Retinoic acid directs neuronal differentiation of human pluripotent stem cell lines in a non-cell-autonomous manner

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3β activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.     

Tracing estuarine organic matter sources into the southern North Sea using C and N isotopic signatures

Sources and distribution of particulate organic matter in surface waters of the Humber and Thames estuaries and in the East Anglian plume in the southern North Sea were investigated in winter 2006/2007. Carbon (C) and nitrogen (N) stable isotopes provided evidence for the presence of three particulate organic matter sources; riverine plankton (δ¹³C ?30 ‰ and δ¹⁵N 7.9 ‰) identified in the Thames estuary only, marine plankton (average δ¹³C ?21.4 ‰ and δ¹⁵N 4.5 ‰) and a third source with an enriched ¹³C signature (>?16.7 ‰) and elevated C:N ratio (>12.7). Particulate organic matter with enriched ¹³C values were observed throughout the Humber estuary and at the marine end-member of the Thames estuary. While bacterial cycling of organic carbon undoubtedly takes place within these estuaries, these processes on their own are unlikely to account for the isotopic signatures seen. The ¹³C enriched organic matter source is suggested to be due to particulate organic matter input from marsh plants and seagrasses such as Spartina spp. and Zostera on the adjacent salt marshes and mudflats and/or macroalgae along the banks of the estuaries. This ¹³C enriched signal was also identified approximately 50 km offshore within the southern North Sea, in the East Anglian plume, which transports UK riverine water off-shore in a discrete plume. This plume therefore provides a mechanism to transport this estuarine derived organic matter pool offshore out of the estuaries. These results indicate that estuarine derived organic matter from marsh plants, seagrasses and/or macroalgae contributes to the southern North Sea organic matter pool and is therefore likely to contribute to winter-time shelf sea carbon and nitrogen cycles.     

Conjugation of lipid and CpG-containing oligonucleotide yields an efficient method for liposome incorporation

For optimal stimulation of T cells, protein-based vaccines must deliver protein antigens to antigen-presenting cells while simultaneously providing immunostimulatory signals. Listeriolysin O (LLO)-containing liposomes have been utilized to efficiently deliver protein antigens to the cytosolic pathway for antigen processing and major histocompatibility complex class I-dependent presentation while codelivering immunostimulatory CpG-oligodeoxyribonuceotides (ODNs). In this report, we describe the synthesis of lipid-CpG-ODN conjugates utilizing maleimide-phosphatidylethanolamine (PE) lipids and 5'-sulfhdryl-containing CpG-ODNs as a method for facile incorporation of CpG-ODNs in liposomal vaccine carriers, an alternative to co-encapsulation inside liposomes and as a means to enhance delivery of CpG-ODNs to their major receptor, Toll-like receptor 9 (TLR9), in the endosome. The characterization and biological evaluation of the vaccine delivery system made of liposomes, which contain the lipid-CpG-ODN conjugates inserted in the liposomal membrane, is described. We demonstrate in vitro in bone marrow derived macrophages that the lipid-CpG-ODN conjugates incorporated onto the liposome bilayers interact with their receptor TLR9 as readily as liposome-encapsulated ODNs and exert their immunostimulatory capabilities. The liposomal vaccine delivery systems were evaluated in mice using ovalbumin (OVA) as a model antigen, and the results indicate equally robust OVA-specific cytotoxic T lymphocyte responses and similar Th1 immune skewing capabilities between liposomes containing lipid-conjugated or encapsulated CpG-ODNs. Overall, this work indicates that conjugating PE lipids and CpG-ODNs results in an efficient method that allows facile incorporation of CpG-ODNs into a liposome-based delivery platform while retaining the immune-stimulating capabilities of CpG-ODNs.     

A Role for the Kp Leucine Zipper in Regulating P Element Transposition in Drosophila Melanogaster

The KP element can repress P element mobility in Drosophila melanogaster. Three mutant KP elements were made that had either two amino acid substitutions or a single amino acid deletion in the putative leucine zipper domain found in the KP polypeptide. Each KP element was expressed from the actin 5C proximal promoter. The wild-type control construct strongly repressed P element mobility, measured by the GD sterility and sn(w) mutability assays, in a position-independent manner. The single amino acid deletion mutant failed to repress P mobility regardless of its insertion site, while repression of P element mobility by the double amino acid substitution mutants was position dependent. The results show that the leucine zipper of the KP polypeptide is important for P element regulation. This supports the multimer-poisoning model of P element repression, because leucine zipper motifs are involved in protein-protein interactions.     

Burying Beetle Larvae Discriminate Between Individual Parents and Between Some Classes of Adults

Offspring begging can be triggered by a variety of acoustic, visual or chemical cues from the parents. In many birds, nestlings use information derived from these cues to discriminate between individual parents or different classes of adults. Although begging occurs in some insects, we know very little about discrimination between adults by insect larvae. Here, we examine whether begging larvae in the burying beetle Nicrophorus vespilloides can discriminate between individual parents or different classes of adults. We found that larvae showed no discrimination between male and female beetles, but that they begged more towards breeding beetles than towards non‐breeding ones. These results were robust regardless of whether larvae had been reared in presence or absence of adult beetles, thus suggesting that larval discrimination is based on an innate template that requires no prior exposure to adult beetles. We also found that larvae begged more towards unfamiliar beetles than towards familiar ones, suggesting that they can learn to discriminate between individual parents based on cues about familiarity. We conclude that insect larvae may benefit from discriminating between different classes of adult beetles, as it allows them to lower the costs associated with begging in response to irrelevant environmental cues (costly in terms of wasted effort) and with not begging in response to the presence of caring parents (costly in terms of lost feeding opportunities).     

The interaction of the upper airway and thermo-metabolism on respiratory rhythm during non-REM sleep in the developing lamb.

This study investigates the role of metabolic rate and of vagal airway mechanisms in sustaining rhythmic breathing in the developing lamb. Fifteen lambs were prepared, at 2 days of age under fluothane anaesthesia, for sequential studies at 4, 14, 30, 45, and 55 days of age. At each age they were maintained at an ambient temperature of 5, 10, 15, 20, 25 and 30 degrees C for at least one hour before measurements were made during N-REM sleep. In 6 lambs at 4 days and in all lambs at older ages the upper airway was by-passed (by opening a tracheal window) for 10-15 minutes at each ambient temperature. Oxygen consumption was unaffected by upper airway by-pass and there were no consistent changes in mean breathing frequency or amplitude, with the exception of shifts to panting at warm ambient temperatures. Breathing pattern was unaffected by upper airway by-pass in lambs at 4 days of age, but at older ages loss of regularity of breathing frequently occurred (up to 47% of 30 days-old lambs at 25 degrees C). This was related to the fall in oxygen consumption with age and to basal values at thermoneutrality, and coincided with lower respiratory rates and increased use of expiratory laryngeal braking. Periodic breathing (and apnea) of a fixed cycle length (9.3 +/- 0.36 s) was a common feature (62%) of the observed breathing dysrhythmia. In young lambs high metabolic rate sustained high frequency rhythmic breathing which was unaffected by upper airway by-pass.(ABSTRACT TRUNCATED AT 250 WORDS)