Spinal neurons that express NK-1 receptors modulate descending controls that project through the dorsolateral funiculus

Selective ablation of spinal neurons possessing substance P receptors (NK-1 receptors) using the selective cytotoxin conjugate substance P-saporin (SP-SAP) decreases hyperalgesia and central sensitization. The mechanisms by which NK-1 expressing neurons modulate the excitability of other dorsal horn neurons are unclear. Because the majority of NK-1 expressing spinal neurons project rostrally, it is possible that they are part of a spinal-supraspinal circuitry that contributes to descending modulation of excitability of spinal nociceptive neurons. We therefore determined whether ablation of spinal neurons that possess the NK-1 receptor altered descending systems that travel via the dorsolateral funiculus (DLF). Spontaneous activity and responses of dorsal horn neurons evoked by mechanical (von Frey monofilaments) and heat (35-51 degrees C) stimuli were determined before and after transection of the DLF and were compared in rats pretreated with intrathecal application of vehicle or SP-SAP. In vehicle-treated rats, transection of the DLF caused a 233% increase in mean spontaneous activity of neurons and enhanced their responses to mechanical and heat stimuli, whereas these increases in excitation were blocked in rats pretreated with SP-SAP. Importantly, SP-SAP alone had no effect on spontaneous or evoked activity in the absence of DLF transection. These results demonstrate that spinal neurons expressing the NK-1 receptor appear to play a pivotal role in regulating descending systems that modulate activity of nociceptive dorsal horn neurons.     

Sensitivity of sup35 and sup45 suppressor mutants in Saccharomyces cerevisiae to the anti-microtubule drug benomyl

 SUP35 and SUP45 genes determine the accuracy of translation at the stage of termination. We present indirect evidence indicating that these genes may also control some cellular process mediated by microtubules. A majority of sup35 and sup45 suppressor mutations confer supersensitivity to benomyl, the drug which de-polymerizes microtubules. In addition, data correlating phenotypic manifestations of sup45 suppressor mutations, involving sensitivity to benomyl, respiratory deficiency and a suppressor effect, are also presented. Received: 16 October 1995/7 December 1995     

Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro

We investigated effects of the paracrine factors secreted by human mesenchymal stem cells (hMSCs) on endothelial cell migration, extracellular matrix invasion, proliferation, and survival in vitro. Human mesenchymal stem cells were cultured as a monolayer or as three-dimensional aggregates in hanging drops (hMSC spheroids). We performed analysis of paracrine factors in medium conditioned by a monolayer of hMSCs and hMSC spheroids. Concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiogenin, procathepsin B, interleukin (IL)-11, and bone morphogenic protein 2 were increased 5-20 times in medium conditioned by hMSC spheroids, whereas concentrations of IL-6, IL-8, and monocyte hemoattractant protein-1 were not increased. Concentrations of VEGF and angiogenin in medium conditioned by hMSC spheroids showed a weak dependence on the presence of serum, which allows serum-free conditioned medium with elevated concentrations of angiogenic cytokines to be obtained. Medium conditioned by hMSC spheroids was more effective in stimulation of umbilical vein endothelial cell proliferation, migration, and basement membrane invasion than medium conditioned by a monolayer of hMSCs. This medium also promotes endothelial cell survival in vitro. We suggest that culturing of hMSCs as three-dimensional cellular aggregates provides a method to concentrate proangiogenic factors secreted by hMSCs and allows for reduction of serum concentration in conditioned medium. Our data support the hypothesis that hMSCs serve as trophic mediators for endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.     

Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins

Development of vaccines against cytomegalovirus (CMV) is an important public health priority. We used a propagation-defective, single-cycle RNA replicon vector system derived from an attenuated strain of an alphavirus, Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRP) expressing various combinations of pp65, IE1, or gB proteins of human CMV. Protein expression in VRP-infected cells was highest with single-promoter replicons expressing pp65, IE1, a pp65/IE1 fusion protein, or the extracellular domain of gB and with double-promoter replicons expressing pp65 and IE1. Protein expression was lower with double- and triple-promoter replicons expressing gB, especially the full-length form of gB. BALB/c mice immunized with VRP expressing gB developed high titers of neutralizing antibody to CMV, and mice immunized with VRP expressing pp65, IE1, or a pp65/IE1 fusion protein developed robust antigen-specific T-cell responses as measured by gamma interferon enzyme-linked immunospot assay. Three overlapping immunodominant pp65 peptides contained a nine-amino-acid sequence (LGPISGHVL) that matches the consensus binding motif for a major histocompatibility complex H2-D(d) T-cell epitope. These data provide the basis for further development and clinical evaluation of an alphavirus replicon vaccine for CMV expressing the pp65, IE1, and gB proteins.     

Biphasic, triphasic and multiphasic calcium orthophosphates

Biphasic, triphasic and multiphasic (polyphasic) calcium orthophosphates have been sought as biomaterials for reconstruction of bone defects in maxillofacial, dental and orthopedic applications. In general, this concept is determined by advantageous balances of more stable (frequently hydroxyapatite) and more resorbable (typically tricalcium orthophosphates) phases of calcium orthophosphates, while the optimum ratios depend on the particular applications. Therefore, all currently known biphasic, triphasic and multiphasic formulations of calcium orthophosphate bioceramics are sparingly soluble in water and, thus, after being implanted they are gradually resorbed inside the body, releasing calcium and orthophosphate ions into the biological medium and, hence, seeding new bone formation. The available formulations have already demonstrated proven biocompatibility, osteoconductivity, safety and predictability in vitro, in vivo, as well as in clinical models. More recently, in vitro and in vivo studies have shown that some of them might possess osteoinductive properties. Hence, in the field of tissue engineering biphasic, triphasic and multiphasic calcium orthophosphates represent promising biomaterials to construct various scaffolds capable of carrying and/or modulating the behavior of cells. Furthermore, such scaffolds are also suitable for drug delivery applications. This review summarizes the available information on biphasic, triphasic and multiphasic calcium orthophosphates, including their biomedical applications. New formulations are also proposed.     

Kinetic methods to study isothermal and nonisothermal epoxy-anhydride cure

The kinetics of epoxy-anhydride cure were studied by differential scanning calorimetry under isothermal and nonisothermal conditions. The data were fitted to single-step and to two-step kinetic models. In both cases the activation energies derived from nonisothermal measurements differ from the values obtained for the isothermal cure data. Unlike the model-fitting methods, a model-free isoconversional method of kinetic analysis yields matching dependencies of the activation energy on the extent of conversion for isothermal and nonisothermal experiments. For both dependencies, the effective activation energy increases from 20 kJ·mol^–1 at the beginning of cure to 70 kJ·mol^–1 when the process is near completion. The occurrence of the dependence suggests that the cure kinetics is determined by competing reactions. The model-fitting methods have not been capable of reliably detecting the complex character of the cure kinetics.     

Inorganic coatings for cardiovascular stents: In vitro and in vivo studies

The in-vitro and in-vivo biocompatibility of two oxides (TiO and ZrO) and diamond-like carbon (D) coated stents has been assessed and compared with uncoated stainless steel (St) stents. In vitro studies demonstrated that both fibrinogen adsorption and platelet adhesion were significantly higher on D coating compared to those on oxide coatings and uncoated stainless steel. In addition TiO and ZrO coatings showed evidence of a minor inflammatory response and more complete endothelialization of the aorta than that seen around D coated and uncoated St stents. The resulting neointimal growth in the aorta with TiO, ZrO, and D coated and uncoated St stents, measured 8 weeks after stenting (the ratio of the neointima in the stented artery to the non-stented artery) was 1.03 + 0.28, 0.85 + 0.36, 1.78 + 1.26, and 1.15 + 0.56, accordingly. From the data obtained it could be concluded that the increased neointima measured around D-coated stents, may be due to both, the inferior haemocompatibility of the diamond-like carbon coating and mechanical instability of D coating observed in an in vivo environment.     

Novel Polymeric Nanoparticles Assembled by Metal Ion Addition

Potentially biodegradable graft polymer core‐shell nanoparticles assembling by addition of metal ions intended to be used for radionuclide and drug delivery purposes are described. With Cu²⁺ ions, the self‐assembly is slow (within minutes) with low efficiency. With Fe³⁺ ions the nanoparticles are formed immediately and are of convenient size for passive tumor accumulation by the enhanced permeation and retention effect. Full in vitro degradation of these particles is achieved with deferoxamine as a model of in vivo transchelation capacity.     

Assemblathon 1: a competitive assessment of de novo short read assembly methods

Low-cost short read sequencing technology has revolutionized genomics, though it is only just becoming practical for the high-quality de novo assembly of a novel large genome. We describe the Assemblathon 1 competition, which aimed to comprehensively assess the state of the art in de novo assembly methods when applied to current sequencing technologies. In a collaborative effort, teams were asked to assemble a simulated Illumina HiSeq data set of an unknown, simulated diploid genome. A total of 41 assemblies from 17 different groups were received. Novel haplotype aware assessments of coverage, contiguity, structure, base calling, and copy number were made. We establish that within this benchmark: (1) It is possible to assemble the genome to a high level of coverage and accuracy, and that (2) large differences exist between the assemblies, suggesting room for further improvements in current methods. The simulated benchmark, including the correct answer, the assemblies, and the code that was used to evaluate the assemblies is now public and freely available from http://www.assemblathon.org/.