Protein microarrays are versatile tools for parallel, miniaturized screening of binding events involving large numbers of immobilized proteins in a time- and cost-effective manner. They are increasingly applied for high-throughput protein analyses in many research areas, such as protein interactions, expression profiling and target discovery. While conventionally made by the spotting of purified proteins, recent advances in technology have made it possible to produce protein microarrays through in situ cell-free synthesis directly from corresponding DNA arrays. This article reviews recent developments in the generation of protein microarrays and their applications in proteomics and diagnostics.

The molecular processes that govern the first cell lineage decisions after fertilization also dictate the developmental potency of stem cells derived from the early mouse embryo. Our understanding of these mechanisms is therefore instrumental for stem cell biology and regenerative medicine. A number of transcription factors are known that determine a cell's fate towards either the embryonic or extraembryonic trophoblast lineages. Recent insights have shown that the definitive fixation of cell lineage fate is achieved by an epigenetic restriction through DNA methylation of the transcription factor Elf5. Lineage crossover can be induced, however, by manipulation of lineage determinants and gatekeepers, or their epigenetic regulation. Here we summarize the accumulating number of experimental conditions where such 'transdifferentiation' is observed that shed light onto the genetic and epigenetic pathways involved in lineage separation and the developmental potential of stem cells.

A key component of any synthetic biology effort is the use of quantitative models. These models and their corresponding simulations allow optimization of a system design, as well as guiding their subsequent analysis. Once a domain mostly reserved for experts, dynamical modelling of gene regulatory and reaction networks has been an area of growth over the last decade. There has been a concomitant increase in the number of software tools and standards, thereby facilitating model exchange and reuse. We give here an overview of the model creation and analysis processes as well as some software tools in common use. Using markup language to encode the model and associated annotation, we describe the mining of components, their integration in relational models, formularization and parametrization. Evaluation of simulation results and validation of the model close the systems biology 'loop'.

The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, which are important components of calcium influx pathways in virtually all cells. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PIP(2) hydrolysis through a mechanism in which IP(3) receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that one or more PIP(2) generated lipid messengers, as well as PIP(2) itself, are essential for regulating TRP and TRPL activity. Evidence on the role of these lipid elements in regulating TRP and TRPL activity is discussed in this review.

Tau inclusions play a key role in the pathogenesis of tauopathies. Altered tau levels have been detected in retina and optic nerve of patients with glaucoma, suggesting the possibility of shared pathogenic mechanisms with tauopathies. Here we report that hyperphosphorylated transgenic tau accumulates in the nerve fibre layer and, from 2 months of age, aggregates into filamentous inclusions in retinal ganglion cells of human P301S tau transgenic mice. Axonopathy and accumulation of hyperphosphorylated tau in the nerve fibre layer preceded inclusion formation. Hyperphosphorylated tau and tau inclusions were also detected in cultured retinal explants from 5-month-old transgenic mice. Axonal outgrowth was similar in transgenic and wild-type retinal explants under basal conditions. However, when exposed to growth-promoting stimuli, axon elongation was enhanced in explants from wild-type but not transgenic mice, indicating that the presence of abnormal tau can impair stimulated axonal outgrowth. These findings suggest that the retina is a good model system for investigating tau-driven neurodegeneration and for assessing potential pharmacological modifiers for tauopathies.

Animals modify food-seeking behavior and metabolism according to perceived food availability. Here we show that, in the roundworm C. elegans, release of neuropeptides from interneurons that are directly postsynaptic to olfactory, gustatory, and thermosensory neurons coordinately regulates behavior and metabolism. Animals lacking these neuropeptides, encoded by the flp-18 gene, are defective in chemosensation and foraging, accumulate excess fat, and exhibit reduced oxygen consumption. Two G protein-coupled receptors of the NPY/RFamide family, NPR-4 and NPR-5, are activated by FLP-18 peptides in vitro and exhibit mutant phenotypes that recapitulate those of flp-18 mutants. Our data suggest that sensory input can coordinately regulate behavior and metabolism via NPY/RFamide-like receptors. They suggest that peptidergic feedback from interneurons regulates sensory neuron activity, and that at least some of this communication occurs extrasynaptically. Extrasynaptic neuropeptide signaling may greatly increase the computational capacity of neural circuits.

There is increasing concern about the neurodegenerative and behavioral consequences of ozone pollution in industrialized urban centers throughout the world and that women may be more susceptible to brain neurodegenerative disorders. In the present study we have investigated the effects of chronic (30 or 60 days) exposure to ozone on olfactory perception and memory and on levels of lipid peroxidation, alpha and beta estrogen receptors and dopamine beta-hydroxylase in the olfactory bulb in ovariectomized female rats. The ability of 17beta-estradiol to prevent these effects was then assessed. Results showed that ozone exposure for 30 or 60 days impaired formation/retention of a selective olfactory recognition memory 120 min after exposure to a juvenile stimulus animal with the effect at 60 days being significantly greater than at 30 days. They also showed impaired speed in locating a buried chocolate reward after 60 days of ozone exposure indicating some loss of olfactory perception. These functional impairments could all be prevented by coincident estradiol treatment. In the olfactory bulb, levels of lipid peroxidation were increased at both 30- and 60-day time-points and numbers of cells with immunohistochemical staining for alpha and beta estrogen receptors, and dopamine beta-hydroxylase were reduced as were alpha and beta estrogen receptor protein levels. These effects were prevented by estradiol treatment. Oxidative stress damage caused by chronic exposure to ozone does therefore impair olfactory perception and social recognition memory and may do so by reducing noradrenergic and estrogen receptor activity in the olfactory bulb. That these effects can be prevented by estradiol treatment suggests increased susceptibility to neurodegenerative disorders in aging women may be contributed to by reduced estrogen levels post-menopause.

Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. Historically, much PLD work has been conducted in mammalian settings although genes encoding enzymes of this family have been identified in all eukaryotic organisms. Recently, important insights on PLD function are emerging from work in yeast, but much less is known about PLD in other organisms. In this review we will summarize what is known about phospholipase D in several model organisms, including C. elegans, D. discoideum, D. rerio and D. melanogaster. In the cases where knockouts are available (C. elegans, Dictyostelium and Drosophila) the PLD gene(s) appear not to be essential for viability, but several studies are beginning to identify pathways where this activity has a role. Given that the proteins in model organisms are very similar to their mammalian counterparts, we expect that future studies in model organisms will complement and extend ongoing work in mammalian settings. At the end of this review we will also provide a short update on phosphatidic acid targets, a topic last reviewed in 2006.

In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR agonists to boost endogenous analgesia.

Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz. an increased beta-sheet secondary structure, congophilic structures indicative of amyloid and proteinase K resistance which could be templated onto normal unmodified prion protein. In the light of these findings, a potential role of post-translational modifications to prion protein in disease initiation or propagation is discussed.

We describe how recent advances in stem cell research may be interpreted by various regulatory regimes and use Canada as a model to demonstrate how broad-based prohibitive legislation can unintentionally restrict research direction. We encourage scientists and policymakers to collaborate to ensure a clear regulatory framework that accommodates future advances.

GIMAP (GTPase of the immunity-associated protein family) proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi.

IGF activity is regulated tightly by a family of IGF binding proteins (IGFBPs). IGFBP-5 is the most conserved of these and is up-regulated significantly during differentiation of several key lineages and in some cancers. The function of IGFBP-5 in these physiological and pathological situations is unclear, however, several IGFBP-5 sequence motifs and studies in vitro suggest IGF-independent actions. Therefore, we aimed to compare the phenotypes of mice overexpressing wild-type Igfbp5 or an N-terminal mutant Igfbp5 with negligible IGF binding affinity. Both significantly inhibited growth, even at low expression levels. Even though wild-type IGFBP-5 severely disrupted the IGF axis, we found no evidence for interaction of mutant IGFBP-5 with the IGF system. Further, overexpression of wild-type IGFBP-5 rescued the lethal phenotype induced by "excess" IGF-II in type 2 receptor-null mice; mutant IGFBP-5 overexpression could not. Therefore, wild-type IGFBP-5 provides a very effective mechanism for the inhibition of IGF activity and a powerful in vivo mechanism to inhibit IGF activity in pathologies such as cancer. This study is also the first to suggest significant IGF-independent actions for IGFBP-5 during development.

Studies in animals have reported that normalized or elevated Cu levels can inhibit or even remove Alzheimer's disease-related pathological plaques and exert a desirable amyloid-modifying effect. We tested engineered nanocarriers composed of diverse core-shell architectures to modulate Cu levels under physiological conditions through bypassing the cellular Cu uptake systems. Two different nanocarrier systems were able to transport Cu across the plasma membrane of yeast or higher eukaryotic cells, CS-NPs (core-shell nanoparticles) and CMS-NPs (core-multishell nanoparticles). Intracellular Cu levels could be increased up to 3-fold above normal with a sublethal dose of carriers. Both types of carriers released their bound guest molecules into the cytosolic compartment where they were accessible for the Cu-dependent enzyme SOD1. In particular, CS-NPs reduced Abeta levels and targeted intracellular organelles more efficiently than CMS-NPs. Fluorescently labeled CMS-NPs unraveled a cellular uptake mechanism, which depended on clathrin-mediated endocytosis in an energy-dependent manner. In contrast, the transport of CS-NPs was most likely driven by a concentration gradient. Overall, nanocarriers depending on the nature of the surrounding shell functioned by mediating import of Cu across cellular membranes, increased levels of bioavailable Cu, and affected Abeta turnover. Our studies illustrate that Cu-charged nanocarriers can achieve a reasonable metal ion specificity and represent an alternative to metal-complexing agents. The results demonstrate that carrier strategies have potential for the treatment of metal ion deficiency disorders.

HuR is an RNA-binding protein implicated in a diverse array of pathophysiological processes due to its effects on the posttranscriptional regulation of AU- and U-rich mRNAs. Here we reveal HuR's requirement in embryonic development through its genetic ablation. Obligatory HuR-null embryos exhibited a stage retardation phenotype and failed to survive beyond midgestation. By means of conditional transgenesis, we restricted HuR's mutation in either embryonic or endothelial compartments to demonstrate that embryonic lethality is consequent to defects in extraembryonic placenta. HuR's absence impaired the invagination of allantoic capillaries into the chorionic trophoblast layer and the differentiation of syncytiotrophoblast cells that control the morphogenesis and vascularization of the placental labyrinth and fetal support. HuR-null embryos rescued from these placental defects proceeded to subsequent developmental stages but displayed defects in skeletal ossification, fusions in limb elements, and asplenia. By coupling gene expression measurements, data meta-analysis, and HuR-RNA association assays, we identified transcription and growth factor mRNAs controlled by HuR, primarily at the posttranscriptional level, to guide morphogenesis, specification, and patterning. Collectively, our data demonstrate the dominant role of HuR in organizing gene expression programs guiding placental labyrinth morphogenesis, skeletal specification patterns, and splenic ontogeny.

Beckwith-Wiedemann syndrome (BWS) is a fetal overgrowth and human imprinting disorder resulting from the deregulation of a number of genes, including IGF2 and CDKN1C, in the imprinted gene cluster on chromosome 11p15.5. Most cases are sporadic and result from epimutations at either of the two 11p15.5 imprinting centres (IC1 and IC2). However, rare familial cases may be associated with germline 11p15.5 deletions causing abnormal imprinting in cis. We report a family with BWS and an IC2 epimutation in which affected siblings had inherited different parental 11p15.5 alleles excluding an in cis mechanism. Using a positional-candidate gene approach, we found that the mother was homozygous for a frameshift mutation in exon 6 of NLRP2. While germline mutations in NLRP7 have previously been associated with familial hydatidiform mole, this is the first description of NLRP2 mutation in human disease and the first report of a trans mechanism for disordered imprinting in BWS. These observations are consistent with the hypothesis that NLRP2 has a previously unrecognised role in establishing or maintaining genomic imprinting in humans.

The mechanisms that regulate NK cell trafficking are unclear. Phosphoinositide-3 kinases (PI3K) control cell motility and the p110gamma and p110delta isoforms are mostly expressed in leukocytes, where they transduce signals downstream of G protein coupled receptors (GPCR) or tyrosine kinase receptors, respectively. Here, we set out to determine the relative contribution of p110gamma and p110delta to NK cell migration in mice. Using a combination of single-cell imaging analysis of transgenic cells reporting on PI3K activity in real time and small molecule inhibitors of p110gamma and p110delta, we show here that the tyrosine-kinase coupled p110delta is linked to GPCR signaling and, depending on the GPCR, may even be preferentially activated over p110gamma. Using gene-targeted mice, we showed that both isoforms were essential for NK cell chemotaxis to CXCL12 and to CCL3 and, in vivo, for normal NK cell migration during pregnancy and to the inflamed peritoneum. By contrast, only p110delta was indispensable for chemotaxis to S1P and CXCL10 and for NK cell distribution throughout lymphoid and nonlymphoid tissues and for extravasation to tumors. These results implicate p110delta downstream of GPCRs in NK cells and highlight its nonredundant role as a key regulator of NK cell trafficking in health and disease.

The elucidation of the crucial differentiation factors for the new Th17 CD4 effector T-cell subset spurred an explosive growth in research publications focused on these cells and led to rapid advances in knowledge concerning their regulation and functional activities. Here we discuss recent discoveries linking the development and functional potential of Th17 cells to a transcription factor that mediates the response to exogenous and endogenous environmental signals.

Ca(2+) elevations are fundamental to cardiac physiology-stimulating contraction and regulating the gene transcription that underlies hypertrophy. How Ca(2+) specifically controls gene transcription on the background of the rhythmic Ca(2+) increases required for contraction is not fully understood. Here we identify a hypertrophy-signaling module in cardiac myocytes that explains how Ca(2+) discretely regulates myocyte hypertrophy and contraction. We show that endothelin-1 (ET-1) stimulates InsP(3)-induced Ca(2+) release (IICR) from perinuclear InsP(3)Rs, causing an elevation in nuclear Ca(2+). Significantly, we show that IICR, but not global Ca(2+) elevations associated with myocyte contraction, couple to the calcineurin (CnA)/NFAT pathway to induce hypertrophy. Moreover, we found that activation of the CnA/NFAT pathway and hypertrophy by isoproterenol and BayK8644, which enhance global Ca(2+) fluxes, was also dependent on IICR and nuclear Ca(2+) elevations. The activation of IICR by these activity-enhancing mediators was explained by their ability to stimulate secretion of autocrine/paracrine ET-1.

Growth factor-stimulated expression and activation of c-Fos is regulated by the ERK1/2 pathway. However, recent reports have also suggested a prominent role for the closely related ERK5 pathway in regulating the expression, transcriptional activation and nuclear localization of c-Fos. Here we have compared the role of ERK1/2 and ERK5 in regulating c-Fos using a combination of conditional protein kinases, selective biochemical inhibitors and ERK5 null fibroblasts. We demonstrate that activation of the ERK1/2 pathway, but not ERK5, is sufficient for c-Fos phosphorylation and transcriptional activation. Furthermore, growth factor-dependent expression of c-Fos is blocked by low doses of PD184352 that selectively inhibit the ERK1/2 pathway but proceeds normally in ERK5-/- 3T9 cells; in addition, nuclear localization of c-Fos is normal in ERK5-/- cells. ERK5-/- cells are, however, defective for c-Jun expression but this is reversed by re-expression of ERK5. In addition to ERK5, neither the JNK nor p38 pathways can substitute for ERK1/2 in the regulation of c-Fos transcriptional activity. These results demonstrate that c-Fos transcriptional activity is not regulated by the ERK5 pathway; rather, of all the MAPKs and SAPKs, c-Fos activation appears to be predominantly linked to the ERK1/2 pathway.

Genomic imprinting is a normal process that causes genes to be expressed according to parental origin. The selective advantage conferred by imprinting is not understood but is hypothesised to act on dosage-critical genes. Here, we report a unique model in which the consequences of a single, double, and triple dosage of the imprinted Dlk1/Pref1, normally repressed on the maternally inherited chromosome, can be assessed in the growing embryo. BAC-transgenic mice were generated that over-express Dlk1 from endogenous regulators at all sites of embryonic activity. Triple dosage causes lethality associated with major organ abnormalities. Embryos expressing a double dose of Dlk1, recapitulating loss of imprinting, are growth enhanced but fail to thrive in early life, despite the early growth advantage. Thus, any benefit conferred by increased embryonic size is offset by postnatal lethality. We propose a negative correlation between gene dosage and survival that fixes an upper limit on growth promotion by Dlk1, and we hypothesize that trade-off between growth and lethality might have driven imprinting at this locus.

From the earliest comparisons of RNA production with steady-state levels, it has been clear that cells transcribe more RNA than they accumulate, implying the existence of active RNA degradation systems. In general, RNA is degraded at the end of its useful life, which is long for a ribosomal RNA but very short for excised introns or spacer fragments, and is closely regulated for most mRNA species. RNA molecules with defects in processing, folding, or assembly with proteins are identified and rapidly degraded by the surveillance machinery. Because RNA degradation is ubiquitous in all cells, it is clear that it must be carefully controlled to accurately recognize target RNAs. How this is achieved is perhaps the most pressing question in the field.