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

Cardiorespiratory Fitness in Individuals Post-stroke:Reference Values and Determinants

Objective: To provide reference values of cardiorespiratory fitness for individuals post-stroke in clinical rehabilitation and to gain insight in characteristics related to cardiorespiratory fitness post stroke. Design: A retrospective cohort study. Reference equations of cardiopulmonary fitness corrected for age and sex for the fifth, 25th, 50th, 75th, and 95th percentile were constructed with quantile regression analysis. The relation between patient characteristics and cardiorespiratory fitness was determined by linear regression analyses adjusted for sex and age. Multivariate regression models of cardiorespiratory fitness were constructed. Setting: Clinical rehabilitation center. Participants: Individuals post-stroke who performed a cardiopulmonary exercise test as part of clinical rehabilitation between July 2015 and May 2021 (N=405). Main Outcome Measures: Cardiorespiratory fitness in terms of peak oxygen uptake (V˙O2peak) and oxygen uptake at ventilatory threshold (V˙O2-VT). Results: References equations for cardiorespiratory fitness stratified by sex and age were provided based on 405 individuals post-stroke. Median V˙O2peak was 17.8[range 8.4-39.6] mL/kg/min and median V˙O2-VT was 9.7[range 5.9-26.6] mL/kg/min. Cardiorespiratory fitness was lower in individuals who were older, women, using beta-blocker medication, and in individuals with a higher body mass index and lower motor ability. Conclusions: Population specific reference values of cardiorespiratory fitness for individuals post-stroke corrected for age and sex were presented. These can give individuals post-stroke and health care providers insight in their cardiorespiratory fitness compared with their peers. Furthermore, they can be used to determine the potential necessity for cardiorespiratory fitness training as part of the rehabilitation program for an individual post-stroke to enhance their fitness, functioning and health. Especially, individuals post-stroke with more mobility limitations and beta-blocker use are at a higher risk of low cardiorespiratory fitness.</p

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

Relative Aerobic Load of Daily Activities After Stroke

Objective: Individuals after stroke are less active, experience more fatigue, and perform activities at a slower pace than peers with no impairments. These problems might be caused by an increased aerobic energy expenditure during daily tasks and a decreased aerobic capacity after stroke. The aim of this study was to quantify relative aerobic load (ie, the ratio between aerobic energy expenditure and aerobic capacity) during daily-life activities after stroke. Methods: Seventy-nine individuals after stroke (14 in Functional Ambulation Category [FAC] 3, 25 in FAC 4, and 40 in FAC 5) and 22 peers matched for age, sex, and body mass index performed a maximal exercise test and 5 daily-life activities at a preferred pace for 5 minutes. Aerobic energy expenditure (mL O2/kg/min) and economy (mL O2/kg/unit of distance) were derived from oxygen uptake (V˙O2). Relative aerobic load was defined as aerobic energy expenditure divided by peak aerobic capacity (%V˙O2peak) and by V˙O2 at the ventilatory threshold (%V˙O2-VT) and compared in individuals after stroke and individuals with no impairments. Results: Individuals after stroke performed activities at a significantly higher relative aerobic load (39%-82% V˙O2peak) than peers with no impairments (38%-66% V˙O2peak), despite moving at a significantly slower pace. Aerobic capacity in individuals after stroke was significantly lower than that in peers with no impairments. Movement was less economical in individuals after stroke than in peers with no impairments. Conclusion: Individuals after stroke experience a high relative aerobic load during cyclic daily-life activities, despite adopting a slower movement pace than peers with no impairments. Perhaps individuals after stroke limit their movement pace to operate at sustainable relative aerobic load levels at the expense of pace and economy. Impact: Improving aerobic capacity through structured aerobic training in a rehabilitation program should be further investigated as a potential intervention to improve mobility and functioning after stroke.</p

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

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

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 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