Methods to create a stringent selection system for mammalian cell lines

The efficient establishment of high protein producing recombinant mammalian cell lines is facilitated by the use of a stringent selection system. Here, we describe two methods to create a stringent selection system based on the Zeocin resistance marker. First, we cloned increasingly longer stretches of DNA, encoding a range of 8–131 amino acids immediately upstream of the Zeocin selection marker gene. The DNA stretches were separated from the open reading frame of the selection marker gene by a stopcodon. The idea behind this was that the translation machinery will first translate the small peptide, stop and then restart at the AUG of the Zeocin marker. This process, however, will become less efficient with increasingly longer stretches of DNA upstream of the Zeocin marker that has to be translated first. This would result in lower levels of the Zeocin selection marker protein and thus a higher selection stringency of the system. Secondly, we performed a genetic screen to identify PCR induced mutations in the Zeocin selection protein that functionally impair the selection marker protein. Both the insertion of increasingly longer peptides and several Zeocin selection protein mutants resulted in a decreasing number of stably transfected colonies that concomitantly displayed higher protein expression levels. When the Zeocin mutants were combined with very short small peptides (8–14 amino acids long), this created a flexible, high stringency selection system. The system allows the rapid establishment of few, but high protein producing mammalian cell lines

Measurement of the Casimir force between parallel metallic surfaces

We report on the measurement of the Casimir force between conducting surfaces in a parallel configuration. The force is exerted between a silicon cantilever coated with chromium and a similar rigid surface and is detected looking at the shifts induced in the cantilever frequency when the latter is approached. The scaling of the force with the distance between the surfaces was tested in the 0.5 - 3.0 $\mu$m range, and the related force coefficient was determined at the 15% precision level.Comment: 4 Figure

Measurements of the Casimir-Lifshitz force in fluids: the effect of electrostatic forces and Debye screening

In this work, we present detailed measurements of the Casimir-Lifshitz force between two gold surfaces (a sphere and a plate) immersed in ethanol and study the effect of residual electrostatic forces, which are dominated by static fields within the apparatus and can be reduced with proper shielding. Electrostatic forces are further reduced by Debye screening through the addition of salt ions to the liquid. Additionally, the salt leads to a reduction of the Casimir-Lifshitz force by screening the zero-frequency contribution to the force; however, the effect is small between gold surfaces at the measured separations and within experimental error. An improved calibration procedure is described and compared to previous methods. Finally, the experimental results are compared to Lifshitz's theory and found to be consistent for the materials used in the experiment.Comment: 11 figures. PRA in pres

Improved Precision Measurement of the Casimir Force

We report an improved precision measurement of the Casimir force. The force is measured between a large Al coated sphere and flat plate using an Atomic Force Microscope. The primary experimental improvements include the use of smoother metal coatings, reduced noise, lower systematic errors and independent measurement of surface separations. Also the complete dielectric spectrum of the metal is used in the theory. The average statistical precision remains at the same 1% of the forces measured at the closest separation

Complete roughness and conductivity corrections for the recent Casimir force measurement

We consider detailed roughness and conductivity corrections to the Casimir force in the recent Casimir force measurement employing an Atomic Force Microscope. The roughness of the test bodies-a metal plate and a sphere- was investigated with the Atomic Force Microscope and the Scanning Electron Microscope respectively. It consists of separate crystals of different heights and a stochastic background. The amplitude of roughness relative to the zero roughness level was determined and the corrections to the Casimir force were calculated up to the fourth order in a small parameter (which is this amplitude divided by the distance between the two test bodies). Also the corrections due to finite conductivity were found up to the fourth order in relative penetration depth of electromagnetic zero point oscillations into the metal. The theoretical result for the configuration of a sphere above a plate taking into account both corrections is in excellent agreement with the measured Casimir force

Casimir and van der Waals force between two plates or a sphere (lens) above a plate made of real metals

The Casimir and van der Waals forces acting between two metallic plates or a sphere (lens) above a plate are calculated accounting for the finite conductivity of the metals. The simple formalism of surface modes is briefly presented which allows the possibility to obtain the generalization of Lifshitz results for the case of two semi-spaces covered by the thin layers. Additional clarifications of the regularization procedure provides the means to obtain reliable results not only for the force but also for the energy density. This, in turn, leads to the value of the force for the configuration of a sphere (lens) above a plate both of which are covered by additional layers. The Casimir interaction between Al and Au test bodies is recalculated using the optical tabulated data for the complex refractive index of these metals. The computations turn out to be in agreement with the perturbation theory up to the fourth order in relative penetration depth of electromagnetic zero point oscillations into the metal. The disagreements between the results recently presented in the literature are resolved. The Casimir force between Al bodies covered by the thin Au layers is computed and the possibility to neglect spatial dispersion effects is discussed as a function the layer thickness. The van der Waals force is calculated including the transition region to the Casimir force. The pure non-retarded van der Waals force law between Al and Au bodies is shown to be restricted to a very narrow distance interval from 0.5 nm to (2--4) nm. New, more exact, values of the Hamaker constant for Al and Au are determined.Comment: 5 figure

Casimir forces and non-Newtonian gravitation

The search for non-relativistic deviations from Newtonian gravitation can lead to new phenomena signalling the unification of gravity with the other fundamental interactions. Various recent theoretical frameworks indicate a possible window for non-Newtonian forces with gravitational coupling strength in the micrometre range. The major expected background in the same range is attributable to the Casimir force or variants of it if dielectric materials, rather than conducting ones, are considered. Here we review the measurements of the Casimir force performed so far in the micrometre range and how they determine constraints on non-Newtonian gravitation, also discussing the dominant sources of false signals. We also propose a geometry-independent parameterization of all data in terms of the measurement of the constant c. Any Casimir force measurement should lead, once all corrections are taken into account, to a determination of the constant c which, in order to assess the accuracy of the measurement, can be compared with its more precise value known through microscopic measurements. Although the last decade of experiments has resulted in solid demonstrations of the Casimir force, the situation is not conclusive with respect to being able to discover new physics. Future experiments and novel phenomenological analysis will be necessary to discover non-Newtonian forces or to push the window for their possible existence into regions of the parameter space which theoretically appear unnatural.Comment: Also available at http://www.iop.org/EJ/abstract/1367-2630/8/10/23

Late-Onset Stargardt Disease Due to Mild, Deep-Intronic ABCA4 Alleles

PURPOSE. To investigate the role of two deep-intronic ABCA4 variants, that showed a mild splice defect in vitro and can occur on the same allele as the low penetrant c.5603A>T, in Stargardt disease (STGD1). METHODS. Ophthalmic data were assessed of 18 STGD1 patients who harbored c.769-784C>T or c.4253+43G>A in combination with a severe ABCA4 variant. Subjects carrying c.[769784C>T; 5603A>T] were clinically compared with a STGD1 cohort previously published carrying c.5603A>T noncomplex. We calculated the penetrances of the intronic variants using ABCA4 allele frequency data of the general population and investigated the effect of c.769-784C>T on splicing in photoreceptor progenitor cells (PPCs). RESULTS. Mostly, late-onset, foveal-sparing STGD1 was observed among subjects harboring c.769-784C>T or c.4253+43G>A (median age of onset, 54.5 and 52.0 years, respectively). However, ages of onset, phenotypes in fundo, and visual acuity courses varied widely. No significant clinical differences were observed between the c.[769-784C>T; 5603A>T] cohort and the c.4253+43G>A or the c.5603A>T cohort. The penetrances of c.769-784C>T (20.5%-39.6%) and c.4253+43G>A (35.8%-43.1%) were reduced, when not considering the effect of yet unidentified or known factors in cis, such as c.5603A>T (identified in 7/7 probands with c.769-784C>T; 1/8 probands with c.4253+43G>A). Variant c.769-784C>T resulted in a pseudo-exon insertion in 15% of the total mRNA (i.e., similar to 30% of the c.769-784C>T allele alone). CONCLUSIONS. Two mild intronic ABCA4 variants could further explain missing heritability in late-onset STGD1, distinguishing it from AMD. The observed clinical variability and calculated reduced penetrance urge research into modifiers within and outside of the ABCA4 gene

Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10–12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCβ levels (5–20 µg/kg) after 5–7 weeks. When treatment is repeated two times, the CCβ levels are reached after 9–11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but—in contrast with the pour-on application—after i.m. injection, significant increase of 17β-testosterone and 17β-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood

Normal and Lateral Casimir Forces between Deformed Plates

The Casimir force between macroscopic bodies depends strongly on their shape and orientation. To study this geometry dependence in the case of two deformed metal plates, we use a path integral quantization of the electromagnetic field which properly treats the many-body nature of the interaction, going beyond the commonly used pairwise summation (PWS) of van der Waals forces. For arbitrary deformations we provide an analytical result for the deformation induced change in Casimir energy, which is exact to second order in the deformation amplitude. For the specific case of sinusoidally corrugated plates, we calculate both the normal and the lateral Casimir forces. The deformation induced change in the Casimir interaction of a flat and a corrugated plate shows an interesting crossover as a function of the ratio of the mean platedistance H to the corrugation length \lambda: For \lambda \ll H we find a slower decay \sim H^{-4}, compared to the H^{-5} behavior predicted by PWS which we show to be valid only for \lambda \gg H. The amplitude of the lateral force between two corrugated plates which are out of registry is shown to have a maximum at an optimal wavelength of \lambda \approx 2.5 H. With increasing H/\lambda \gtrsim 0.3 the PWS approach becomes a progressively worse description of the lateral force due to many-body effects. These results may be of relevance for the design and operation of novel microelectromechanical systems (MEMS) and other nanoscale devices.Comment: 20 pages, 5 figure