Absence Of Other Signals Research Articles

Optogenetic control of the Dab1 signaling pathway

The Reelin-Dab1 signaling pathway regulates development of the mammalian brain, including neuron migrations in various brain regions, as well as learning and memory in adults. Extracellular Reelin binds to cell surface receptors and activates phosphorylation of the intracellular Dab1 protein. Dab1 is required for most effects of Reelin, but Dab1-independent pathways may contribute. Here we developed a single-component, photoactivatable Dab1 (opto-Dab1) by using the blue light-sensitive dimerization/oligomerization property of A. thaliana Cryptochrome 2 (Cry2). Opto-Dab1 can activate downstream signals rapidly, locally, and reversibly upon blue light illumination. The high spatiotemporal resolution of the opto-Dab1 probe also allows us to control membrane protrusion, retraction and ruffling by local illumination in both COS7 cells and in primary neurons. This shows that Dab1 activation is sufficient to orient cell movement in the absence of other signals. Opto-Dab1 may be useful to study the biological functions of the Reelin-Dab1 signaling pathway both in vitro and in vivo.

Abstract B017: A soluble form of CD80 inhibits PD-L1 immune suppression and stimulates T cells through CD28-specific pathways indicating potential for increased therapeutic activity over checkpoint inhibition alone

Abstract Antibodies that block the PD-1/PD-L1 pathway have demonstrated therapeutic success against several cancer types, but do not induce a response in the majority of patients. The development of treatments that provide activation signals to T cells by targeting co-stimulatory receptors, in addition to checkpoint inhibition, may increase anti-tumor immunity and the percent of responding patients. CD80/B7-1 is a co-stimulatory molecule that also interacts with PD-L1. CD80 is expressed by antigen presenting cells and ligates to CD28 providing co-stimulation during T cell activation. Furthermore, PD-L1 interacts with T cell CD80 in a reverse signaling pathway that promotes T cell anergy. The ability of CD80 to block inhibitory and promote stimulatory signaling pathways in T cell activation suggests potential therapeutic value. We have shown that soluble CD80 fused to the Fc region of IgG (CD80-Fc) blocks PD-1/PD-L1 interactions thus restoring activation of CD4+ and CD8+ T cells. Moreover, CD80-Fc was more effective in vitro than PD-1/PD-L1 monoclonal antibodies at restoring T cell activation, which suggests that CD80-Fc may be stimulating CD28 in addition to blocking PD-1/PD-L1 signaling. Stimulation of T cell CD28 in the absence of other signals induces downstream transcription, and signaling pathway activation. In fact, agonist CD28 antibody can induce functions in T cells in both naïve and activated cells, even in the absence of TCR activation. We now demonstrate that CD80-Fc does indeed stimulate T cells via CD28 activation by demonstrating comparable downstream results of CD80-Fc and agonist CD28 antibody treatment of both human and mouse T cells. Therefore, CD80-Fc may provide a novel therapeutic approach to target T cell checkpoint inhibition in addition to providing stimulatory signaling to improve T cell activation and anti-tumor immunity. Citation Format: Tiha M. Long, Suzanne Ostrand-Rosenberg. A soluble form of CD80 inhibits PD-L1 immune suppression and stimulates T cells through CD28-specific pathways indicating potential for increased therapeutic activity over checkpoint inhibition alone. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B017.

Coherence dynamics in light-harvesting complexes with two-colour spectroscopy

We investigate coherent dynamics in the cryptophyte light-harvesting complex Phycocyanin-645 (PC-645). A two-colour four-wave mixing experiment allows us to isolate a coherence pathway and observe its evolution in the absence of other signals. We measured a decoherence time of 540fs for the coherence [1]. Additionally oscillations in the signal pathway give evidence for the coherent excitation of states outside the bandwidth of the laser pulse. This suggests strong coupling between the excited states and phonon modes [1].

B cells do not take up bacterial DNA: an essential role for antigen in exposure of DNA to toll‐like receptor‐9

Murine dendritic cells (DC) and macrophages respond to bacterial CpG DNA through toll-like receptor 9 (TLR9). Although it is frequently assumed that bacterial DNA is a direct stimulus for B cells, published work does not reliably show responses of purified B cells. Here we show that purified splenic B cells did not respond to Escherichia coli DNA with induction of CD86, despite readily responding to single-stranded (ss) phosphodiester CpG oligodeoxynucleotides (ODN). This was due to a combination of weak responses to both long and double-stranded (ds) DNA. B-cell DNA uptake was greatly reduced with increasing DNA length. This contrasts with macrophages where DNA uptake and subsequent responses were enhanced with increasing DNA length. However, when DNA was physically linked to hen egg lysozyme (HEL), HEL-specific B cells showed efficient uptake of DNA, and limited proliferation in response to the HEL-DNA complex. We propose that, in the absence of other signals, B cells have poor uptake and responses to long dsDNA to prevent polyclonal activation. Conversely, when DNA is physically linked to a B-cell receptor (BCR) ligand, its uptake is increased, allowing TLR9-dependent B-cell activation in an antigen-specific manner. We could not generate fragments of E. coli DNA by limited DNaseI digestion that could mimic the stimulatory effect of ss CpG ODN on naïve B cells. We suggest that the frequently studied polyclonal B-cell responses to CpG ODN are relevant to therapeutic applications of phosphorothioate-modified CpG-containing ODN, but not to natural responses to foreign or host dsDNA.

Autocrine production of TGF-beta1 promotes myofibroblastic differentiation of neonatal lung mesenchymal stem cells.

We have isolated mesenchymal stem cells (MSCs) from tracheal aspirates of premature infants with respiratory distress. We examined the capacity of MSCs to differentiate into myofibroblasts, cells that participate in lung development, injury, and repair. Gene expression was measured by array, qPCR, immunoblot, and immunocytochemistry. Unstimulated MSCs expressed mRNAs encoding contractile (e.g., ACTA2, TAGLN), extracellular matrix (COL1A1 and ELN), and actin-binding (DBN1, PXN) proteins, consistent with a myofibroblast phenotype, although there was little translation into immunoreactive protein. Incubation in serum-free medium increased contractile protein (ACTA2, MYH11) gene expression. MSC-conditioned medium showed substantial levels of TGF-beta1, and treatment of serum-deprived cells with a type I activin receptor-like kinase inhibitor, SB-431542, attenuated the expression of genes encoding contractile and extracellular matrix proteins. Treatment of MSCs with TGF-beta1 further induced the expression of mRNAs encoding contractile (ACTA2, MYH11, TAGLN, DES) and extracellular matrix proteins (FN1, ELN, COL1A1, COL1A2), and increased the protein expression of alpha-smooth muscle actin, myosin heavy chain, and SM22. In contrast, human bone marrow-derived MSCs failed to undergo TGF-beta1-induced myofibroblastic differentiation. Finally, primary cells from tracheal aspirates behaved in an identical manner as later passage cells. We conclude that human neonatal lung MSCs demonstrate an mRNA expression pattern characteristic of myofibroblast progenitor cells. Autocrine production of TGF-beta1 further drives myofibroblastic differentiation, suggesting that, in the absence of other signals, fibrosis represents the "default program" for neonatal lung MSC gene expression. These data are consistent with the notion that MSCs play a key role in neonatal lung injury and repair.

Individual effects of product quality signals in the presence versus absence of other signals: differential effects across brick‐and‐mortar and online settings

Purpose – The purpose of this paper is to study the effectiveness of multiple signals. Specifically, the paper investigates how the individual strength of a marketplace signal varies as a function of whether consumers are exposed to that signal alone or in combination with another signal.Design/methodology/approach – The research uses experimental designs to empirically address the research questions. Hypotheses are formulated primarily based on signaling theory and these hypotheses are tested with laboratory experiments using real consumers.Findings – The key finding is that a signal's stand‐alone credibility largely determines whether its individual strength would be diluted or augmented by the coexistence of another signal. Further, when signals with different stand‐alone strengths coexist, the individual strength of the weaker signal is higher than when that signal is present alone. These effects are observed in brick‐and‐mortar and online shopping media.Originality/value – Past research reports mixed...

Elevated ATF4 Expression, in the Absence of Other Signals, Is Sufficient for Transcriptional Induction via CCAAT Enhancer-binding Protein-activating Transcription Factor Response Elements

Protein limitation in vivo or amino acid deprivation of cells in culture causes a signal transduction cascade consisting of activation of the kinase GCN2 (general control nonderepressible 2), phosphorylation of eukaryotic initiation factor 2, and increased synthesis of activating transcription factor (ATF) 4 by a translational control mechanism. In a self-limiting transcriptional program, ATF4 transiently activates a wide range of downstream target genes involved in transport, cellular metabolism, and other cell functions. Simultaneous activation of other signal transduction pathways by amino acid deprivation led to the question of whether or not the increased abundance of ATF4 alone was sufficient to trigger the transcriptional control mechanisms. Using 293 cells that ectopically express ATF4 in a tetracycline-inducible manner showed that ATF4 target genes were activated in the absence of amino acid deprivation. Ectopic expression of ATF4 alone resulted in effective recruitment of the general transcription machinery, but some reduction in histone modification was observed. These data document that ATF4 alone is sufficient to trigger the amino acid-responsive transcriptional control program. However, the absolute amount of ectopic ATF4 required to achieve the same degree of transcriptional activation observed after amino acid limitation was greater, suggesting that other factors may serve to enhance ATF4 function.

The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.

Ras Induces Chromosome Instability and Abrogation of the DNA Damage Response

Ras mutations are frequent in thyroid tumors, the most common endocrine malignancy. The ability of Ras to transform thyroid cells is thought to rely on its mitogenic activity. Unexpectedly, acute expression of activated Ras in normal rat thyroid cells induced a DNA damage response, followed by apoptosis. Notably, a subpopulation of cells evaded apoptosis and emerged with features of transformation, including the loss of epithelial morphology, dedifferentiation, and the acquisition of hormone- and anchorage-independent proliferation. Strikingly, the surviving cells showed marked chromosomal instability. Acutely, Ras stimulated replication stress as evidenced by the induction of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR) activity (Chk1 phosphorylation) and of gammaH2A.X, a marker of DNA damage. Despite the activation of a checkpoint, cells continued through mitosis in the face of DNA damage, resulting in an increase in cells harboring micronuclei, an indication of defects in chromosome segregation and other forms of chromosome damage. Cells that survived exposure to Ras continued to exhibit replication stress (ATR activation) but no longer exhibited gammaH2A.X or full activation of p53. When rechallenged with Ras or DNA-damaging agents, the surviving cells were more resistant to apoptosis than parental cells. These data show that acute expression of activated Ras is sufficient to induce chromosomal instability in the absence of other signals, and suggest that Ras-induced chromosomal instability arises as a consequence of defects in the processing of DNA damage. Hence, abrogation of the DNA damage response may constitute a novel mechanism for Ras transformation.

Uncommitted Xenopus blastula cells can be directed to uniform muscle gene expression by gradient interpretation and a community effect.

The animal cap cells of Xenopus blastulae behave as multi-potent stem cells in so far as they can differentiate along many unrelated pathways according to the kind and amount of signal factor that they experience. At first, animal cap cells activate early zygotic genes across a broad range of TGFbeta concentrations; soon after this, they activate later genes more intensely and over a narrow concentration range. Here we show that uncommitted blastula cells can be directed, by the sequential influence of a particular concentration of a TGFbeta morphogen and an FGF-mediated community effect, to form a homogeneous single cell type. As a result of these two signalling processes, an entire population of animal cap cells can be converted, in the absence of other signals, to a uniform population of one tissue type. Mesoderm cells that experience a particular concentration of activin increase their XMyoD expression by 10-fold and become distinct from neighbouring cells that received lower or higher concentrations of activin. The signalling processes that we employ here may be important in normal development and useful in guiding stem cell differentiation.

Neurotrophin-3 Promotes the Differentiation of Muscle Spindle Afferents in the Absence of Peripheral Targets

The neurons of the dorsal root ganglia (DRG) that supply muscle spindles require target-derived factors for survival. One necessary factor for these neurons is neurotrophin-3 (NT3). To determine whether NT3 can promote the survival of these neurons in the absence of other target-derived factors, we analyzed the effects of exogenous NT3 after early limb bud deletion in the chick. In control embryos, limb bud deletion eliminated approximately 90% of the trkC-positive (trkC+) neurons in lumbar DRG on the deleted side. In addition, the deletion led to a dramatic loss of collateral sensory projections to motoneurons. Exogenous NT3 restored a normal population of trkC+ neurons in lumbar DRG on the deleted side and increased the number of trkC+ neurons in DRG with normal targets (contralateral lumbar and thoracic). The effect was highly selective; NT3 increased the number of trkC+ neurons without significantly changing the number of either trkA+ or trkB+ neurons. The effect of NT3 was attributable to the rescue of DRG neurons from cell death, because exogenous NT3 reduced the number of pyknotic nuclei without significantly altering proliferation. Analysis of spinal projections showed further that many of the trkC+ neurons rescued by NT3 projected to the ventral spinal cord. These neurons thus had central projections characteristic of muscle spindle afferents. Together, our results indicate that NT3 signaling is both necessary and sufficient for the development of the proprioceptive phenotype, even in the absence of other signals from limb muscle.

Extracellular matrix proteins, regulators of T-cell functions in healthy and diseased individuals

Antigen-dependentT-cellactivationrequiresengagementof the T-cell receptor (TCR)-CD3 complex with peptides presented by major histocompatibility complex molecules. However, stimulation of T cells through the complex of T-cell antigen receptor and CD3 in the absence of other signals is insufficient for optimal interleukin-2 (IL-2) production and proliferation and induces T-cell anergy. Therefore, additional costimulatory signals are needed for an effective T-cell-mediatedimmuneresponse.Suchsignalscanbeprovidedbyvarious interactions between T cells and ligands on antigen-presenting cells (e.g., CD2-CD58 [LFA-3] and CD28-CD80 [B7] cells [2, 59]). Furthermore, recent data indicate that the extracellular matrix (ECM) proteins can mediate strong costimulation in theprocessofT-cellactivationintheabsenceofmonocytes,so one could view the ECM protein as an equivalent of an antigen-presenting cell. This view is further strengthened by observations that ECM proteins can interact with both an integrin and other T-cell-activating agents (e.g., cytokines) (26, 29). Lymphocyte extravasation into the lymphatic tissue constitutes a normal physiological process by which the cells return finally to blood through the thoracic duct. This migration is associated with lymphocyte interaction with the ECM proteins present in the basement membranes (collagen type IV [C-IV] and laminin), because once the cells reach the underlying tissue they come into contact with interstitial ECM proteins (collagentypeI[C-I]andfibronectin[FN]).Thelatterinteractions appear to be especially relevant at sites of tissue invasion by inflammatory cells, because both C-I and FN are ubiquitous in interstitialECMproteins.Inaddition,FNhasbeenreportedto be produced by mononuclear cells themselves (monocytes and T cells), and its increased concentrations are found at the foci of inflammation and in sites of delayed-type hypersensitivity (34, 58). Therefore, it could be expected that the ECM proteins may regulate T-cell functions under both physiological and pathological conditions. (The biochemical structures of major ECM proteins were recently reviewed [48].) Evidence that integrins mediate information transfer into T cells is accumulating: occupation of integrin receptors by their ligands leads to tyrosine phosphorylation, elevation of cytoplasmic pH and Ca 21 concentration, activation of protein kinases, and inositol release. Moreover, FN binding via VLA-5 induces the AP-1 transcription factor necessary for IL-2 transcription.Integrinreceptors,byrecognizingECMproteins,can provide the signal for the stimulation of cytokine genes and subsequent cytokine production by T cells (this was demonstrated for IL-1, IL-2, and IL-4 and tumor necrosis factor alpha). Recent minireviews have provided a detailed analysis of ‘‘inside-to-out’’ and ‘‘outside-to-in’’ signaling by integrins (10, 29, 32, 48). Interestingly, recent data suggest that ECM protein-mediated cytokine gene activation can also occur in the absence of TCR engagement. This pathway of antigen-independent cytokine production by T cells interacting with the ECM proteins could be particularly important for gdT cells, frequently identified at sites of inflammation and thought to play an immunoregulatory rather than an effector role in the immune response (56). FewstudieshaveinvestigatedthefunctionsofECMproteins and integrins involved in T-cell development. These proteins are present in the thymic parenchyma and basement membranes and may be found to be associated with the epithelial cells which secrete them. Up to 15% of unseparated thymocytes adhere to FN, but 70% of the CD4 2 CD8 2 (double negative) cells do. In addition, thymocytes adhere to thymic stromal cells interacting with the FN present on their surfaces. Blocking of this adhesion (with anti-FN antibody and antiVLA-4 and anti-VLA-5 reagents) causes a potent inhibition of thymocyte differentiation in vitro. Thus, thymocyte-FN interactions may be critical in inducing and supporting the differentiation of immature T cells. In addition, another integrin (a6b4), with unknown ligand specificity, may play a role in intrathymic T-cell differentiation (63, 64). Recently, it was shown that human thymocyte proliferation induced by CD3 can be enhanced by ECM proteins in the presence of an additional signal: IL-2 or phorbol myristate acetate (61).

Ventral ectoderm of Xenopus forms neural tissue, including hindbrain, in response to activin.

The peptide growth factor Activin A has been shown to induce complete axial structures in explanted blastula animal caps. However, it is not understood how much this response to activin depends upon early signals that prepattern the ectoderm. We have therefore asked what tissues can be induced in blastula animal caps by activin in the absence of early dorsal signals. Using whole-mount in situ hybridization, we compare the expression of three neural markers, N-CAM, En-2 and Krox-20 in activin-treated ectoderm from control and ventralized embryos. In response to activin, both normal and ventralized animal caps frequently form neural tissue (and express N-CAM) and express the hindbrain marker Krox-20. However, the more anterior marker, En-2, is expressed in only a small fraction of normal animal caps and rarely in ventralized animal caps; the frequency of expression does not increase with higher doses of activin. In all cases En-2 and Krox-20 are expressed in coherent patches or stripes in the induced caps. Although mesoderm is induced in both control and ventralized animal caps, notochord is found in response to activin at moderate frequency in control caps, but rarely in ventralized animal caps. These results support the idea that in the absence of other signals, activin treatment elicits hindbrain but not notochord or anterior neural tissue; and thus, the anterior and dorsal extent of tissues formed in response to activin depends on a prior prepatterning or previous inductions.

The N-terminal (pre-S2) domain of a hepatitis B virus surface glycoprotein is translocated across membranes by downstream signal sequences

The coding region for the hepatitis B virus surface antigens contains three in-phase ATG codons which direct the synthesis of three related polypeptides. The 24-kilodalton major surface (or S) glycoprotein is initiated at the most distal ATG and is a transmembrane protein whose translocation across the bilayer is mediated by at least two uncleaved signal sequences. The product of the next upstream ATG is the 31-kilodalton pre-S2 protein, which contains 55 additional amino acids attached to the N terminus of the S protein. This pre-S2-specific domain is translocated into the endoplasmic reticulum. Using a coupled in vitro translation-translocation system, we showed that (i) the pre-S2 domain itself lacks functional signal sequence activity, (ii) its translocation across the endoplasmic reticulum membrane is mediated by downstream signals within the S domain, and (iii) the N-terminal signal sequence of the S protein can translocate upstream protein domains in the absence of other signals. The hepatitis B virus pre-S2 protein is an example of a natural protein which displays upstream domain translocation, a phenomenon whose existence was originally inferred from the behavior of synthetic fusion proteins in vitro.