Modeling of angioadaptation: insights for vascular development

Vascular beds are generated by vasculogenesis and sprouting angiogenesis, and these processes have strong stochastic components. As a result, vascular patterns exhibit significant heterogeneity with respect to the topological arrangement of the individual vessel segments and the characteristics (length, number of segments) of different arterio-venous pathways. This structural heterogeneity tends to cause heterogeneous distributions of flow and oxygen availability in tissue. However, these quantities must be maintained within tolerable ranges to allow normal tissue function. This is achieved largely through adjustment of vascular flow resistance by control of vessel diameters. While short-term diameter control by changes in vascular tone in arterioles and small arteries plays an important role, in the long term an even more important role is played by structural adaptation (angioadaptation), occurring in response to metabolic and hemodynamic signals. The effectiveness, stability and robustness of this angioadaptation depend sensitively on the nature and strength of the vascular responses involved and their interactions with the network structure. Mathematical models are helpful in understanding these complex interactions, and can be used to simulate the consequences of failures in sensing or signal transmission mechanisms. For the tumor microcirculation, this strategy of combining experimental observations with theoretical models, has led to the hypothesis that dysfunctional information transport via vascular connexins is a major cause of the observed vascular pathology and increased heterogeneity in oxygen distribution.     

Rejection of brood‐parasitic shiny cowbird Molothrus bonariensis nestlings by the firewood‐gatherer Anumbius annumbi?

Costs imposed by brood parasitic birds exert strong selection on their hosts to avoid parasitism. While egg rejection is a common defence, nestling rejection is rarer and less well understood. Theoretical models suggest that among non‐evicting parasites such as cowbirds nestling rejection can only evolve when levels of parasitism are high. Here we describe a possible case of early rejection of cowbird nestlings, by an infrequently parasitised host, the firewood‐gatherer Anumbius annumbi. Firewood‐gatherers accepted most shiny cowbird Molothrus bonariensis eggs despite clear differences in coloration. Cowbird eggs usually hatched 4–5 d before host eggs. All parasitic nestlings died within 48 h, and hosts continued their breeding attempts. Nestling death was most likely due to neglect since little food was found in the stomach of dead nestlings. Feeding neglect could be due to differences in visual or acoustic appearance between host and parasite hatchlings. Alternatively, hosts may refrain from feeding nestlings that hatch too early compared to their normal incubation time. At the moment our data do not allow distinction between active nestling recognition or cowbird nestling failure due to the unsuitability of the firewood‐gatherer as a host (i.e. too long incubation). Experiments are needed to tease these alternatives apart.     

Detection of monochlorobenzene metabolizing bacteria under anoxic conditions by DNA-stable isotope probing

Cultivation-independent analyses were applied to study the structural diversity of the bacterial community which developed in groundwater inoculated microcosms actively metabolizing monochlorobenzene (MCB) under anaerobic conditions. Addition of ¹³C-labelled MCB demonstrated that the community produced ¹³CO₂ as a metabolite at slightly increasing rates over a period of 1,051 days while no ¹³C-methane evolved. Genetic profiles of partial 16S rRNA genes generated with the single-strand conformation polymorphism (SSCP) technique by PCR from directly extracted total DNA revealed that, despite the long incubation period, six replicate microcosms were characterized by almost the same microbial members. Nine distinguishable contributors to the SSCP-profiles were characterized by DNA sequencing, revealing the presence of different members from the phyla Proteobacteria, Fibrobacteres and from the candidate division OD1. DNA-stable isotope probing (SIP) was applied to distinguish the actual MCB metabolizing bacteria from the other community members. This study reveals for the first time the structural diversity of an anaerobic MCB metabolizing bacterial community. However, it also demonstrates the limitations of SIP to detect bacteria slowly metabolizing carbon sources under anaerobic conditions.     

Novel estrogen-related genes and potential biomarkers of ovarian endometriosis identified by differential expression analysis

In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.     

Analysis of short-term changes in the Arabidopsis thaliana glycerolipidome in response to temperature and light

Although the influence of temperature, particularly cold, on lipid metabolism is well established, previous studies have focused on long-term responses and have largely ignored the influence of other interacting environmental factors. Here, we present a time-resolved analysis of the early responses of the glycerolipidome of Arabidopsis thaliana plants exposed to various temperatures (4, 21 and 32°C) and light intensities (darkness, 75, 150 and 400 μmol m(-2) s(-1)), including selected combinations. Using a UPLC/MS-based lipidomic platform, we reproducibly measured most glycerolipid species reported for Arabidopsis leaves, including the classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). In addition to known lipids, we have identified previously unobserved compounds, such as 36-C PGs and eukaryotic phospholipids containing 16:3 acyl chains. Occurrence of these lipid species implies the action of new biochemical mechanisms. Exposition of Arabidopsis plants to various light and temperature regimes results in two major effects. The first is the dependence of the saturation level of PC and MGDG pools on light intensity, likely arising from light regulation of de novo fatty acid synthesis. The second concerns an immediate decrease in unsaturated species of PG at high-temperature conditions (32°C), which could mark the first stages of adaptation to heat-stress conditions. Observed changes are discussed in the context of current knowledge, and new hypotheses have been formulated concerning the early stages of the plant response to changing light and temperature conditions.     

Geographic variation in otolith morphology among freshwater populations of Aphanius dispar (Teleostei,Cyprinodontiformes) from the southeastern Arabian Peninsula

Aphanius Nardo is a large genus of teleost fishes in the Old World, with 19 described species. Several of these species have only recently been recognized and additional species can be expected from isolated populations in remote areas. We show here that otolith morphology and statistical analyses of otolith variables can contribute to the detection of genetic differentiation in Aphanius. We studied samples of eight Aphanius dispar populations from the southeastern part of the Arabian Peninsula. Two populations originate from freshwater habitats far inland and probably have been isolated since the late Holocene some 4,000 years ago, three populations come from freshwater habitats with occasional connections to the sea, one population originates from a coastal site, and two were artificially introduced populations. The coastal population is interpreted as possessing the basic otolith type of A. dispar. The basic otolith type also occurs in fishes from the freshwater habitat which is located closest to the coast. Otoliths from the two other freshwater populations with occasional connections to the coast differ slightly from the basic type. However, the two populations from the long‐term isolated freshwater habitats far inland show distinct morphological changes. Our results are consistent with the hypotheses that i) otolith morphology is primarily genetically determined and is little influenced by physical parameters of the habitat, and ii) isolated A. dispar populations may be capable of evolving into new species within short periods of time. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Object-oriented image analysis for high content screening: detailed quantification of cells and sub cellular structures with the Cellenger software.

BACKGROUND: Detailed image analysis still is a considerable bottleneck for many cellular assays, and automated solutions to the problem are desirable. However, dealing with the complexity and variability of structures in cellular images makes detailed and reliable analysis a nontrivial task. METHODS: Therefore, based on the object-oriented image analysis approach, a novel image analysis technology, a flexible and reliable system for image analysis in cellular assays was developed. It contains a library of predefined, adaptable modules, each of them developed for a specific analysis task. The system can be configured easily by combining appropriate modules and adapting them interactively to the specific image data, if necessary. By representing cells and sub cellular structures within a network of interlinked image objects, a large number of parameters can be derived that describe shape, intensity, and relevant structural and relational aspects of any chosen class of structures. RESULTS: Thus, multi-parameter analysis and multiplexing are supported. A sample application based on this approach demonstrates that GFP signals can be distinguished based on their properties and the relative location within the cell.     

Pulmonary Hypoplasia Associated with Congenital Heart Diseases: A Fetal Study

Background

Abnormalities of the fetal pulmonary vasculature may affect lung morphogenesis. Postnatal studies have suggested that pulmonary hypoplasia (PH) may be associated with congenital heart diseases (CHDs).

Objective

To determine the prevalence of PH associated with CHDs, and to evaluate whether CHDs with right outflow obstruction were associated with the highest risk of lung growth impairment.

Methods

Between January 2006 and December 2010, fetuses with CHD obtained following the termination of pregnancies due to fetal abnormalities were examined in a prospective manner for the detection of heart and lung defects. CHDs were classified into five pathophysiological groups. Lung weight (LW), body weight (BW), and LW/BW ratio were analyzed for each case. The expression of CD31 and VEGF in the lung was evaluated by immunohistochemistry.

Results

Fetuses with CHDs and right outflow obstruction had significantly lower LW for a given BW, and significantly lower LW/BW ratios for a given gestational age. When defining PH as a fetal LW/BW ratio <0.015 before 28 weeks, and <0.012 after 28 weeks, PH was detected in 15 of the 119 fetuses analyzed (13%). It was significantly associated with CHD with right outflow obstruction, independently of chromosomal abnormalities and associated extracardiac abnormalities (p<0.03). Right outflow obstruction was detected in 60% of the fetuses with CHD and PH, but in only 32% of those with CHD but no PH. In fetuses with right outflow obstruction, no difference was observed between those with PH and those without PH, in terms of the ratio of pulmonary artery diameter to aortic diameter, lung CD31 expression, or lung VEGF expression.

Conclusion

CHDs with right outflow obstruction are a significant risk factor for prenatally acquired PH. The occurrence of fetal PH is not correlated with abnormalities of the pulmonary vasculature, suggesting the involvement of perfusion-independent mechanisms.     

LAMTOR2-Mediated Modulation of NGF/MAPK Activation Kinetics during Differentiation of PC12 Cells

LAMTOR2 (p14), a part of the larger LAMTOR/Ragulator complex, plays a crucial role in EGF-dependent activation of p42/44 mitogen-activated protein kinases (MAPK, ERK1/2). In this study, we investigated the role of LAMTOR2 in nerve growth factor (NGF)-mediated neuronal differentiation. Stimulation of PC12 (rat adrenal pheochromocytoma) cells with NGF is known to activate the MAPK. Pharmacological inhibition of MEK1 as well as siRNA–mediated knockdown of both p42 and p44 MAPK resulted in inhibition of neurite outgrowth. Contrary to expectations, siRNA–mediated knockdown of LAMTOR2 effectively augmented neurite formation and neurite length of PC12 cells. Ectopic expression of a siRNA-resistant LAMTOR2 ortholog reversed this phenotype back to wildtype levels, ruling out nonspecific off-target effects of this LAMTOR2 siRNA approach. Mechanistically, LAMTOR2 siRNA treatment significantly enhanced NGF-dependent MAPK activity, and this effect again was reversed upon expression of the siRNA-resistant LAMTOR2 ortholog. Studies of intracellular trafficking of the NGF receptor TrkA revealed a rapid colocalization with early endosomes, which was modulated by LAMTOR2 siRNA. Inhibition of LAMTOR2 and concomitant destabilization of the remaining members of the LAMTOR complex apparently leads to a faster release of the TrkA/MAPK signaling module and nuclear increase of activated MAPK. These results suggest a modulatory role of the MEK1 adapter protein LAMTOR2 in NGF-mediated MAPK activation required for induction of neurite outgrowth in PC12 cells.