High Incidence of Non-Random Template Strand Segregation and Asymmetric Fate Determination In Dividing Stem Cells and their Progeny

Decades ago, the “immortal strand hypothesis” was proposed as a means by which stem cells might limit acquiring mutations that could give rise to cancer, while continuing to proliferate for the life of an organism. Originally based on observations in embryonic cells, and later studied in terms of stem cell self-renewal, this hypothesis has remained largely unaccepted because of few additional reports, the rarity of the cells displaying template strand segregation, and alternative interpretations of experiments involving single labels or different types of labels to follow template strands. Using sequential pulses of halogenated thymidine analogs (bromodeoxyuridine [BrdU], chlorodeoxyuridine [CldU], and iododeoxyuridine [IdU]), and analyzing stem cell progeny during induced regeneration in vivo, we observed extraordinarily high frequencies of segregation of older and younger template strands during a period of proliferative expansion of muscle stem cells. Furthermore, template strand co-segregation was strongly associated with asymmetric cell divisions yielding daughters with divergent fates. Daughter cells inheriting the older templates retained the more immature phenotype, whereas daughters inheriting the newer templates acquired a more differentiated phenotype. These data provide compelling evidence of template strand co-segregation based on template age and associated with cell fate determination, suggest that template strand age is monitored during stem cell lineage progression, and raise important caveats for the interpretation of label-retaining cells

Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA

BACKGROUND: Therapeutic drug monitoring of immunosuppressive drugs in organ-transplanted patients is crucial to prevent intoxication or transplant rejection due to inadequate dosage. The commonly used immunoassays have been gradually undergoing replacement by mass spectrometry, since this physical method offers both a higher sensitivity and specificity. However, a switch should be carefully considered because it is a challenging procedure and needs to be thoroughly validated. From an economic perspective it is reasonable to include mycophenolic acid into the assay, because this saves the necessity for an additional measurement. However, to date very few validation protocols for the measurement of immunosuppressants, including mycophenolic acid, are available. In order to adequately compensate for matrix effects, the use of stable isotope labeled internal standards is advisable. Here, the authors describe a single method suitable for the quantification of cyclosporine A, tacrolimus, sirolimus, everolimus and mycophenolic acid, based on deuterated internal standards. METHODS: Plasma proteins were precipitated with zinc-sulfate, followed by an online solid phase extraction in the flow-through direction. Chromatographic separation was performed by a c18-phenyl-hexyl column. For subsequent mass spectrometric analysis stable-isotope-labeled internal standards were used. Results were available after 3.5 minutes. RESULTS: Low quantification limits (accuracy: 104 - 118%) and linearity resulted in 2 -1250 ng/ml for cyclosporine A; 0.5 - 42.2 ng/ml for tacrolimus; 0.6 - 49.2 ng/ml for sirolimus; 0.5 - 40.8 ng/ml for everolimus and 0.01 - 7.5 μg/ml for mycophenolic acid. Intra-assay precision revealed a coefficient of variation (CV) of 0.9 - 14.7%, with an accuracy of 89 - 138%. The CV of inter-assay precision was 2.5 - 12.5%, with an accuracy of 90 - 113%. Recovery ranged from 76.6 to 84%. Matrix effects were well compensated by deuterated internal standards. CONCLUSIONS: The authors present a fast, economical and robust method for routine therapeutic drug monitoring comprising five immunosuppressants including mycophenolic acid

Interpersonal Cognitive Differentiation and Drug of Choice

The relationship between drug abusers' level of interpersonal cognitive differentiation and preferred drug of abuse was explored. 30 subjects completed a drug-history questionnaire which provided information on the pattern and extent of drug use. Interpersonal cognitive differentiation was assessed with a modified form of Kelly's grid repertory test. Depressant abusers were significantly less differentiated than stimulant abusers. Cross-validation with a larger sample would make more interesting the implications which are discussed in the context of earlier studies. </jats:p

Karstification beneath the Birs weir in Basel/Switzerland: A 3D modeling approach

We present a three-dimensional model describing the evolution of a gypsum karst aquifer along the Birs river near Basel in Switzerland. Here, a small dam-site was built in 1890 to generate hydro-electric power. After subsidence of a nearby highway, geophysical and geological field studies have been carried out and a detailed hydrogeological model has been developed. These observations identified a karstified weathered zone as the reason for the subsidence. To prevent any further damage, remedial construction measures have been carried out. We employ our numerical model KARSTAQUIFER, which simulates the temporal evolution of a karst system by dissolution, increasing the porosity in the aquifer. We used results from geological and geophysical investigations as a priori information for implementing the initial boundary conditions into our three-dimensional numerical model. Our model accounts for topography, precipitation and geology and especially for the anisotropy of the local bedrock. It can successfully reproduce the weathered zone in its current horizontal and vertical extent. We cm also simulate the possible evolution of the aquifer following a remedial construction phase. Therefore, taken together with the geophysical and geological observations and the groundwater model, our three-dimensional karst aquifer evolution model enhances the understanding of the development of this heterogeneous karst aquifer system

Karstification of an aquifer along the Birs river, Switzerland - a modeling approach

The impact, caused by a construction of a dam site on the Birs river close to Basel (Switzerland), on the evolution of a gypsum-karst aquifer in the vicinity of the hydraulic building, is studied. Several sinkholes provoked subsidence of the dam and the highway nearby. Extensive technical measures had to be conducted in order to prevent further karstification. The numerous geophysical and geological field studies executed in the area, together with a 3D hydrogeological model of the aquifer, provide a very detailed information about the boundary conditions, and the local properties determining the karstification. In this work, we present a 2D karst evolution model of the aquifer in the vicinity of the dam structure. In contrast to older studies, this time the focus of the research is not the basic processes governing the karst evolution, but modeling the temporal development of the real aquifer. Using the large amount of information about the location, we demonstrate that a detailed knowledge of the local properties of the rock (hydraulic conductivity, solubility) is of crucial importance when modeling real aquifers. From a wide range of possible evolution scenarios, we deduce a warning that solutional features, such as sinkholes, can develop far away from the hydraulic structure and endanger facilities at the surface. Our model is able to reproduce and successfully explain the main geological features revealed by field studies. We suggest a workflow to combine the data from field observations, groundwater modeling, and karst evolution modeling and to study the karstification of real aquifers. We propose a scenario for the evolution of the aquifer and a reasonable range for the values of the basic parameters governing the karstification. (C) 2012 Elsevier B.V. All rights reserved. Hydrogeologie der Nordwestschweiz, Grundwasse