Molecular signature of recent thymic selection events on effector and regulatory CD4+ T lymphocytes

Natural CD4+CD25+ regulatory T lymphocytes (Treg) are key protagonists in the induction and maintenance of peripheral T cell tolerance. Their thymic origin and biased repertoire continue to raise important questions about the signals that mediate their development. We validated analysis of MHC class II capture by developing thymocytes from thymic stroma as a tool to study quantitative and qualitative aspects of the cellular interactions involved in thymic T cell development and used it to analyze Treg differentiation in wild-type mice. Our data indicate that APCs of bone marrow origin, but, surprisingly and importantly, not thymic epithelial cells, induce significant negative selection among the very autoreactive Treg precursors. This fundamental difference between thymic development of regulatory and effector T lymphocytes leads to the development of a Treg repertoire enriched in cells specific for a selected subpopulation of self-Ags, i.e., those specifically expressed by thymic epithelial cells.     

SETAC Europe Young Scientist LCA award 2019 for P. James Joyce

The International Journal of Life Cycle Assessment - At the 29th SETAC Europe annual meeting, May 26–30, 2019, Helsinki (Finland), P. James Joyce received the SETAC Europe Young Scientist LCA...     

Adenoviral vector-directed expression of neurotrophin-3 in rat dorsal root ganglion explants results in a robust neurite outgrowth response

The neurotrophins are a family of proteins that promote neuronal survival and neurite outgrowth during development and can also enhance the regeneration of injured adult neurons. The local and continuous delivery of these proteins at the site of injury is problematic, since this requires repeated intraparenchymal injections or the use of invasive canula-micropump devices. In the present study we report the generation and characterization of an adenoviral vector for a member of the neurotrophins, neurotrophin-3 (Ad-NT-3). Using Ad-NT-3, we examined the expression and biological activity of NT-3 in dorsal root ganglia (DRG) explant cultures. Gene transfer with Ad-NT-3 results in the synthesis of genuine NT-3 and in a dosage-dependent neurite outgrowth response in DRG explants. Transduction of DRG explants with a viral vector dosage of 5 × 10⁵ to 5 × 10⁶ plaque-forming units induced the formation of a dense halo of neurites comparable to outgrowth observed following the addition of 100 ng/mL exogenous NT-3. In addition, a single infection with Ad-NT-3 produced biologically active NT-3 for at least 20 days in culture, as evidenced by continued neurite extension. This indicates that adenoviral vector-mediated expression of NT-3 results in high-level production of biologically active NT-3 and could therefore be used as a strategy to promote the regeneration of injured peripheral and central nerve projections. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 172–184, 1997     

Construction of repeat-free fluorescence in situ hybridization probes

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes.     

Temporin/VesCP (T/V)-like antibiotic peptides,derived from frogs and wasps,induce migration of human monocytes and neutrophils

Summary Several naturally occurring antimicrobial peptides, from mammals and insects, have previously been shown to be chemotactic for human inflammatory cells. Based on this evidence, ten synthetic analogs of naturally occurring antibiotic peptides from the skin secretions of three species of Ranid frogs and the venom of one species of Vespid wasp (i.e., T/V-like peptides) were tested for their abilities to induce migration of human neutrophils and monocytes. These included temporin A (TA fromRana temporaria), temporin 1P (T1P fromR. pipens), ranateurin 6 (Rana-6 fromR. catesbeiana)], three TA analogs [all D-amino acids (D-TA), reversed sequence (Rev-TA), and Pro3→Gly (G3-TA)], two frog skin-related T/V-like peptide consensus sequences (I4S10-Con and I4G10-Con), VesCP-M (VCP-M fromVespa mandarinia), and a hybrid peptide composed of portions of the insect antibiotic peptide, cecropin A (CA), and TA (CATA). TA, T1P, Rana-6, VCP-M, G3-TA, I4S10-Con, I4G10-Con, and CATA all induced cell migration at micromolar concentrations. D-TA and Rev-TA did not induce cell migration, suggesting that this process involves a chiral interaction, such as receptor binding, and also depends on the order of amino acids within TA. The results demonstrate, for the first time, that certain T/V-like antibiotic peptides are capable of inducing chemotaxis of human phagocytes and suggest that these peptides are multifunctional molecules with antimicrobial, hemolytic, and chemotactic capabilities.     

Dynamic Localization of Tat Protein Transport Machinery Components in Streptomyces coelicolor

The Tat pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the Streptomyces genus of bacteria. In this study, we have examined the localization of Tat components in the model organism Streptomyces coelicolor by constructing enhanced green fluorescent protein (eGFP) and mCherry fusions with the TatA, TatB, and TatC proteins. All three components colocalized dynamically in the vegetative hyphae, with foci of each tagged protein being prominent at the tips of emerging germ tubes and of the vegetative hyphae, suggesting that this may be a primary site of Tat secretion. Time-lapse imaging revealed that localization of the Tat components was highly dynamic during tip growth and again demonstrated a strong preference for apical sites in growing hyphae. During aerial hypha formation, TatA-eGFP and TatB-eGFP fusions relocalized to prespore compartments, indicating repositioning of Tat components during the Streptomyces life cycle.     

Learning and colonization of new niches: a first step toward speciation

Learning processes potentially play a role in speciation but are often ignored in speciation models. Learning may, for instance, play a role when a new niche is being colonized, because the learning of niche features may cause niche-specific assortative mating and a tendency to produce young in this niche. Several animal species learn about their environmental features that may be important in finding or attracting mates. We use a gene-culture coevolutionary model to look into the effect of such learning on the colonization of new niches and on the genetic divergence between groups using different niches, which are steps necessary in achieving speciation. We assume that density is regulated separately in each of the two niches and that the viability of an individual depends on its genotype as well as on which niche it exploits. Our results show that genetic adaptation to the new niche is enhanced by a high female fecundity and a low viability selection against heterozygotes. Furthermore, when initial colonization (without genetic adaptation) fails, genetic divergence is more difficult when the mating preference is stronger. In contrast, when colonization without genetic adaptation is successful, a stronger mating preference makes genetic divergence easier. An increase in the number of egg-laying mistakes by females can have a positive or negative effect on the success of genetic adaptation depending on other parameters. We show that genetic divergence can be prevented by a niche shift, which can occur only if viabilities in the two niches are asymmetrical.