Multidimensional spectroscopy of magneto-excitons at high magnetic fields.

We perform two-dimensional Fourier transform spectroscopy on magneto-excitons in GaAs at magnetic fields and observe Zeeman splitting of the excitons. The Zeeman components are clearly resolved as separate peaks due to the two-dimensional nature of the spectra, leading to a more accurate measurement of the Zeeman splitting and the Landé g factors. Quantum coherent coupling between Zeeman components is observed using polarization dependent one-quantum two-dimensional spectroscopy. We use two-quantum two-dimensional spectroscopy to investigate higher four-particle correlations at high magnetic fields and reveal the role of the Zeeman splitting on the two-quantum transitions. The experimental two-dimensional spectra are simulated using the optical Bloch equations, where many-body effects are included phenomenologically.

Optical Coherence in Atomic-Monolayer Transition-Metal Dichalcogenides Limited by Electron-Phonon Interactions.

We systematically investigate the excitonic dephasing of three representative transition-metal dichalcogenides, namely, MoS_{2}, MoSe_{2}, and WSe_{2} atomic monolayer thick and bulk crystals, in order to gain a proper understanding of the factors that determine the optical coherence in these materials. Coherent nonlinear optical spectroscopy and temperature dependent absorption, combined with theoretical calculations of the phonon spectra, indicate electron-phonon interactions, to be the limiting factor. Surprisingly, the excitonic dephasing, differs only slightly between atomic monolayers and high quality bulk crystals, which indicates that material imperfections are not the limiting factor in atomically thin monolayer samples. The temperature dependence of the electronic band gap and the excitonic linewidth combined with "ab initio" calculations of the phonon energies and the phonon density of states reveal a strong interaction with the E' and E" phonon modes.

Strong Quantum Coherence between Fermi Liquid Mahan Excitons.

In modulation doped quantum wells, the excitons are formed as a result of the interactions of the charged holes with the electrons at the Fermi edge in the conduction band, leading to the so-called "Mahan excitons." The binding energy of Mahan excitons is expected to be greatly reduced and any quantum coherence destroyed as a result of the screening and electron-electron interactions. Surprisingly, we observe strong quantum coherence between the heavy hole and light hole excitons. Such correlations are revealed by the dominating cross-diagonal peaks in both one-quantum and two-quantum two-dimensional Fourier transform spectra. Theoretical simulations based on the optical Bloch equations where many-body effects are included phenomenologically reproduce well the experimental spectra. Time-dependent density functional theory calculations provide insight into the underlying physics and attribute the observed strong quantum coherence to a significantly reduced screening length and collective excitations of the many-electron system.

Biexciton formation and exciton coherent coupling in layered GaSe.

Nonlinear two-dimensional Fourier transform (2DFT) and linear absorption spectroscopy are used to study the electronic structure and optical properties of excitons in the layered semiconductor GaSe. At the 1s exciton resonance, two peaks are identified in the absorption spectra, which are assigned to splitting of the exciton ground state into the triplet and singlet states. 2DFT spectra acquired for co-linear polarization of the excitation pulses feature an additional peak originating from coherent energy transfer between the singlet and triplet. At cross-linear polarization of the excitation pulses, the 2DFT spectra expose a new peak likely originating from bound biexcitons. The polarization dependent 2DFT spectra are well reproduced by simulations using the optical Bloch equations for a four level system, where many-body effects are included phenomenologically. Although biexciton effects are thought to be strong in this material, only moderate contributions from bound biexciton creation can be observed. The biexciton binding energy of ∼2 meV was estimated from the separation of the peaks in the 2DFT spectra. Temperature dependent absorption and 2DFT measurements, combined with "ab initio" theoretical calculations of the phonon spectra, indicate strong interaction with the A1 (') phonon mode. Excitation density dependent 2DFT measurements reveal excitation induced dephasing and provide a lower limit for the homogeneous linewidth of the excitons in the present GaSe crystal.

Exploring two-dimensional electron gases with two-dimensional Fourier transform spectroscopy.

The dephasing of the Fermi edge singularity excitations in two modulation doped single quantum wells of 12 nm and 18 nm thickness and in-well carrier concentration of ∼4 × 10(11) cm(-2) was carefully measured using spectrally resolved four-wave mixing (FWM) and two-dimensional Fourier transform (2DFT) spectroscopy. Although the absorption at the Fermi edge is broad at this doping level, the spectrally resolved FWM shows narrow resonances. Two peaks are observed separated by the heavy hole/light hole energy splitting. Temperature dependent "rephasing" (S1) 2DFT spectra show a rapid linear increase of the homogeneous linewidth with temperature. The dephasing rate increases faster with temperature in the narrower 12 nm quantum well, likely due to an increased carrier-phonon scattering rate. The S1 2DFT spectra were measured using co-linear, cross-linear, and co-circular polarizations. Distinct 2DFT lineshapes were observed for co-linear and cross-linear polarizations, suggesting the existence of polarization dependent contributions. The "two-quantum coherence" (S3) 2DFT spectra for the 12 nm quantum well show a single peak for both co-linear and co-circular polarizations.

Superradiant emission in a high-mobility two-dimensional electron gas.

We use terahertz time-domain spectroscopy to study gallium arsenide two-dimensional electron gas samples in external magnetic field. We measure cyclotron decay as a function of temperature from 0.4 to10Kand a quantum confinement dependence of the cyclotron decay time belowT0=1.2K. In the wider quantum well, we observe a dramatic enhancement in the decay time due to the reduction in dephasing and the concomitant enhancement of superradiant decay in these systems. We show that the dephasing time in 2DEG's depends on both the scatteringrateand also on the distribution of scattering angles.

In vivo clonal tracking reveals evidence of haemangioblast and haematomesoblast contribution to yolk sac haematopoiesis.

During embryogenesis, haematopoietic and endothelial lineages emerge closely in time and space. It is thought that the first blood and endothelium derive from a common clonal ancestor, the haemangioblast. However, investigation of candidate haemangioblasts in vitro revealed the capacity for mesenchymal differentiation, a feature more compatible with an earlier mesodermal precursor. To date, no evidence for an in vivo haemangioblast has been discovered. Using single cell RNA-Sequencing and in vivo cellular barcoding, we have unravelled the ancestral relationships that give rise to the haematopoietic lineages of the yolk sac, the endothelium, and the mesenchyme. We show that the mesodermal derivatives of the yolk sac are produced by three distinct precursors with dual-lineage outcomes: the haemangioblast, the mesenchymoangioblast, and a previously undescribed cell type: the haematomesoblast. Between E5.5 and E7.5, this trio of precursors seeds haematopoietic, endothelial, and mesenchymal trajectories.

The role of gp130-mediated signals in osteoclast development: regulation of interleukin 11 production by osteoblasts and distribution of its receptor in bone marrow cultures.

Interleukin (IL)-11 is a multifunctional cytokine whose role in osteoclast development has not been fully elucidated. We examined IL-11 production by primary osteoblasts and the effects of rat monoclonal anti-mouse glycoprotein 130 (gp130) antibody on osteoclast formation, using a coculture of mouse osteoblasts and bone marrow cells. IL-1, TNF alpha, PGE2, parathyroid hormone (PTH) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) similarly induced production of IL-11 by osteoblasts, but IL-6, IL-4, and TGF beta did not. Primary osteoblasts constitutively expressed mRNAs for both IL-11 receptor (IL-11R alpha) and gp130. Osteotropic factors did not modulate IL-11R alpha mRNA at 24 h, but steady-state gp130 mRNA expression in osteoblasts was upregulated by 1 alpha,25(OH)2D3, PTH, or IL-1. In cocultures, the formation of multinucleated osteoclast-like cells (OCLs) in response to IL-11, or IL-6 together with its soluble IL-6 receptor was dose-dependently inhibited by rat monoclonal anti-mouse gp130 antibody. Furthermore, adding anti-gp130 antibody abolished OCL formation induced by IL-1, and partially inhibited OCL formation induced by PGE2, PTH, or 1 alpha,25(OH)2D3. During osteoclast formation in marrow cultures, a sequential relationship existed between the expression of calcitonin receptor mRNA and IL-11R alpha mRNA. Osteoblasts as well as OCLs expressed transcripts for IL-11R alpha, as indicated by RT-PCR analysis and in situ hybridization. These results suggest a central role of gp130-coupled cytokines, especially IL-11, in osteoclast development. Since osteoblasts and mature osteoclasts expressed IL-11R alpha mRNA, both bone-forming and bone-resorbing cells are potential targets of IL-11.

Direct measurement of cyclotron coherence times of high-mobility two-dimensional electron gases.

We have observed long-lived (approximately 30 ps) coherent oscillations of charge carriers due to cyclotron resonance (CR) in high-mobility two-dimensional electrons in GaAs in perpendicular magnetic fields using time-domain terahertz spectroscopy. The observed coherent oscillations were fitted well by sinusoids with exponentially-decaying amplitudes, through which we were able to provide direct and precise measures for the decay times and oscillation frequencies simultaneously. This method thus overcomes the CR saturation effect, which is known to prevent determination of true CR linewidths in high-mobility electron systems using Fourier-transform infrared spectroscopy.

LIF: lots of interesting functions.

Leukaemia inhibitory factor (LIF) is one of a growing number of cytokines that cannot be readily categorized according to its functions. Rather, these pleiotropic hormones have diverse and often overlapping effects on a multitude of cell types: for example, LIF can inhibit the differentiation of embryonal stem cells on one hand and induce the differentiation of M1 leukaemic cells on the other. Recent work has shed light on the physiological effects of LIF, how these are limited, and the biochemical and biological properties of LIF and its receptor.

Suckling defect in mice lacking the soluble haemopoietin receptor NR6.

Cytokines control a variety of cellular responses including proliferation, differentiation, survival and functional activation, via binding to specific receptors expressed on the surface of target cells [1]. The cytokine receptors of the haemopoietin family are defined by the presence of a conserved 200 amino acid extracellular domain known as the haemopoietin domain [2]. We report here the isolation of NR6, a haemopoietin receptor that, like the p40 subunit of interleukin-12 (IL-12) [3] and the EBI3 gene induced by Epstein-Barr virus infection in lymphocytes [4], contains a typical haemopoietin domain but lacks transmembrane and cytoplasmic domains. Although in situ hybridisation revealed NR6 expression at multiple sites in the developing embryo, mice lacking NR6 did not display obvious abnormalities and were born in the expected numbers. Neonatal NR6(-/-) mice failed to suckle, however, and died within 24 hours of birth, suggesting that NR6 is necessary for the recognition or processing of pheromonal signals or for the mechanics of suckling itself. In addition, NR6(-/-) mice had reduced numbers of haemopoietic progenitor cells, suggesting a potential role in the regulation of primitive haemopoiesis.

Activation and inhibition of erythropoietin receptor function: role of receptor dimerization.

Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.

Functional analysis of Asb-1 using genetic modification in mice.

The Asbs are a family of ankyrin repeat proteins that, along with four other protein families, contain a C-terminal SOCS box motif, which was first identified in the suppressor of cytokine signaling (SOCS) proteins. While it is clear that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological roles of the other SOCS box-containing families are unknown. We have investigated Asb-1 function by generating mice that lack this protein, as well as mice that overexpress full-length or truncated Asb-1 in a wide range of tissues. Although Asb-1 is expressed in multiple organs, including the hematopoietic compartment in wild-type mice, Asb-1(-/-) mice develop normally and exhibit no anomalies of mature blood cells or their progenitors. While most organs in these mice appear normal, the testes of Asb-1(-/-) mice display a diminution of spermatogenesis with less complete filling of seminiferous tubules. In contrast, the widespread overexpression of Asb-1 in the mouse has no apparent deleterious effects.

The human IL-11 receptor requires gp130 for signalling: demonstration by molecular cloning of the receptor.

We describe the molecular cloning of a cDNA for the alpha chain of the human IL-11 receptor (IL-11R alpha) and demonstrate the requirement of either the human or mouse gp130 molecule for signalling. cDNA clones encoding IL-11R alpha were isolated from a bone marrow cDNA library using a fragment from the murine IL-11R alpha as a probe. The human receptor was predicted to consist of 422 amino acids and was found to share 84% identity with the murine protein. In the extra-cellular region it exhibited a single hemopoietin domain with conserved cysteine residues and WSTWS motif. The transmembrane region was followed by a short cytoplasmic tail which did not contain a tyrosine kinase domain. Interaction of the human IL-11R alpha with murine gp130 was demonstrated: expression of the human IL-11R alpha in murine M1 cells which constitutively express murine gp130 (and murine LIF receptor), resulted in the generation of specific high-affinity binding sites for IL-11 (Kd = 250 pM). In addition, expression of the human IL-11R alpha in these cells permitted the induction of macrophage differentiation in response to IL-11. These results suggested that the human IL-11R alpha chain was able to form a functional receptor complex in association with murine gp130. The requirement of gp130 for signalling was confirmed by expression of the human IL-11R alpha in Ba/F3 cells. BaF3 cells that expressed the human IL-11R alpha alone showed binding of radiolabelled IL-11 but no proliferative response. Introduction of human gp130 into these cells resulted in high-affinity IL-11 binding sites and IL-11 dependent cellular proliferation. Thus these results demonstrated the absolute requirement of gp130 for signalling.

Expression and function of members of the cytokine receptor superfamily on breast cancer cells.

Receptors for the cytokines leukemia inhibitory factor (LIF), interleukin-6 (IL-6), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and interleukin-11 (IL-11) are members of the structurally conserved hemopoietin receptor superfamily. In addition, they all share the transmembrane signalling protein gp130. In this paper the expression and function of this family of receptors in breast cancer cells was examined. RT-PCR analyses demonstrated that gp130 was expressed in 12/12 breast cell lines and the specific receptor alpha-chains for IL-6, LIF, IL-11 and CNTF were expressed in the majority of these cell lines. This was in contrast to other hemopoietin receptors. Examination of 50 clinical samples of malignant breast tissue by RT-PCR showed a similar pattern of expression of gp130 associated receptors. Treatment of breast cancer cell lines with OSM resulted in changes in cellular morphology. Cellular proliferation was inhibited following exposure to OSM (3/4 cell lines), IL-11 (2/4 cell lines), and by IL-6 and LIF (1/4 cell lines). Cell surface binding of LIF and OSM was also documented. The expression of these receptors in 12/12 cell lines and greater than 95% of clinical samples suggests that these molecules may be important in regulating the growth of breast cells.

Clonal analysis of the actions of the murine leukemia inhibitory factor on leukemic and normal murine hemopoietic cells.

The in vitro actions of leukemia inhibitory factor (LIF) purified from Krebs tumor conditioned medium, were analyzed on murine leukemic M1 and WEHI-3B D+ cells and on normal hemopoietic progenitor cells. LIF has no observable effects on WEHI-3B D+ cells but rapidly induced macrophage differentiation and loss of clonogenicity in M1 cells, resulting in the formation of abortive clones or differentiating colonies of reduced size and number. These effects were observable within one to two cell divisions in the presence of LIF and were irreversible. Addition of macrophage-colony-stimulating factor (CSF) but not granulocyte/macrophage-CSF, granulocyte-CSF, or multi-CSF reduced the LIF-induced suppression of colony numbers and size. G-CSF had a slower differentiation-inducing action on M1 cells than LIF but potentiated the differentiation-inducing effects of low concentrations of LIF. LIF had no colony-stimulating activity for normal granulocyte-macrophage progenitor cells and did not alter their quantitative responsiveness to CSF. However, culture of normal progenitor cells in the presence of LIF, but initial absence of CSF, reduced the survival of these cells. The differing actions of LIF and G-CSF on M1 leukemic cells suggest the existence of distinct mechanisms for inducing macrophage differentiation in these leukemic cells.

Production of hematopoietic regulatory factors in cultures of adult and fetal mouse organs: measurement by specific bioassays.

Factor-specific cell line bioassays were used to monitor the production in vitro by adult and fetal mouse organs of GM-CSF, G-CSF, M-CSF, Multi-CSF (IL-3), IL-6 and leukemia inhibitory factor (LIF). No tissue was observed to produce Multi-CSF. Highest producers of the other regulators were lung, muscle, thymus, heart and bone shaft and all tissues producing detectable growth factors produced all five with the same rank order of activity. Adult tissues produced more GM-CSF than G-CSF and less M-CSF than either, no differences being observed between male, female and pregnant female tissues. In contrast, the pregnant uterus produced high levels of M-CSF as did the fetal membranes and tissues with only low GM-CSF and no G-CSF production. Pre-irradiation did not alter the pattern of regulator production by adult tissues. The intravenous injection of endotoxin elevated serum levels of GM-CSF, G-CSF, M-CSF and IL-6 but the dominant rise was in G-CSF levels. The data indicating that multiple organs produce the regulators monitored in a common rank order suggest some overall linkage in their production with major differences between adult and fetal tissues.

Aberrant hematopoiesis in mice with inactivation of the gene encoding SOCS-1.

Mice with homozygous inactivation of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) protein die within 21 days of birth with low body weight, fatty degeneration and necrosis of the liver, infiltration of the lung, pancreas, heart and skin by macrophages and granulocytes and a profound depletion of T- and B-lymphocytes. In the present study, SOCS-1 -/- mice were found to have a moderate neutrophilia, and reduced platelet and hematocrit levels. Replacement of the SOCS-1 gene by a lac-Z reporter gene allowed documentation by FACS sorting that at least a proportion of granulocyte-macrophage progenitor cells transcribe SOCS-1. Most hematopoietic progenitor cell frequencies were normal in -/- marrow as were the size and cellular content of colonies formed by -/- progenitor cells in response to various stimulating factors. However, there was an increased frequency of macrophage progenitor cells in -/- mice and, abnormally, one quarter of all progenitor cells were located in the liver. Progenitor cells from -/- mice were hyper-responsive to stimulation by GM-CSF but not by M-CSF or Multi-CSF (IL-3). Progenitor cells from -/- mice were also hypersensitive to inhibition by interferon-gamma (IFN-gamma), the degree of inhibition varying markedly with the stimulating factor used. The suppressive effects of IFN-gamma therefore appear to involve interactions with particular growth factor-initiated signals in -/- cells--interactions that are strongly modulated by the action of the SOCS-1 protein.

Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins.

In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS proteins it was found that the ability of SOCS proteins to inhibit GH-mediated transcription was located in the amino-terminal 40-80 amino acids. In SOCS-3, 46 amino acids C-terminal to the SH2 domain were required for the inhibitory activity, while a truncated SOCS-1 having only 2 amino acids C-terminal to the SH2 domain was able to inhibit GH-mediated transcription. Both SOCS-1 and SOCS-3 were able to inhibit GH-induced STAT5 (signal transducer and activator of transcription) activation. SOCS-1 inhibited the tyrosine kinase activity of Janus kinase 2 (JAK2) directly, while SOCS-3 only inhibited JAK2 when stimulated by the GH receptor. All four SOCS proteins were able to bind to a tyrosine-phosphorylated glutathione-S-transferase-GH receptor fusion protein, and SOCS-3 required the same 46 C-terminal amino acids for GH receptor binding as it did for inhibition of GH-mediated transcription and STAT5 activation. These data suggest that SOCS-1 and -3 can suppress GH-induced transcriptional activity, presumably by inhibiting the kinase activity of JAK2 either directly in the case of SOCS-1 or via binding to the tyrosine-phosphorylated GH receptor in the case of SOCS-3.

Negative regulation of cytokine signaling.

Cytokines use complex signaling cascades to elicit their biological effects, many of which involve phosphorylation as a mechanism of activation. Rapid and efficient attenuation of cytokine signals is crucial to maintaining regulation of these processes and to preventing toxic side effects. Phosphatases have been shown to be involved in these regulatory processes, but more recent research has seen the discovery of two new families of negative regulators, the suppressor of cytokine signaling (SOCS) and protein inhibitors of signal transducer and activator of transcription (STAT) (PIAS) protein families. SOCS proteins are induced by and inhibit many cytokine-signaling systems in a classic negative-feedback loop, and the generation of transgenic and knockout models has greatly increased our understanding of their physiological functions. PIAS proteins interact with the transcriptional mediators of cytokine action, the STATs, to suppress their DNA-binding activity. These three classes of molecules form what is now emerging as an integrated system for deactivating cytokine signaling at a number of levels, from the receptor to the transcription factor.