In the beginning: Generating neural crest cell diversity

Neural crest cells (NCCs) are migratory cells that delaminate from the neural tube early in development and then disseminate throughout the embryo to give rise to a wide variety of cell types that are key to the vertebrate body plan. During their journey from the neural tube to their peripheral targets, NCCs progressively differentiate, raising the question of when the fate of an individual NCC is sealed. One hypothesis suggests that the fate of a NCC is specified by target-derived signals emanating from the environment they migrate through, while another hypothesis proposes that NCCs are already specified to differentiate along select lineages at the time they are born in the neural tube, with environmental signals helping them to realize their prespecified fate potential. Alternatively, both mechanisms may cooperate to drive NCC diversity. This review highlights recent advances in our understanding of prespecification during trunk NCC development.Key words: neural crest cell, multipotent, prespecification, neuropilin, semaphorin, migration, cell fate     

Mitochondrial Complex II Prevents Hypoxic but Not Calcium- and Proapoptotic Bcl-2 Protein-induced Mitochondrial Membrane Potential Loss

Mitochondrial membrane potential loss has severe bioenergetic consequences and contributes to many human diseases including myocardial infarction, stroke, cancer, and neurodegeneration. However, despite its prominence and importance in cellular energy production, the basic mechanism whereby the mitochondrial membrane potential is established remains unclear. Our studies elucidate that complex II-driven electron flow is the primary means by which the mitochondrial membrane is polarized under hypoxic conditions and that lack of the complex II substrate succinate resulted in reversible membrane potential loss that could be restored rapidly by succinate supplementation. Inhibition of mitochondrial complex I and F₀F₁-ATP synthase induced mitochondrial depolarization that was independent of the mitochondrial permeability transition pore, Bcl-2 (B-cell lymphoma 2) family proteins, or high amplitude swelling and could not be reversed by succinate. Importantly, succinate metabolism under hypoxic conditions restores membrane potential and ATP levels. Furthermore, a reliance on complex II-mediated electron flow allows cells from mitochondrial disease patients devoid of a functional complex I to maintain a mitochondrial membrane potential that conveys both a mitochondrial structure and the ability to sequester agonist-induced calcium similar to that of normal cells. This finding is important as it sets the stage for complex II functional preservation as an attractive therapy to maintain mitochondrial function during hypoxia.     

Most Personal Exposure to House Dust Mite Aeroallergen Occurs during the Day

Background

The bed is commonly regarded as the main site of house dust mite exposure; however this has not been directly established by continuous measurements. The objective of this study was to determine the pattern of personal exposure to mite aeroallergen over 24 hours.

Methods

12 adults each collected 9 sequential samples (8 during the day, mean 115 mins, and one overnight, mean 514 mins) over 24 hours using a portable air-pump (2L/min) connected to an IOM filter located on the shoulder during the day and on the bed head overnight. Samples were analysed for mite allergen Der p 1 by ELISA. Location and activity were recorded. A mixed model analysis was performed to determine exposure as a function of 14 categories of activity.

Results

Personal aeroallergen exposure differed widely over time, both within and between subjects. The highest average exposure (1117 pg/m³, 95% CI: 289-4314) occurred on public transport and the lowest overnight in bed (45 pg/m³, 95% CI: 17-17), which contributed only 9.8% (95% CI: 4.4%-15.1%) of total daily exposure. Aeroallergens were not related to bed reservoirs.

Conclusion

The study challenges the current paradigm that the bed is the main site of HDM exposure and instead suggests most exposure occurs in association with domestic activity and proximity to other people. Effective mite interventions, designed to improve asthma outcomes, need to first identify and then address the multiple sources of aeroallergen exposure.     

The cytoplasmic domain of neuropilin 1 is dispensable for angiogenesis, but promotes the spatial separation of retinal arteries and veins

Neuropilin 1 (NRP1) is a transmembrane glycoprotein that is essential for blood vessel development in vertebrates. Best known for its ability to bind members of the vascular endothelial growth factor (VEGF) and class 3 semaphorin families through its extracellular domain, it also has a highly conserved cytoplasmic domain, which terminates in a SEA motif that binds the PDZ protein synectin/GIPC1/NIP. Previous studies in zebrafish embryos and tissue culture models raised the possibility that the SEA motif of NRP1 is essential for angiogenesis. Here, we describe the generation of mice that express a form of NRP1 that lacks the cytoplasmic domain and, therefore, the SEA motif (Nrp1(cyto)(Δ)(/)(Δ) mice). Our analysis of pre- and perinatal vascular development revealed that vasculogenesis and angiogenesis proceed normally in these mutants, demonstrating that the membrane-anchored extracellular domain is sufficient for vessel growth. By contrast, the NRP1 cytoplasmic domain is required for normal arteriovenous patterning, because arteries and veins crossed each other at an abnormally high frequency in the Nrp1(cyto)(Δ)(/)(Δ) retina, as previously reported for mice with haploinsufficient expression of VEGF in neural progenitors. At crossing sites, the artery was positioned anteriorly to the vein, and both vessels were embedded in a shared collagen sleeve. In human eyes, similar arteriovenous crossings are risk factors for branch retinal vein occlusion (BRVO), an eye disease in which compression of the vein by the artery disrupts retinal blood flow, causing local tissue hypoxia and impairing vision. Nrp1(cyto)(Δ)(/)(Δ) mice may therefore provide a suitable genetic model to study the aetiology of BRVO.     

Diverse roles for VEGF-A in the nervous system

Vascular endothelial growth factor A (VEGF-A) is best known for its essential roles in blood vessel growth. However, evidence has emerged that VEGF-A also promotes a wide range of neuronal functions, both in vitro and in vivo, including neurogenesis, neuronal migration, neuronal survival and axon guidance. Recent studies have employed mouse models to distinguish the direct effects of VEGF on neurons from its indirect, vessel-mediated effects. Ultimately, refining our knowledge of VEGF signalling pathways in neurons should help us to understand how the current use of therapeutics targeting the VEGF pathway in cancer and eye disease might be expanded to promote neuronal health and nerve repair.     

In Situ Activity of Suspended and Immobilized Microbial Communities as Measured by Fluorescence Lifetime Imaging

In this study, the feasibility of fluorescence lifetime imaging (FLIM) for measurement of RNA:DNA ratios in microorganisms was assessed. The fluorescence lifetime of a nucleic acid-specific probe (SYTO 13) was used to directly measure the RNA:DNA ratio inside living bacterial cells. In vitro, SYTO 13 showed shorter fluorescence lifetimes in DNA solutions than in RNA solutions. Growth experiments with bacterial monocultures were performed in liquid media. The results demonstrated the suitability of SYTO 13 for measuring the growth-phase-dependent RNA:DNA ratio in Escherichia coli cells. The fluorescence lifetime of SYTO 13 reflected the known changes of the RNA:DNA ratio in microbial cells during different growth phases. As a result, the growth rate of E. coli cells strongly correlated with the fluorescence lifetime. Finally, the fluorescence lifetimes of SYTO 13 in slow- and fast-growing biofilms were compared. For this purpose, biofilms developed from activated sludge were grown as autotrophic and heterotrophic communities. The FLIM data clearly showed a longer fluorescence lifetime for the fast-growing heterotrophic biofilms and a shorter fluorescence lifetime for the slow-growing autotrophic biofilms. Furthermore, starved biofilms showed shorter lifetimes than biofilms supplied with glucose, indicating a lower RNA:DNA ratio in starved biofilms. It is suggested that FLIM in combination with SYTO 13 represents a useful tool for the in situ differentiation of active and inactive bacteria. The technique does not require radioactive chemicals and may be applied to a broad range of sample types, including suspended and immobilized microorganisms.     

Design and synthesis of a novel series of 4-heteroarylamino-1′-azaspiro[oxazole-5,3′-bicyclo[2.2.2]octanes as α7 nicotinic receptor agonists 2. Development of 4-heteroaryl SAR

Quinuclidine-containing spirooxazolines, as described in the previous report in this series, were demonstrated to have utility as α7 nicotinic acetylcholine receptor (α7 nAChR) partial agonists. In this work, the SAR of this chemotype was expanded to include an array of diazine heterocyclic substitutions. Many of the heterocyclic analogs were potent partial agonists of the α7 receptor, selective against other nicotinic receptors and the serotinergic 5HT_3A receptor. (1′S,3′R,4′S)-N-(6-phenylpyrimidin-4-yl)-4H-1′-azaspiro[oxazole-5,3′-bicyclo[2.2.2]octan]-2-amine, a potent and selective α7 nAChR partial agonist, was demonstrated to improve cognition in the mouse novel object recognition (NOR) model of episodic memory.     

Disparity between levels of in vitro neutralization of vaccinia virus by antibody to the A27 protein and protection of mice against intranasal challenge

Immunization with recombinant proteins may provide a safer alternative to live vaccinia virus for prophylaxis of poxvirus infections. Although antibody protects against vaccinia virus infection, the mechanism is not understood and the selection of immunogens is daunting as there are dozens of surface proteins and two infectious forms known as the mature virion (MV) and the enveloped virion (EV). Our previous studies showed that mice immunized with soluble forms of EV membrane proteins A33 and B5 and MV membrane protein L1 or passively immunized with antibodies to these proteins survived an intranasal challenge with vaccinia virus. The present study compared MV protein A27, which has a role in virus attachment to glycosaminoglycans on the cell surface, to L1 with respect to immunogenicity and protection. Although mice developed similar levels of neutralizing antibody after immunizations with A27 or L1, A27-immunized mice exhibited more severe disease upon an intranasal challenge with vaccinia virus. In addition, mice immunized with A27 and A33 were not as well protected as mice receiving L1 and A33. Polyclonal rabbit anti-A27 and anti-L1 IgG had equivalent MV-neutralizing activities when measured by the prevention of infection of human or mouse cells or cells deficient in glycosaminoglycans or by adding antibody prior to or after virus adsorption. Nevertheless, the passive administration of antibody to A27 was poorly protective compared to the antibody to L1. These studies raise questions regarding the basis for antibody protection against poxvirus disease and highlight the importance of animal models for the early evaluation of vaccine candidates.