Nuclear staining and relative distance for quantifying epidermal differentiation in biomarker expression profiling

Background  

The epidermal physiology results from a complex regulated homeostasis of keratinocyte proliferation, differentiation and death and is tightly regulated by a specific protein expression during cellular maturation. Cellular in silico models are considered a promising and inevitable tool for the understanding of this complex system. Hence, we need to incorporate the information of the differentiation dependent protein expression in cell based systems biological models of tissue homeostasis. Such methods require measuring tissue differentiation quantitatively while correlating it with biomarker expression intensities.     

Remodelling of the angular collagen fiber distribution in cardiovascular tissues

Understanding collagen fiber remodelling is desired to optimize the mechanical conditioning protocols in tissue-engineering of load-bearing cardiovascular structures. Mathematical models offer strong possibilities to gain insight into the mechanisms and mechanical stimuli involved in these remodelling processes. In this study, a framework is proposed to investigate remodelling of angular collagen fiber distribution in cardiovascular tissues. A structurally based model for collagenous cardiovascular tissues is extended with remodelling laws for the collagen architecture, and the model is subsequently applied to the arterial wall and aortic valve. For the arterial wall, the model predicts the presence of two helically arranged families of collagen fibers. A branching, diverging hammock-type fiber architecture is predicted for the aortic valve. It is expected that the proposed model may be of great potential for the design of improved tissue engineering protocols and may give further insight into the pathophysiology of cardiovascular diseases.     

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.     

Repeatability and individual correlates of basal metabolic rate and total evaporative water loss in birds: a case study in European stonechats

Basal metabolic rate (BMR) and total evaporative water loss (TEWL) are thought to have evolved in conjunction with life history traits and are often assumed to be characteristic features of an animal. Physiological traits can show large intraindividual variation at short and long timescales, yet natural selection can only act on a trait if it is a characteristic feature of an individual. The repeatability of a trait, a measure of the portion of variance that is caused by differences among individuals, indicates if it is a characteristic feature of an individual. We measured repeatability of BMR and TEWL of 18 captive European stonechats (Saxicola torquata rubicola) within the winter season. Repeatability was 0.56 for BMR and 0.60 for mass-specific BMR. Age and body mass had a significant effect on variation in BMR. Also after accounting for this variation, BMR remained repeatable. TEWL and mass-specific TEWL showed nonsignificant repeatabilities of 0.11 and 0.12, respectively. We conclude that BMR is a characteristic feature of an individual in our population of European stonechats, whereas TEWL is not. We discuss our results in the context of a review of currently available estimates of repeatability of BMR and TEWL for birds.     

Dimerization of endogenous MT1-MMP is a regulatory step in the activation of the 72-kDa gelatinase MMP-2 on fibroblasts and fibrosarcoma cells

The secreted gelatinase matrix metalloprotease-2 (MMP-2) and the membrane-anchored matrix metalloprotease MT1-MMP (MMP-14), are central players in pericellular proteolysis in extracellular matrix degradation. In addition to possessing a direct collagenolytic and gelatinolytic activity, these enzymes take part in a cascade pathway in which MT1-MMP activates the MMP-2 proenzyme. This reaction occurs in an interplay with the matrix metalloprotease inhibitor, TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex with one TIMP-2 molecule. We provide positive evidence that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of fibroblasts and fibrosarcoma cells. Even in the absence of transfection and overexpression, dimerization of MT1-MMP markedly stimulated the formation of active MMP-2 products. The effect demonstrated here was brought about by a monoclonal antibody that binds specifically to MT1-MMP as shown by immunofluorescence experiments. The antibody has no effect on the catalytic activity. The effect on proMMP-2 activation involves MT1-MMP dimerization because it requires the divalent monoclonal antibody, with no effect obtained with monovalent Fab fragments. Since only a negligible level of proMMP-2 activation was obtained with MT1-MMP-expressing cells in the absence of dimerization, our results identify the dimerization event as a critical level of proteolytic cascade regulation.     

Oxygen isotope fractionation between human phosphate and water revisited

The oxygen isotope composition of human phosphatic tissues (δ¹⁸O_P) has great potential for reconstructing climate and population migration, but this technique has not been applied to early human evolution. To facilitate this application we analyzed δ¹⁸O_P values of modern human teeth collected at 12 sites located at latitudes ranging from 4°N to 70°N together with the corresponding oxygen composition of tap waters (δ¹⁸O_W) from these areas. In addition, the δ¹⁸O of some raw and boiled foods were determined and simple mass balance calculations were performed to investigate the impact of solid food consumption on the oxygen isotope composition of the total ingested water (drinking water + solid food water). The results, along with those from three, smaller published data sets, can be considered as random estimates of a unique δ¹⁸O_W/δ¹⁸O_P linear relationship: δ¹⁸O_W = 1.54(±0.09) × δ¹⁸O_P−33.72(±1.51) (R² = 0.87: p [H₀:R² = 0] = 2 × 10⁻¹⁹). The δ¹⁸O of cooked food is higher than that of the drinking water. As a consequence, in a modern diet the δ¹⁸O of ingested water is +1.05 to 1.2‰ higher than that of drinking water in the area. In meat-dominated and cereal-free diets, which may have been the diets of some of our early ancestors, the shift is a little higher and the application of the regression equation would slightly overestimate δ¹⁸O_W in these cases.     

The growth factor environment defines distinct pluripotent ground states in novel blastocyst-derived stem cells

Pluripotent stem cell lines can be derived from blastocyst embryos, which yield embryonic stem cell lines (ES cells), as well as the postimplantation epiblast, which gives rise to epiblast stem cell lines (EpiSCs). Remarkably, ES cells and EpiSCs display profound differences in the combination of growth factors that maintain their pluripotent state. Molecular and functional differences between these two stem cell types demonstrate that the tissue of origin and/or the growth factor milieu may be important determinants of the stem cell identity. We explored how developmental stage of the tissue of origin and culture growth factor conditions affect the stem cell pluripotent state. Our findings indicate that novel stem cell lines, with unique functional and molecular properties, can be generated from murine blastocyst embryos. We demonstrate that the culture growth factor environment and cell-cell interaction play a critical role in defining several unique and stable stem cell ground states.     

A protein domain-based interactome network for C. elegans early embryogenesis

Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or "interactome" networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early-embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.     

Do associated microbial abundances impact marine demosponge pumping rates and tissue densities?

The evolution of marine demosponges has led to two basic life strategies: one involving close associations with large and diverse communities of microorganisms, termed high microbial abundance (HMA) species, and one that is essentially devoid of associated microorganisms, termed low microbial abundance (LMA) species. This dichotomy has previously been suggested to correlate with morphological differences, with HMA species having a denser mesohyl and a more complex aquiferous systems composed of longer and narrower water canals that should necessitate slower seawater filtration rates. We measured mesohyl density for a variety of HMA and LMA sponges in the Florida Keys, and seawater pumping rates for a select group of these sponges using an in situ dye technique. HMA sponges were substantially denser than LMA species, and had per unit volume pumping rates 52–94% slower than the LMA sponges. These density and pumping rate differences suggest that evolutionary differences between HMA and LMA species may have resulted in profound morphological and physiological differences between the two groups. The LMA sponge body plan moves large quantities of water through their porous tissues allowing them to rapidly acquire the small particulate organic matter (POM) that supplies the majority of their nutritional needs. In contrast, the HMA sponge body plan is suited to host large and tightly packed communities of microorganisms and has an aquiferous system that increases contact time between seawater and the sponge/microbial consortium that feeds on POM, dissolved organic matter and the raw inorganic materials for chemolithotrophic sponge symbionts. The two evolutionary patterns represent different, but equally successful patterns and illustrate how associated microorganisms can potentially have substantial effects on host evolution. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sinapis phylogeny and evolution of glucosinolates and specific nitrile degrading enzymes

Levels of sinalbin (4-hydroxybenzylglucosinolate) and 28 other glucosinolates were determined in leaves and roots of 20 species that were either phylogenetically close to Sinapis alba, Sinapis arvensis, or Sinapis pubescens (tribe Brassiceae, Brassicaceae), or were expected to contain arylalkyl nitrilase activity. Comparison with a molecular phylogenetic tree based on ITS DNA sequences identified two separate occurrences of sinalbin. The first in a group of species related to S. alba (including members of the genera Coincya and Kremeriella); and the second in S. arvensis, nested among sinalbin deficient species. Significant 4-hydroxyphenylacetonitrile degrading enzyme activity was found in both S. alba and S. arvensis, but in S. alba the major product was the corresponding carboxylic acid, while in S. arvensis the major product was the amide. Both investigated enzyme activities, nitrilase and nitrile hydratase, were specific, accepting only certain arylacetonitriles such as 4-hydroxy and 4-methoxyphenylacetonitrile. Only the S. alba enzyme required an oxygen in para position of the substrate, as found in sinalbin. Indole-3-acetonitrile, arylcyanides, and arylpropionitriles were poor substrates. The nitrilase activity of S. alba was quantitatively comparable to that reported in the monocot Sorghum bicolor (believed to be involved in cyanogenic glycoside metabolism). Glucosinolates derived from methionine were found in all Sinapis clades. Glucosinolate patterns suggested a complex evolution of glucosinolates in the investigated species, with several apparent examples of abrupt changes in glucosinolate profiles including chain length variation and appearance of glucosinolates derived from branched-chain amino acids. NMR data for desulfated homosinalbin, 9-methylsulphonylnonylglucosinolate, 3-methylpentylglucosinolate and related glucosinolates are reported, and a facultative connection between sinalbin and specific nitrilases is suggested.