Integration of wings and their eyespots in the speckled wood butterfly Pararge aegeria

We investigated both the phenotypic and developmental integration of eyespots on the fore- and hindwings of speckled wood butterflies Pararge aegeria. Eyespots develop within a framework of wing veins, which may not only separate eyespots developmentally, but may at the same time also integrate them by virtue of being both signalling sources and barriers during eyespot development. We therefore specifically investigated the interaction between wing venation patterns and eyespot integration. Phenotypic covariation among eyespots was very high, but only eyespots in neighbouring wing cells and in homologous wing cells on different wing surfaces were developmentally integrated. This can be explained by the fact that the wing cells of these eyespots share one or more wing veins. The wing venation patterns of fore- and hindwings were highly integrated, both phenotypically and developmentally. This did not affect overall developmental integration of the eyespots. The adaptive significance of integration patterns is discussed and more specifically we stress the need to conduct studies on phenotypic plasticity of integration.     

A handheld NASBA analyzer for the field detection and quantification of Karenia brevis

Blooms of Karenia brevis, the red tide forming dinoflagellate in the Gulf of Mexico, cause a myriad of ecological and economic problems for coastal communities, including massive fish and mammal mortalities, and damage to tourism and fisheries/shellfish harvesting industries. There is a need for accurate detection and prediction of K. brevis blooms, including rapid and inexpensive monitoring of both water and shellfish meats to ensure the safety of shellfish harvested for human consumption. To address this issue, we have developed a protocol for easy field extraction of cellular RNA from water samples and coupled it with a handheld nucleic acid sequence-based amplification (NASBA) sensor that amplifies and detects target mRNA specific to the rbcL gene of K. brevis. This extraction protocol is a modified version of the Qiagen RNeasy Mini Kit spin protocol and requires no specialized equipment or training. Once extracted, the RNA is amplified and detected by NASBA in an in-house designed and produced handheld sensor that provides a real-time fluorescence plotting of the amplification. Both the field RNA extraction protocol and the handheld NASBA analyzer compared favorably to laboratory-based technologies. In duplicate reactions, the amplification curves generated with the handheld detector closely mirrored the curves generated with the bench top Nuclisens EasyQ NASBA analyzer and there was no difference in the sensitivity obtained using the handheld device versus the bench top models. This extraction protocol and detection sensor will be a valuable tool for rapidly monitoring K. brevis in field environments.     

Neural progenitors, neurons and oligodendrocytes from human umbilical cord blood cells in a serum-free, feeder-free cell culture

We have previously demonstrated that lineage negative cells (Lin^neg) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone, muscle, endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin^neg UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin, neurofilament, A2B5 and Sox2) are expressed in Lin^neg cells cultured in FGF4, SCF, Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase, GalC, Olig2 and the late-stage marker MOSP are observed, as is the Schwann cell marker PMP22. In summary, Lin^neg UCB cells, when appropriately cultured, are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation.     

Pyrimidine-based antagonists of h-MCH-R1 derived from ATC0175: In vitro profiling and in vivo evaluation

A series of pyrimidine analogues derived from ATC0175 were potent antagonists of human MCH-R1 in vitro. Significantly improved receptor selectivity was achieved with several analogues from this series, but no improvement in brain partitioning was noted. One example from this series was shown to inhibit food intake and decrease body weight in a chronic study. However no clear correlation between the pharmacodynamic effect and the pharmacokinetic data with respect to brain concentration was discernible leading us to conclude that the observed effect was most likely not due to interaction with the MCH-R1.     

Endoplasmic reticulum stress-induced apoptosis in the development of diabetes: is there a role for adipose tissue and liver?

Diabetes mellitus (DM) is a multifactorial chronic metabolic disease characterized by hyperglycaemia. Several different mechanisms have been implicated in the development of the disease, including endoplasmic reticulum (ER) stress. ER stress is increasingly acknowledged as an important mechanism in the development of DM, not only for β-cell loss but also for insulin resistance. Accumulating evidence suggests that ER stress-induced apoptosis may be an important mode of β-cell loss and therefore important in the development of diabetes. Recent data also suggest a role of ER stress-induced apoptosis in liver and adipose tissue in relation to diabetes, but more extensive studies on human adipocyte and hepatocyte (patho)physiology and ER stress are needed to identify the exact interactions between environmental signals, ER stress and apoptosis in these organs.     

Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes

Background

Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD) on the expression stability of the following frequently used reference genes in brain and blood: beta-actin (b-actin), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine guanine phosphoribosyl transferase (HPRT).

Results

Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that b-actin, B2M, GAPDH and HPRT are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of brain derived neurotrophic factor (BDNF) and glycerol-3-phosphate dehydrogenase1 (GPD1) was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance.

Conclusion

This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results.     

Histone Deacetylase 3 Depletion in Osteo/Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat

Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.