Novel insights into relationships between egg corticosterone and timing of breeding revealed by LC‐MS/MS

Inter‐ and intra‐clutch variation in egg corticosterone (CORT), the major glucocorticoid in birds, may provide insights into how maternal stress levels vary with the timing of breeding and with laying order. Common analytical methods (e.g. immunoassays), however, suffer from cross‐reaction with other steroids, leading to potential overestimation of CORT concentrations which can obscure true hormone–environment relationships and complicate among‐study comparisons. We here apply a new LC‐MS/MS technique, which has recently been shown to avoid the problem of cross‐reactivity due to its high specificity, to quantify CORT concentrations in yolk and albumen in clutches of lesser black‐backed gulls Larus fuscus. We found that CORT concentration exhibited a previously unreported U‐shaped relationship with time of breeding, which we explain as a potential interplay of two forces exerting extra strain on the early and late breeders. Furthermore, results showed an increase in CORT with laying order indicating the energetic expense of egg production. The levels of CORT assessed in this study were significantly lower than those previously reported in studies using immunoassays for CORT analysis. This supports the fact that incorporating chromatography effectively reduces overestimation of CORT due to cross‐reactivity with other steroid hormones, particularly in egg yolk.     

Gene expression profiling revealed specific spermatogonial stem cell genes in mouse

Mammalian spermatogenesis originates from spermatogonial stem cells (SSCs), which undergo mitosis, meiosis and spermiogenesis in order to generate mature spermatozoa. SSCs are adult stem cells that can both self‐renew and differentiate. To maintain pluripotency, SSCs are regulated by both extrinsic factors secreted from surrounding somatic cells and intrinsic factors including specific gene expression programs. Using fluorescent labeled germ line stem cells, mouse gonocytes and SSCs were purified up to 97% by improved FACS method. Through microarray analyses, global gene expression profiles of gonocytes, SSCs, and differentiated cells were compared. A large number of distinctive genes were found to be enriched in respective cell populations, indicating different functional requirements of each cell type. Functional clustering analyses revealed that while gonocytes and SSCs preferentially express genes implicated in gene expression regulation and epigenetic modifications, differentiated cells including somatic cells are enriched with genes encoding proteins involved in various cellular activities. Further in situ hybridization and RT‐PCR experiments confirmed SSC specific expression of several genes of which functions have not been characterized in SSCs. The comparative gene expression profiling provides a useful resource for gene discovery in relation to SSC regulation and opens new avenues for the study of molecular mechanisms underlying SSC self‐renewal and differentiation. genesis 51:83–96, 2013. © 2012 Wiley Periodicals, Inc.     

Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast

Haploinsufficiency is defined as a dominant phenotype in diploid organisms that are heterozygous for a loss-of-function allele. Despite its relevance to human disease, neither the extent of haploinsufficiency nor its precise molecular mechanisms are well understood. We used the complete set of Saccharomyces cerevisiae heterozygous deletion strains to survey the genome for haploinsufficiency via fitness profiling in rich (YPD) and minimal media to identify all genes that confer a haploinsufficient growth defect. This assay revealed that approximately 3% of all approximately 5900 genes tested are haploinsufficient for growth in YPD. This class of genes is functionally enriched for metabolic processes carried out by molecular complexes such as the ribosome. Much of the haploinsufficiency in YPD is alleviated by slowing the growth rate of each strain in minimal media, suggesting that certain gene products are rate limiting for growth only in YPD. Overall, our results suggest that the primary mechanism of haploinsufficiency in yeast is due to insufficient protein production. We discuss the relevance of our findings in yeast to human haploinsufficiency disorders.     

Differential endocrine regulation of genes enriched in initial segment and distal caput of the mouse epididymis as revealed by genome-wide expression profiling

We have performed genome-wide expression profiling of endocrine regulation of genes expressed in the mouse initial segment (IS) and distal caput of the epididymis by using Affymetrix microarrays. The data revealed that of the 15 020 genes expressed in the epididymis, 35% were enriched in one of the two regions studied, indicating that differential functions can be attributed to the IS and the more distal caput regions. The data, furthermore, showed that 27% of the genes expressed in the IS and/or distal caput epididymidis are under the regulation of testicular factors present in the duct fluid, while bloodborne androgens can regulate for 14% of them. This is in line with the high testis dependency of epididymal physiology. We then focused on genes with moderate or strong expression, showing strict segment enrichment and strong dependency on testicular factors. Analyses of the 59 genes, including upregulated and downregulated genes, fulfilling the criteria indicated that the expression of 18 (17 downregulated genes; 1 upregulated gene) of 19 gonadectomy-responsive genes enriched in the IS was not maintained by the androgen treatment, whereas the expression of all six downregulated genes enriched in the distal caput and the majority of those with no strict segment enrichment of expression (28 of 34; consisting of 23 downregulated and 5 upregulated genes) were maintained by androgens. Hence, it is evident that testicular factors other than androgens are important for the expression of IS-enriched genes, whereas the expression of distal caput-enriched genes is typically regulated by androgens. Identical data were obtained by independent clustering analyses performed for the expression data of 3626 epididymal genes. Several novel genes with putative involvement in epididymal sperm maturation, such as a disintegrin and metallopeptidase domain 28 (Adam28) and a solute carrier organic anion transporter family, member 4C1 (Slco4c1), were identified, indicating that this approach is successful for identifying novel epididymal genes.     

Toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy

Dendritic cells (DCs) are central players of the immune response. To date, DC-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients, predominantly with ex vivo-cultured monocyte-derived DCs (moDCs). However, the extensive culture period and compounds required to differentiate them into DCs may negatively affect their immunological potential. Therefore, it is attractive to consider alternative DC sources, such as blood DCs. Two major types of naturally occurring DCs circulate in peripheral blood, myeloid DCs (mDCs) and plasmacytoid (pDCs). These DC subsets express different surface molecules and are suggested to have distinct functions. Besides scavenging pathogens and presenting antigens, DCs secrete cytokines, all of which is vital for both the acquired and the innate immune system. These immunological functions relate to Toll-like receptors (TLRs) expressed by DCs. TLRs recognize pathogen-derived products and subsequently provoke DC maturation, antigen presentation and cytokine secretion. However, not every TLR is expressed on each DC subset nor causes the same effects when activated. Considering the large amount of clinical trials using DC-based immunotherapy for cancer patients and the decisive role of TLRs in DC maturation, this review summarizes TLR expression in different DC subsets in relation to their function. Emphasis will be given to the therapeutic potential of TLR-matured DC subsets for DC-based immunotherapy.     

Novel insights into the calcium action in cherry fruit development revealed by high-throughput mapping

Plant Molecular Biology - This work provides the first system-wide datasets concerning metabolic changes in calcium-treated fruits, which reveal that exogenously applied calcium may specifically...     

Differential regulation of human blood dendritic cell subsets by IFNs

Based on the relative expression of CD11c and CD1a, we previously identified subsets of dendritic cells (DCs) or DC precursors in human peripheral blood. A CD1a(+)/CD11c(+) population (CD11c(+) DCs), also called myeloid DCs, is an immediate precursor of Langerhans cells, whereas a CD1a(-)/CD11c(-) population (CD11c(-) DCs), sometimes called lymphoid DCs but better known as plasmacytoid DCs, is composed of type I IFN (IFN-alpha beta)-producing cells. Here, we investigate the effects of IFN-alpha beta and IFN-gamma as well as other cytokines on CD11c(+) and CD11c(-) DC subsets, directly isolated from the peripheral blood, instead of in vitro-generated DCs. IFN-gamma and IFN-alpha, rather than GM-CSF, were the most potent cytokines for enhancing the maturation of CD11c(+) DCs. Incubation of CD11c(+) DCs with IFN-gamma also resulted in increased IL-12 production, and this IL-12 allowed DCs to increase Th1 responses by alloreactive T cells. In contrast, IFN-alpha did not induce IL-12 but, rather, augmented IL-10 production. IFN-alpha-primed matured CD11c(+) DCs induced IL-10-producing regulatory T cells; however, this process was independent of the DC-derived IL-10. On the other hand, IFN-alpha by itself neither matured CD11c(-) DCs nor altered the polarization of responding T cells, although this cytokine was a potent survival factor for CD11c(-) DCs. Unlike IFN-alpha, IL-3 was a potent survival factor and induced the maturation of CD11c(-) DCs. The IL-3-primed CD11c(-) DCs activated T cells to produce IL-10, IFN-gamma, and IL-4. Thus, CD11c(+) and CD11c(-) DC subsets play distinct roles in the cytokine network, especially their responses to IFNs.     

Relationships among murine CD11c(high) dendritic cell subsets as revealed by baseline gene expression patterns

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11c(high) DC subsets. CD4(+), CD8alpha(+), and CD8alpha(-) CD4(-) (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4(+) and CD8alpha(+) DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4(+) DC, differing from them by <25 genes. This suggests that CD4(+) and DN DC are closely related phylogenetically, whereas CD8alpha(+) DC represent a more distant lineage, supporting the historical distinction between CD8alpha(+) and CD8alpha(-) DC. However, staining patterns revealed that in contrast to CD4(+) DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11c(high) DC subsets that to date have been difficult to define as rigid distinctions.     

Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.     

Common gene expression strategies revealed by genome-wide analysis in yeast

Background  

Gene expression is a two-step synthesis process that ends with the necessary amount of each protein required to perform its function. Since the protein is the final product, the main focus of gene regulation should be centered on it. However, because mRNA is an intermediate step and the amounts of both mRNA and protein are controlled by their synthesis and degradation rates, the desired amount of protein can be achieved following different strategies.     

Novel insights into the phylogenetic relationships of the endangered marsupial genus Potorous

The three extant potoroo species of the marsupial genus Potorous -Potorous tridactylus, P. longipes and P. gilbertii - are all of conservation concern due to introduced predators and habitat loss associated with the European settlement of Australia. Robust phylogenies can be useful to inform conservation management, but past phylogenetic studies on potoroos have been unable to fully resolve relationships within the genus. Here, a multi-locus approach was employed, using three mitochondrial DNA (mtDNA): NADH dehydrogenase subunit 2, cytochrome c oxidase subunit 1 and 12S rRNA and four nuclear DNA (nuDNA) gene regions: breast and ovarian cancer susceptibility gene, recombination activating gene-1, apolipoprotein B and omega globin. This was coupled with widespread geographic sampling of the broadly distributed P. tridactylus, to investigate the phylogenetic relationships within this genus. Analyses of the mtDNA identified five distinct and highly divergent lineages including, P. longipes, P. gilbertii and three distinct lineages within P. tridactylus (northern mainland, southern mainland and Tasmanian). P. tridactylus was paraphyletic with the P. gilbertii lineage, suggesting that cryptic taxa may exist within P. tridactylus. NuDNA sequences lacked the resolution of mtDNA. Although they resolved the three currently recognised species, they were unable to differentiate lineages within P. tridactylus. Current management of P. tridactylus as two sub-species (mainland and Tasmania) does not recognise the full scope of genetic diversity within this species, especially that of the mainland populations. Until data from more informative nuDNA markers are available, we recommend this species be managed as the following three subspecies: Potorous tridactylus tridactylus (southern Queensland and northern New South Wales); Potorous tridactylus trisulcatus (southern New South Wales and Victoria) Potorous tridactylus apicalis (Tasmania). Molecular dating estimated that divergences within Potorous occurred in the late Miocene through to the early Pliocene.     

Heterogeneity of respiratory dendritic cell subsets and lymphocyte populations in inbred mouse strains

Background

Inbred mouse strains are used in different models of respiratory diseases but the variation of critical respiratory leukocyte subpopulations across different strains is unknown.

Methods

By using multiparameter flow cytometry we have quantitated respiratory leukocyte subsets including dendritic cells subpopulations, macrophages, classical T and B cells, natural killer cells, γδTCR+ T cells and lineage-negative leukocytes in the five most common inbred mouse strains BALB/c, C57BL/6, DBA/2, 129SV and C3H. To minimize confounding environmental factors, age-matched animals were received from the same provider and were housed under identical specific-pathogen-free conditions.

Results

Results revealed significant strain differences with respect to respiratory neutrophils (p=0.005; up to 1.4 fold differences versus C57BL/6 mice), eosinophils (p=0.029; up to 2.7 fold), certain dendritic cell subsets (p≤0.0003; up to 3.4 fold), T (p<0.001; up to 1.6 fold) and B lymphocyte subsets (p=0.005; up to 0.4 fold), γδ T lymphocytes (p=0.003; up to 1.6 fold), natural killer cells (p<0.0001; up to 0.6 fold) and lineage-negative innate leukocytes (p≤0.007; up to 3.6 fold). In contrast, total respiratory leukocytes, macrophages, total dendritic cells and bronchoalveolar lavage leukocytes did not differ significantly. Stimulation of respiratory leukocytes via Toll-like receptor 4 and 9 as well as CD3/CD28 revealed significant strain differences of TNF-α and IL-10 production.

Conclusion

Our study demonstrates significant strain heterogeneity of respiratory leukocyte subsets that may impact respiratory immunity in different disease models. Additionally, the results may help identification of optimal strains for purification of rare respiratory leukocyte subsets for ex vivo analyses.     

Differential production of IL-12, IFN-alpha, and IFN-gamma by mouse dendritic cell subsets

Dendritic cells (DC) not only stimulate T cells effectively but are also producers of cytokines that have important immune regulatory functions. In this study we have extended information on the functional differences between DC subpopulations to include differences in the production of the major immune-directing cytokines IL-12, IFN-alpha, and IFN-gamma. Splenic CD4(-)8(+) DC were identified as the major IL-12 producers in response to microbiological or T cell stimuli when compared with splenic CD4(-)8(-) or CD4(+)8(-) DC; however, all three subsets of DC showed similar IL-12 regulation and responded with increased IL-12 p70 production if IL-4 was present during stimulation. High level CD8 expression also correlated with extent of IL-12 production for DC isolated from thymus and lymph nodes. By using gene knockout mice we ruled out any role for CD8alpha itself, or of priming by T cells, on the superior IL-12-producing capacity of the CD8(+) DC. Additionally, CD8(+) DC were identified as the major producers of IFN-alpha compared with the two CD8(-) DC subsets, a finding that suggests similarity to the human plasmacytoid DC lineage. In contrast, the CD4(-)8(-) DC produced much more IFN-gamma than the CD4(-)8(+) or the CD4(+)8(-) DC under all conditions tested.     

Gene expression profiling defines ATP as a key regulator of human dendritic cell functions

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPgammaS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPgammaS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPgammaS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPgammaS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPgammaS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPgammaS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPgammaS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPgammaS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.