap junctional coupling needed for homeostasis and growth. In mice, deletion of Cx46 produced severe cataracts, whereas knockout of Cx50 resulted in significantly reduced lens growth and milder cataracts. Genetic replacement of Cx50 with Cx46 by knockin rescued clarity but not growth. By mating knockin and knockout mice, we show that heterozygous replacement of Cx50 with Cx46 rescued growth but produced dominant cataracts that resulted from disruption of lens fiber morphology and crystallin precipitation. Impedance measurements revealed normal levels of ionic gap junctional coupling, whereas the passage of fluorescent dyes that mimic biochemical coupling was altered in heterozygous knockin lenses. In addition, double heterozygous knockout lenses retained normal growth and clarity, whereas knockover lenses, where native Cx46 was deleted and homozygously knocked into the Cx50 locus, displayed significantly deficient growth but maintained clarity. Together, these findings suggest that unique biochemical modes of gap junctional communication influence lens clarity and lens growth, and this biochemical coupling is modulated by the connexin composition of the gap junction channels

SDSS J092455.87+021924.9: an Interesting Gravitationally Lensed Quasar from the Sloan Digital Sky Survey

We report the discovery of a new gravitationally lensed quasar from the Sloan Digital Sky Survey, SDSS J092455.87+021924.9 (SDSS J0924+0219). This object was selected from among known SDSS quasars by an algorithm that was designed to select another known SDSS lensed quasar (SDSS 1226-0006A,B). Five separate components, three of which are unresolved, are identified in photometric follow-up observations obtained with the Magellan Consortium's 6.5m Walter Baade telescope at Las Campanas Observatory. Two of the unresolved components (designated A and B) are confirmed to be quasars with z=1.524; the velocity difference is less than 100 km sec^{-1} according to spectra taken with the W. M. Keck Observatory's Keck II telescope on Mauna Kea. A third stellar component, designated C, has the colors of a quasar with redshift similar to components A and B. The maximum separation of the point sources is 1.78". The other two sources, designated G and D, are resolved. Component G appears to be the best candidate for the lensing galaxy. Although component D is near the expected position of the fourth lensed component in a four image lens system, its properties are not consistent with being the image of a quasar at z~1.5. Nevertheless, the identical redshifts of components A and B and the presence of component C strongly suggest that this object is a gravitational lens. Our observations support the idea that a foreground object reddens the fourth lensed component and that another unmodeled effect (such as micro- or milli-lensing) demagnificates it, but we cannot rule out the possibility that SDSS0924+0219 is an example of the relatively rare class of ``three component'' lens systems.Comment: 24 pages, 6 figures, accepted by A

A Survey of z>5.7 Quasars in the Sloan Digital Sky Survey II: Discovery of Three Additional Quasars at z>6

We present the discovery of three new quasars at z>6 in 1300 deg^2 of SDSS imaging data, J114816.64+525150.3 (z=6.43), J104845.05+463718.3 (z=6.23) and J163033.90+401209.6 (z=6.05). The first two objects have weak Ly alpha emission lines; their redshifts are determined from the positions of the Lyman break. They are only accurate to 0.05 and could be affected by the presence of broad absorption line systems. The last object has a Ly alpha strength more typical of lower redshift quasars. Based on a sample of six quasars at z>5.7 that cover 2870 deg^2 presented in this paper and in Paper I, we estimate the comoving density of luminous quasars at z 6 and M_{1450} < -26.8 to be (8 +/- 3)x10^{-10} Mpc^{-3} (for H_0 = 50 km/s/Mpc, Omega = 1). HST imaging of two z>5.7 quasars and high-resolution ground-based images (seeing 0.4'') of three additional z>5.7 quasars show that none of them is gravitationally lensed. The luminosity distribution of the high-redshfit quasar sample suggests the bright end slope of the quasar luminosity function at z 6 is shallower than Psi L^{-3.5} (2-sigma), consistent with the absence of strongly lensed objects.Comment: AJ in press (Apr 2003), 26 pages, 9 figure