Uncoupling of 3T3-L1 gene expression from lipid accumulation during adipogenesis

Adipocyte differentiation comprises altered gene expression and increased triglyceride storage. To investigate the interdependency of these two events, 3T3-L1 cells were differentiated in the presence of glucose or pyruvate. All adipocytic proteins examined were similarly increased between the two conditions. In contrast, 3T3-L1 adipocytes differentiated with glucose exhibited significant lipid accumulation, which was largely suppressed in the presence of pyruvate. Subsequent addition of glucose to the latter cells restored lipid accumulation and acute rates of insulin-stimulated lipogenesis. These data indicate that extracellular energy is required for induction of adipocytic proteins, while only glucose sustained the parallel increase in triglyceride storage.     

Taking neural crest stem cells to new heights

The carotid body is an organ of the peripheral nervous system that senses oxygen concentration in the blood and responds to changes by regulating breathing. Pardal et al. (2007) now report the discovery of carotid body stem cells, which proliferate in response to hypoxia and generate neurons that secrete dopamine. This new source of adult stem cells may be useful in therapies for treating Parkinson's disease.     

Identification of covalent modifications in P450 2E1 by 1,2-epoxy-3-butene in vitro

1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His(109), His(370)) are clearly associated with the active site, and that their Calpha atoms are located less than 9A from a known inhibitor binding site. In addition, the side chain of His(370) is within 4A of the heme group and its modification is expected to influence the orientation of the heme. The Calpha atom of Tyr(71) is within 14A of the potential inhibitor binding site and within 7A of the flap undergoing conformational change upon ligand binding, potentially placing Tyr(71) near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism.     

Skeletal growth in early and late Neolithic foragers from the Cis‐Baikal region of Eastern Siberia

Skeletal growth is explored between Early Neolithic (EN) (8000 to 6800 BP) and Late Neolithic (LN) (6000 to 5200 BP) foragers from the Cis‐Baikal region of Eastern Siberia. Previous studies suggest that increased systemic stress and smaller adult body size characterize the EN compared to LN. On this basis, greater evidence for stunting and wasting is expected in the EN compared to LN. Skeletal growth parameters assessed here include femoral and tibial lengths, estimated stature and body mass, femoral midshaft cortical thickness, total bone thickness, and medullary width. Forward selection was used to fit polynomial lines to each skeletal growth parameter relative to dental age in the pooled samples, and standardized residuals were compared between groups using t tests. Standardized residuals of body mass and femoral length were significantly lower in the EN compared to LN sample, particularly from late infancy through early adolescence. However, no significant differences in the standardized residuals for cortical thickness, medullary width, total bone thickness, tibial length, or stature were found between the groups. Age ranges for stunting in femoral length and wasting in body mass are consistent with environmental perturbations experienced at the cessation of breast feeding and general resource insecurity in the EN compared to LN sample. Differences in relative femoral but not tibial length may be associated with age‐specific variation in growth‐acceleration for the distal and proximal limb segments. Similarity in cortical bone growth between the two samples may reflect the combined influences of systemic and mechanical factors on this parameter. Am J Phys Anthropol 153:377–386, 2014. © 2013 Wiley Periodicals, Inc.     

Visualization and correction of automated segmentation,tracking and lineaging from 5-D stem cell image sequences

Background

Neural stem cells are motile and proliferative cells that undergo mitosis, dividing to produce daughter cells and ultimately generating differentiated neurons and glia. Understanding the mechanisms controlling neural stem cell proliferation and differentiation will play a key role in the emerging fields of regenerative medicine and cancer therapeutics. Stem cell studies in vitro from 2-D image data are well established. Visualizing and analyzing large three dimensional images of intact tissue is a challenging task. It becomes more difficult as the dimensionality of the image data increases to include time and additional fluorescence channels. There is a pressing need for 5-D image analysis and visualization tools to study cellular dynamics in the intact niche and to quantify the role that environmental factors play in determining cell fate.

Results

We present an application that integrates visualization and quantitative analysis of 5-D (x,y,z,t,channel) and large montage confocal fluorescence microscopy images. The image sequences show stem cells together with blood vessels, enabling quantification of the dynamic behaviors of stem cells in relation to their vascular niche, with applications in developmental and cancer biology. Our application automatically segments, tracks, and lineages the image sequence data and then allows the user to view and edit the results of automated algorithms in a stereoscopic 3-D window while simultaneously viewing the stem cell lineage tree in a 2-D window. Using the GPU to store and render the image sequence data enables a hybrid computational approach. An inference-based approach utilizing user-provided edits to automatically correct related mistakes executes interactively on the system CPU while the GPU handles 3-D visualization tasks.

Conclusions

By exploiting commodity computer gaming hardware, we have developed an application that can be run in the laboratory to facilitate rapid iteration through biological experiments. We combine unsupervised image analysis algorithms with an interactive visualization of the results. Our validation interface allows for each data set to be corrected to 100% accuracy, ensuring that downstream data analysis is accurate and verifiable. Our tool is the first to combine all of these aspects, leveraging the synergies obtained by utilizing validation information from stereo visualization to improve the low level image processing tasks.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-328) contains supplementary material, which is available to authorized users.     

Interaction of Amyloid Inhibitor Proteins with Amyloid Beta Peptides: Insight from Molecular Dynamics Simulations

Knowledge of the detailed mechanism by which proteins such as human αB- crystallin and human lysozyme inhibit amyloid beta (Aβ) peptide aggregation is crucial for designing treatment for Alzheimer''s disease. Thus, unconstrained, atomistic molecular dynamics simulations in explicit solvent have been performed to characterize the Aβ_17–42 assembly in presence of the αB-crystallin core domain and of lysozyme. Simulations reveal that both inhibitor proteins compete with inter-peptide interaction by binding to the peptides during the early stage of aggregation, which is consistent with their inhibitory action reported in experiments. However, the Aβ binding dynamics appear different for each inhibitor. The binding between crystallin and the peptide monomer, dominated by electrostatics, is relatively weak and transient due to the heterogeneous amino acid distribution of the inhibitor surface. The crystallin-bound Aβ oligomers are relatively long-lived, as they form more extensive contact surface with the inhibitor protein. In contrast, a high local density of arginines from lysozyme allows strong binding with Aβ peptide monomers, resulting in stable complexes. Our findings not only illustrate, in atomic detail, how the amyloid inhibitory mechanism of human αB-crystallin, a natural chaperone, is different from that of human lysozyme, but also may aid de novo design of amyloid inhibitors.     

The Niche Factor Syndecan-1 Regulates the Maintenance and Proliferation of Neural Progenitor Cells during Mammalian Cortical Development

Neural progenitor cells (NPCs) divide and differentiate in a precisely regulated manner over time to achieve the remarkable expansion and assembly of the layered mammalian cerebral cortex. Both intrinsic signaling pathways and environmental factors control the behavior of NPCs during cortical development. Heparan sulphate proteoglycans (HSPG) are critical environmental regulators that help modulate and integrate environmental cues and downstream intracellular signals. Syndecan-1 (Sdc1), a major transmembrane HSPG, is highly enriched in the early neural germinal zone, but its function in modulating NPC behavior and cortical development has not been explored. In this study we investigate the expression pattern and function of Sdc1 in the developing mouse cerebral cortex. We found that Sdc1 is highly expressed by cortical NPCs. Knockdown of Sdc1 in vivo by in utero electroporation reduces NPC proliferation and causes their premature differentiation, corroborated in isolated cells in vitro. We found that Sdc1 knockdown leads to reduced levels of β-catenin, indicating reduced canonical Wnt signaling. Consistent with this, GSK3β inhibition helps rescue the Sdc1 knockdown phenotype, partially restoring NPC number and proliferation. Moreover, exogenous Wnt protein promotes cortical NPC proliferation, but this is prevented by Sdc1 knockdown. Thus, Sdc1 in the germinal niche is a key HSPG regulating the maintenance and proliferation of NPCs during cortical neurogenesis, in part by modulating the ability of NPCs to respond to Wnt ligands.     

SCOREM: statistical consolidation of redundant expression measures

Many platforms for genome-wide analysis of gene expression contain ‘redundant’ measures for the same gene. For example, the most highly utilized platforms for gene expression microarrays, Affymetrix GeneChip® arrays, have as many as ten or more probe sets for some genes. Occasionally, individual probe sets for the same gene report different trends in expression across experimental conditions, a situation that must be resolved in order to accurately interpret the data. We developed an algorithm, SCOREM, for determining the level of agreement between such probe sets, utilizing a statistical test of concordance, Kendall''s W coefficient of concordance, and a graph-searching algorithm for the identification of concordant probe sets. We also present methods for consolidating concordant groups into a single value for its corresponding gene and for post hoc analysis of discordant groups. By combining statistical consolidation with sequence analysis, SCOREM possesses the unique ability to identify biologically meaningful discordant behaviors, including differing behaviors in alternate RNA isoforms and tissue-specific patterns of expression. When consolidating concordant behaviors, SCOREM outperforms other methods in detecting both differential expression and overrepresented functional categories.     

The welfare of growing pigs in five different production systems: assessment of feeding and housing

Ninety-one farms were visited over a 2-year period to assess the welfare of growing pigs in five different production systems found either in France or in Spain using the Welfare Quality® protocol. This study focused on animal-based measures as indicators of ‘good feeding’ and ‘good housing’. Multiple Generalized Linear Mixed Models were performed for each measure to evaluate the differences between production systems and to detect possible causal factors. Pigs in the conventional system presented the lowest prevalence of poor body condition, whereas extensive Mallorcan Black pigs and extensive Iberian pigs were associated with a decreased prevalence of bursitis and pig dirtiness. The straw-bedded system presented a lower prevalence of bursitis, but poorer hygiene and more susceptibility of poor body condition than the conventional system. The age of the animals had a significant effect on the appearance of bursitis in the three intensive systems studied. The type of floor was a significant causal factor of bursitis and pig dirtiness in the conventional system and among intensive Iberian pigs. The feeding system was another causal factor of pig dirtiness on more than 50% of the body in the conventional system, whereas pig dirtiness on less than 50% of the body was influenced by the age of the animals. The prevalence of huddling animals in the conventional system was associated with the highest stocking densities and the lowest environmental temperatures. The results indicate that there were important differences between production systems based on animal-based indicators of the good feeding and housing principles. The recording of the age of the animals, type of floor, feeding system, stocking density and environmental temperature can be useful to predict the appearance of a given welfare measure of ‘good housing’ on a farm.     

Variation in limb proportions between Jomon foragers and Yayoi agriculturalists from prehistoric Japan

Variation in limb proportions between prehistoric Jomon and Yayoi people of Japan are explored by this study. Jomon people were the descendents of Pleistocene nomads who migrated to the Japanese Islands around 30,000 yBP. Phenotypic and genotypic evidence indicates that Yayoi people were recent migrants to Japan from continental Northeast Asia who likely interbred with Jomon foragers. Limb proportions of Jomon and Yayoi people were compared using RMA regression and "Quick-Test" calculations to investigate relative variability between these two groups. Cluster and principal components analyses were performed on size-standardized limb lengths and used to compare Jomon and Yayoi people with other groups from various climatic zones. Elongated distal relative to proximal limb lengths were observed among Jomon compared to Yayoi people. Jomon limb proportions were similar to human groups from temperate/tropical climates at lower latitudes, while Yayoi limb proportions more closely resemble groups from colder climates at higher latitudes. Limb proportional similarities with groups from warmer environments among Jomon foragers likely reflect morphological changes following Pleistocene colonization of the Japanese Islands. Cold-derived limb proportions among the Yayoi people likely indicate retention of these traits following comparatively recent migrations to the Japanese Islands. Changes in limb proportions experienced by Jomon foragers and retention of cold-derived limb proportions among Yayoi people conform to previous findings that report changes in these proportions following long-standing evolution in a specific environment.