Healing of sub-critical femoral osteotomies in mice is unaffected by tacrolimus and deletion of recombination activating gene 1.

Clinical management of delayed healing or non-union of long bone fractures and segmental defects poses a substantial orthopaedic challenge. There are suggestions in the literature that bone healing may be enhanced by inhibiting the activities of T and B lymphocytes, but this remains controversial. To examine this matter in more detail, sub-critical-sized segmental defects were created in the femora of mice and it was assessed whether there might be a benefit from the administration of a Food and Drug Administration (FDA)-approved drug that blocks T cell activation (tacrolimus). Defects were stabilised using an internal plate. In certain groups of animals, 1 mg/kg or 10 mg/kg tacrolimus was delivered locally to the defect site for 3 or 7 d using an implanted osmotic pump with a silicon catheter directing drug delivery into the defect area. Healing was monitored by weekly X-ray and assessed at 12 weeks by mechanical testing, µCT and histology. Radiographic and histological evaluations revealed that 100 % of defects healed well regardless of tacrolimus dosage or duration. A comparison of healed C57BL/6 and Rag1-/- femora by µCT and ex vivo torsion testing showed no differences within mouse strains in terms of bone volume, tissue volume, bone volume/tissue volume ratio, shear modulus, torsional rigidity or torsional stiffness. These data failed to support an important role for tacrolimus in modulating the natural healing of segmental defects under those experimental conditions.

The occupied electronic structure of ultrathin boron doped diamond.

Using angle-resolved photoelectron spectroscopy, we compare the electronic band structure of an ultrathin (1.8 nm) δ-layer of boron-doped diamond with a bulk-like boron doped diamond film (3 µm). Surprisingly, the measurements indicate that except for a small change in the effective mass, there is no significant difference between the electronic structure of these samples, irrespective of their physical dimensionality, except for a small modification of the effective mass. While this suggests that, at the current time, it is not possible to fabricate boron-doped diamond structures with quantum properties, it also means that nanoscale boron doped diamond structures can be fabricated which retain the classical electronic properties of bulk-doped diamond, without a need to consider the influence of quantum confinement.

Simultaneous Conduction and Valence Band Quantization in Ultrashallow High-Density Doping Profiles in Semiconductors.

We demonstrate simultaneous quantization of conduction band (CB) and valence band (VB) states in silicon using ultrashallow, high-density, phosphorus doping profiles (so-called Si:P δ layers). We show that, in addition to the well-known quantization of CB states within the dopant plane, the confinement of VB-derived states between the subsurface P dopant layer and the Si surface gives rise to a simultaneous quantization of VB states in this narrow region. We also show that the VB quantization can be explained using a simple particle-in-a-box model, and that the number and energy separation of the quantized VB states depend on the depth of the P dopant layer beneath the Si surface. Since the quantized CB states do not show a strong dependence on the dopant depth (but rather on the dopant density), it is straightforward to exhibit control over the properties of the quantized CB and VB states independently of each other by choosing the dopant density and depth accordingly, thus offering new possibilities for engineering quantum matter.

Ubiquitous formation of bulk Dirac cones and topological surface states from a single orbital manifold in transition-metal dichalcogenides.

Transition-metal dichalcogenides (TMDs) are renowned for their rich and varied bulk properties, while their single-layer variants have become one of the most prominent examples of two-dimensional materials beyond graphene. Their disparate ground states largely depend on transition metal d-electron-derived electronic states, on which the vast majority of attention has been concentrated to date. Here, we focus on the chalcogen-derived states. From density-functional theory calculations together with spin- and angle-resolved photoemission, we find that these generically host a co-existence of type-I and type-II three-dimensional bulk Dirac fermions as well as ladders of topological surface states and surface resonances. We demonstrate how these naturally arise within a single p-orbital manifold as a general consequence of a trigonal crystal field, and as such can be expected across a large number of compounds. Already, we demonstrate their existence in six separate TMDs, opening routes to tune, and ultimately exploit, their topological physics.

Spin-valley locking in the normal state of a transition-metal dichalcogenide superconductor.

Metallic transition-metal dichalcogenides (TMDCs) are benchmark systems for studying and controlling intertwined electronic orders in solids, with superconductivity developing from a charge-density wave state. The interplay between such phases is thought to play a critical role in the unconventional superconductivity of cuprates, Fe-based and heavy-fermion systems, yet even for the more moderately-correlated TMDCs, their nature and origins have proved controversial. Here, we study a prototypical example, 2H-NbSe2, by spin- and angle-resolved photoemission and first-principles theory. We find that the normal state, from which its hallmark collective phases emerge, is characterized by quasiparticles whose spin is locked to their valley pseudospin. This results from a combination of strong spin-orbit interactions and local inversion symmetry breaking, while interlayer coupling further drives a rich three-dimensional momentum dependence of the underlying Fermi-surface spin texture. These findings necessitate a re-investigation of the nature of charge order and superconducting pairing in NbSe2 and related TMDCs.

Rapid and reliable healing of critical size bone defects with genetically modified sheep muscle.

Large segmental defects in bone fail to heal and remain a clinical problem. Muscle is highly osteogenic, and preliminary data suggest that autologous muscle tissue expressing bone morphogenetic protein-2 (BMP-2) efficiently heals critical size defects in rats. Translation into possible human clinical trials requires, inter alia, demonstration of efficacy in a large animal, such as the sheep. Scale-up is fraught with numerous biological, anatomical, mechanical and structural variables, which cannot be addressed systematically because of cost and other practical issues. For this reason, we developed a translational model enabling us to isolate the biological question of whether sheep muscle, transduced with adenovirus expressing BMP-2, could heal critical size defects in vivo. Initial experiments in athymic rats noted strong healing in only about one-third of animals because of unexpected immune responses to sheep antigens. For this reason, subsequent experiments were performed with Fischer rats under transient immunosuppression. Such experiments confirmed remarkably rapid and reliable healing of the defects in all rats, with bridging by 2 weeks and remodelling as early as 3-4 weeks, despite BMP-2 production only in nanogram quantities and persisting for only 1-3 weeks. By 8 weeks the healed defects contained well-organised new bone with advanced neo-cortication and abundant marrow. Bone mineral content and mechanical strength were close to normal values. These data demonstrate the utility of this model when adapting this technology for bone healing in sheep, as a prelude to human clinical trials.

Excitation of coherent phonons in the one-dimensional Bi(114) surface.

We present time-resolved photoemission experiments from a peculiar bismuth surface, Bi(114). The strong one-dimensional character of this surface is reflected in the Fermi surface, which consists of spin-polarized straight lines. Our results show that the depletion of the surface state and the population of the bulk conduction band after the initial optical excitation persist for very long times. The disequilibrium within the hot electron gas along with strong electron-phonon coupling cause a displacive excitation of coherent phonons, which in turn are reflected in coherent modulations of the electronic states. Beside the well-known A(1g) bulk phonon mode at 2.76 THz, the time-resolved photoelectron spectra reveal a second mode at 0.72 THz which can be attributed to an optical surface phonon mode along the atomic rows of the Bi(114) surface.

Direct measurement of electrical conductance through a self-assembled molecular layer.

The self-assembly of organic molecules on surfaces is a promising approach for the development of nanoelectronic devices. Although a variety of strategies have been used to establish stable links between molecules, little is known about the electrical conductance of these links. Extended electronic states, a prerequisite for good conductance, have been observed for molecules adsorbed on metal surfaces. However, direct conductance measurements through a single layer of molecules are only possible if the molecules are adsorbed on a poorly conducting substrate. Here we use a nanoscale four-point probe to measure the conductivity of a self-assembled layer of cobalt phthalocyanine on a silver-terminated silicon surface as a function of thickness. For low thicknesses, the cobalt phthalocyanine molecules lie flat on the substrate, and their main effect is to reduce the conductivity of the substrate. At higher thicknesses, the cobalt phthalocyanine molecules stand up to form stacks and begin to conduct. These results connect the electronic structure and orientation of molecular monolayer and few-layer systems to their transport properties, and should aid in the rational design of future devices.

Nondegenerate metallic States on Bi(114): a one-dimensional topological metal.

The (114) surface of the semimetal Bi is found to support a quasi-one-dimensional, metallic surface state. As required by symmetry, the state is degenerate along the Gamma-Y line of the surface Brillouin zone with a highest binding energy of approximately 150 meV. In the Gamma-X direction the degeneracy is lifted by the strong spin-orbit splitting in Bi, as directly shown by spin-resolved photoemission. This results in a Fermi contour consisting of two closely separated, parallel lines of opposite spin direction. It is argued that similar states on related insulators would give rise to a one-dimensional quantum spin Hall effect.

Interleukin-1beta and tumor necrosis factor alpha inhibit chondrogenesis by human mesenchymal stem cells through NF-kappaB-dependent pathways.

OBJECTIVE: The differentiation of mesenchymal stem cells (MSCs) into chondrocytes provides an attractive basis for the repair and regeneration of articular cartilage. Under clinical conditions, chondrogenesis will often need to occur in the presence of mediators of inflammation produced in response to injury or disease. The purpose of this study was to examine the effects of 2 important inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), on the chondrogenic behavior of human MSCs. METHODS: Aggregate cultures of MSCs recovered from the femoral intermedullary canal were used. Chondrogenesis was assessed by the expression of relevant transcripts by quantitative reverse transcription-polymerase chain reaction analysis and examination of aggregates by histologic and immunohistochemical analyses. The possible involvement of NF-kappaB in mediating the effects of IL-1beta was examined by delivering a luciferase reporter construct and a dominant-negative inhibitor of NF-kappaB (suppressor-repressor form of IkappaB [srIkappaB]) with adenovirus vectors. RESULTS: Both IL-1beta and TNFalpha inhibited chondrogenesis in a dose-dependent manner. This was associated with a marked activation of NF-kappaB. Delivery of srIkappaB abrogated the activation of NF-kappaB and rescued the chondrogenic response. Although expression of type X collagen followed this pattern, other markers of hypertrophic differentiation responded differently. Matrix metalloproteinase 13 was induced by IL-1beta in a NF-kappaB-dependent manner. Alkaline phosphatase activity, in contrast, was inhibited by IL-1beta regardless of srIkappaB delivery. CONCLUSION: Cell-based repair of lesions in articular cartilage will be compromised in inflamed joints. Strategies for enabling repair under these conditions include the use of specific antagonists of individual pyrogens, such as IL-1beta and TNFalpha, or the targeting of important intracellular mediators, such as NF-kappaB.

Suppression of airway inflammation by a natural acute infection of the intestinal epithelium.

Although chronic intestinal helminth infections may suppress allergen-induced airway pathology by inducing a combination of modified T-helper (Th) 2 and immunosuppressive cytokines, a similar capacity of natural acute intestinal infections has remained untested, despite their global prevalence. Here, we show that allergic airway phenotypes including eosinophilia, eotaxin mRNA, and Th2 cytokines are significantly suppressed in animals that were infected by and that have cleared the intestinal parasite Eimeria vermiformis. Unlike in helminth-infected animals, regulation requires temporal coincidence of infection with sensitization; depends on interferon-gamma; and is not associated with an enhanced antigen-specific immunoglobulin G1 response. Moreover, regulation was effective following allergen sensitization in different anatomical sites, and in young and adult mice. These data highlight a transient anatomical dissemination of "functional immunologic dominance" following infection of the gut mucosa. They strongly support the hypothesis that airway allergies are naturally suppressed by both acute and chronic mucosal pathogens, but by different mechanisms.

Suppression of allergic airway inflammation and IgE responses by a class I restricted allergen peptide vaccine.

CD8 T cells are known to deviate CD4 T-cell responses from Th2 toward Th1. Reduction of Th2 cytokines and increased interferon-gamma ameliorates allergic airway disease. We have developed a novel approach to the suppression of allergic airway inflammation, by designing a MHC class I-restricted allergen peptide vaccine, which induces potent and long-lived CD8 T-cell responses. Vaccination of C57BL/6 mice before allergen sensitization completely prevented allergen-specific immunoglobulin E (IgE) antibody responses. Vaccination after sensitization failed to suppress IgE, but inhibited accumulation of eosinophils and neutrophils in airways after subsequent allergen challenge. Vaccination suppressed Th2 airway infiltration and enhanced the lung Th1 response without inducing excessive CD8 cellular infiltration or interleukin-17, and the combination of class I peptide with adjuvant was more effective than adjuvant alone. Airway hyperreactivity was prevented by vaccination in an allergen-specific fashion. Class I peptide vaccines might therefore represent a robust and long-lasting immunotherapeutic strategy in allergic disease.

Use of genetically modified muscle and fat grafts to repair defects in bone and cartilage.

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human bone morphogenetic protein-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess bone healing, the genetically modified ("gene activated") tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as 10 days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed bone were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal bone and cartilage more expeditiously, and at lower cost, than is presently possible.

Surface-sensitive conductance measurements.

Several approaches for surface-sensitive conductance measurements are reviewed. Particular emphasis is placed on nanoscale multi-point probe techniques. The results for two model systems, which have given rise to some dispute, are discussed in detail: Si(111)(7 × 7) and ([Formula: see text])Ag-Si(111). Other recent examples are also given, such as phase transitions in quasi-one-dimensional structures on semiconductor surfaces and the surface sheet conductivity of Bi(111), the surface of a semimetal.

Thermal switching of the electrical conductivity of Si(111)([Formula: see text])Ag due to a surface phase transition.

The temperature-dependent surface conductivity of the Si(111)([Formula: see text])Ag surface was measured using a microscopic four-point probe. The conductivity was found to undergo a sharp increase of about three orders of magnitude when the system was heated above about 220 K. This strong conductivity change is reversible and attributed to the phase transition which is generally believed to occur on this surface. It is also shown that, in order to find the true surface conductivity, it is necessary to separate it from the contribution of the bulk and space charge layer. In this work, this is achieved by using a finite-element model. A percolating network of Ag islands on Si(111) was also studied and a much simpler behaviour (compared to that of Si(111)([Formula: see text])Ag) was found. The temperature-dependent conductivity of this system was found to display typical metallic behaviour. The absolute value of the conductivity is comparable to the value expected by modelling the Ag film as exhibiting the bulk Ag transport properties.

Disentangling surface, bulk, and space-charge-layer conductivity in Si(111)-(7 x 7).

A novel approach for extracting genuine surface conductivities is presented and illustrated using the unresolved example of Si(111)-(7 x 7). Its temperature-dependent conductivity was measured with a microscopic four point probe between room temperature and 100 K. At room temperature the measured conductance corresponds to that expected from the bulk doping level. However, as the temperatures is lowered below approximately 200 K, the conductance decreases by several orders of magnitude in a small temperature range and it saturates at a low temperature value of approximately 4 x 10(-8) Omega(-1), irrespective of bulk doping. This abrupt transition is interpreted as the switching from bulk to surface conduction, an interpretation which is supported by a numerical model for the measured four point probe conductance. The value of the surface conductance is considerably lower than that of a good metal.

Influence of interleukin-4 on the phenotype and function of bone marrow-derived murine dendritic cells generated under serum-free conditions.

Murine bone marrow-derived dendritic cells (DC) can be generated by culture in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) alone or GM-CSF in conjunction with interleukin-4 (IL-4). However, these two culture methods result in the production of heterogeneous DC populations with distinct phenotypic and stimulatory properties. In this study, we investigated the properties of DC generated under serum-free conditions in the presence or absence of IL-4 and compared their yield and phenotype to that of DC generated in the presence of fetal calf serum (FCS) (+/-IL-4). We did not observe a significant difference in the total cell yield between these four culture conditions, although the proportion of CD11c+ DC in cultures that received FCS was higher than that of their counterparts generated under serum-free conditions. Also, the four culture conditions generated CD11c+ DC with comparable levels of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86 expression, with the exception of cells cultured under serum-free conditions in the absence of IL-4, which displayed suboptimal levels of these markers. Moreover, we compared the functional and stimulatory properties of DC generated under serum-free conditions in the presence or absence of IL-4. DC cultured in the presence of IL-4 were stronger stimulators of allogeneic splenocytes in a primary mixed lymphocyte reaction (MLR) and of naive antigen-specific OT-II transgenic T cells when pulsed with the class II ovalbumin (OVA)323-339 peptide or whole OVA protein than DC cultured in the absence of IL-4. However, both DC populations displayed a similar capacity to take up fluorescein isothiocyanate (FITC)-albumin by macropinocytosis and FITC-Dextran by the mannose receptor and to secrete IL-12 in response to stimulation with lipopolysaccharide (LPS) or an agonistic anti-CD40 monoclonal antibody. Therefore, we conclude that although both DC culture methods result in the production of DC with similar functional abilities, under serum-free conditions, DC cultured in GM-CSF and IL-4 show an increased stimulatory potential over DC cultured in GM-CSF alone. This is an important consideration in the design of experiments where DC are being exploited as immunotherapeutic vaccines.

Apparent capacity of cardiac muscarinic receptors for different radiolabeled antagonists.

Muscarinic receptors in sarcolemmal membranes, digitonin-solubilized extracts, and purified preparations from porcine atria have revealed a shortfall in the apparent capacity for N-[3H]methylscopolamine, which was only about 75% of that for [3H]quinuclidinylbenzilate. Since binding at near-saturating concentrations of [3H]quinuclidinylbenzilate was inhibited fully at comparatively low concentrations of unlabeled N-methylscopolamine, the data are inconsistent with the notion that [3H]quinuclidinylbenzilate binds selectively to a subclass of distinct, non-interconverting, and mutually independent sites. The discrepancy is resolved by adjusting the specific activity of N-[3H]methylscopolamine to account for unlabeled scopolamine that was identified in some batches of the radioligand. Also, there was no shortfall in capacity when N-[3H]methylscopolamine was devoid of scopolamine, and the predicted effect was obtained when pure N-[3H]methylscopolamine was supplemented with known amounts of scopolamine. A small discrepancy in the levels of scopolamine estimated pharmacologically and by mass spectrometry can be attributed largely to a difference in the efficiency of ionization between scopolamine and N-methylscopolamine. Different capacities for different radioligands are not uncommon with muscarinic and other G protein-coupled receptors, and in some cases the effect may have been due wholly or in part to an unlabeled impurity. Binding data can be mechanistically ambiguous, particularly when acquired only at graded concentrations of the radioligand. The predicted effects of an unlabeled impurity mimic or resemble those of alternative scenarios such as sequestration behind a hydrophobic barrier, a nucleotide-regulated interconversion from one state of affinity to another, and cooperativity between interacting sites.

Nature of the oligomers formed by muscarinic m2 acetylcholine receptors in Sf9 cells.

Wild-type, FLAG-tagged, and c-myc-tagged muscarinic m2 receptors extracted in digitonin-cholate from singly and co-infected Sf9 (Spodoptera frugiperda) cells were indistinguishable in their binding of [3H]quinuclidinylbenzilate, either before or after purification. The FLAG epitope was found to coimmunoprecipitate with the c-myc epitope when co-infected cells were solubilised in digitonin-cholate, n-dodecyl-beta-D-maltoside or Lubrol-PX. The degree of coprecipitation in digitonin-cholate was unaffected by preincubation of the extract for up to 60 min at 30 degrees C, with or without muscarinic receptor ligands; no coimmunoprecipitation occurred in mixed extracts from singly infected cells. As measured by [3H]quinuclidinylbenzilate, the efficiency of immunoprecipitation from co-infected cells was 87% of that from singly infected cells. The amount of receptor immunoprecipitated from the latter, as determined by densitometry, was 2.3-fold that expected from the loss of binding from the extract. The data suggest that at least some of the receptors were trimeric or larger and that oligomers neither formed nor dissociated under the conditions of the experiments. Also, some receptors appear to be non-functional or latent in digitonin-solubilised extracts.