Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.     

Parameter Estimation for a Generalized Gamma Distribution

It is fairly commonplace in reliability analyses to encounter data which is incompatible with the exponential, Weibull, and other familiar probability models. Such data motivates research to enlarge the group of probability distributions which are useful to the reliability analyst.

In this paper, we examine a three-parameter generalization of the gamma distribution and derive parameter estimation techniques for that distribution. Those techniques, in the general case, depend upon method of moments considerations which lead to simultaneous equations for which closed form solutions are not available. Graphic solution is proposed and aids to the computations are provided. Major concepts in the paper are summarized by means of a numerical example.

Details are given for the special case in which only the scale parameter is unknown. Three unbiased estimators for that parameter are derived along with their variance formulas. Minimum variance considerations are discussed by application of the Cramér-Rao Theorem.     

Engineered Fibrillar Fibronectin Networks as Three‐Dimensional Tissue Scaffolds

Extracellular matrix (ECM) proteins, and most prominently, fibronectin (Fn), are routinely used in the form of adsorbed pre‐coatings in an attempt to create a cell‐supporting environment in both two‐ and three‐dimensional cell culture systems. However, these protein coatings are typically deposited in a form which is structurally and functionally distinct from the ECM‐constituting fibrillar protein networks naturally deposited by cells. Here, the cell‐free and scalable synthesis of freely suspended and mechanically robust three‐dimensional (3D) networks of fibrillar fibronectin (fFn) supported by tessellated polymer scaffolds is reported. Hydrodynamically induced Fn fibrillogenesis at the three‐phase contact line between air, an Fn solution, and a tessellated scaffold microstructure yields extended protein networks. Importantly, engineered fFn networks promote cell invasion and proliferation, enable in vitro expansion of primary cancer cells, and induce an epithelial‐to‐mesenchymal transition in cancer cells. Engineered fFn networks support the formation of multicellular cancer structures cells from plural effusions of cancer patients. With further work, engineered fFn networks can have a transformative impact on fundamental cell studies, precision medicine, pharmaceutical testing, and pre‐clinical diagnostics.     

Memory accessibility and association of alcohol use and its positive outcomes.

Theoretical predictions involving memory associations between alcohol use and its positive outcomes were examined. In 2 preliminary studies, the availability from memory of outcomes from drinking was investigated. In subsequent studies, a measurement paradigm was used that decreased the accessibility of the association between drinking behavior and positive outcomes so that individual differences in drinking frequency might predict the accessibility of memory responses. In a final study, this paradigm was refined further by manipulating various types of alcohol-related cues. The results converge on a class of theory of memory association in which repeated drinking experiences influence the accessibility of thoughts about alcohol use and its most normatively frequent outcomes. The increased association of this behavior and its outcomes in memory has important implications for the cognitive mediation of alcohol use and abuse. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Family structure and age at menarche: A children-of-twins approach.

Girls who grow up in households with an unrelated adult male reach menarche earlier than peers, a finding hypothesized to be an evolutionary strategy for families under stress. The authors tested the alternative hypothesis that nonrandom selection into stepfathering due to shared environmental and/or genetic predispositions creates a spurious relation between stepfathering and early menarche. Using the unique controls for genetic and shared environmental experiences offered by the children-of-twins design, the authors found that cousins discordant for stepfathering did not differ in age of menarche. Moreover, controlling for mother's age of menarche eliminated differences in menarcheal age associated with stepfathering in unrelated girls. These findings strongly suggest selection, and not causation, accounts for the relationship between stepfathering and early menarche. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Indigenous Home Ownership and Community Title Land: A Preliminary Household Survey

Home ownership is currently not an option for Indigenous people living outside of urban centres. This article presents the results of a household survey conducted in four community settlements in Queensland revealing diverse understandings, aspirations and concerns for Indigenous home ownership. Discussion concludes that the development of a home ownership scheme will require rigorous policy analysis and innovative economic and cultural solutions.     

Preparation of functionalized poly(caprolactone diol)/castor oils blends to be applied as photocrosslinkable tissue adhesives

Polycaprolactone (PCL) and PCL-based materials are widely applied in the biomedical field, however, their slow biodegradation profile makes them more suitable to be used in hard tissues, where healing requires longer periods of time. In order to adjust their properties to suit for soft tissues applications, PCL can be blended with other biodegradable materials in order to tune its degradation rate. Herein, polymeric blends of PCL and castor oil (CO) were prepared after their chemical modification with 2-isocyanatoethylmethacrylate (IEMA) in order to be applied as photocrosslinkable tissue adhesives. These functionalized macromers were chemically characterized and used to prepare polymeric blends (PCL-IEMA/CO-IEMA) with variable mass proportions. A biocompatible photoinitiator (Irgacure 2959) was added to these macromers blends which were then irradiated under UV light. The feasibility of the prepared materials as tissue adhesives was evaluated by assessment of their chemical/physical properties as well as their interaction with blood. Moreover, their cytotoxic profile was also evaluated through in vitro studies using human dermal fibroblasts as model cells.     

Assessment of defects in as-grown III-V multilayer structures by differentiated cathodoluminescence topography (DCLT)

We report the use of a SEM in the differentiated cathodoluminescence mode to characterize as-grown LPE III-V multi-layer structures. To display the abilities of DCLT, a thorough comparison was made with anomalous transmission X-ray topography (TXRT). It is shown that DCLT reveals the same and in fact more defects than TXRT. The extra defects are growth related and their origin is investigated with EBIC, CL, chemical etching and TEM. Application of DCLT to GaAs-AlGaAs DH laser structures is shown to facilitate selection of wafer material.