Regulated expression of transgene production and function is of great importance for gene therapy. Such regulation can potentially be used to monitor and control complex biological processes. We report here a regulated stem cell-based system for controlling bone regeneration, utilizing genetically engineered mesenchymal stem cells (MSCs) harboring a tetracycline-regulated expression vector encoding the osteogenic growth factor human BMP-2. We show that doxycycline (a tetracycline analogue) is able to control hBMP-2 expression and thus control MSC osteogenic differentiation both in vitro and in vivo. Following in vivo transplantation of genetically engineered MSCs, doxycycline administration controlled both bone formation and bone regeneration. Moreover, our findings showed increased angiogenesis accompanied by bone formation whenever genetically engineered MSCs were induced to express hBMP-2 in vivo. Thus, our results demonstrate that regulated gene expression in mesenchymal stem cells can be used as a means to control bone healing.
A new method for the treatment of correlation effects between modes in vibrational self-consistent-field (VSCF) calculations is introduced. It is based upon using a partially separable form for the wave function. As a result, some of the modes are treated as mutually fully correlated, while the rest are separable. The modes which are explicitly coupled together in the calculation are chosen on physical grounds. Trial calculations are performed upon H2O, H3O+, and CH3NH2 and indicate that the method performs well. The agreement with experiment for the explicitly coupled modes is improved when compared to both the vibrational self-consistent-field method and its correlation-corrected extension. When interfaced with an electronic structure code this method opens the way for the accurate first-principles prediction of vibrational frequencies of strongly coupled modes. If only a few modes are mutually strongly coupled, the method has a very favorable scaling with system size, as does VSCF itself. (C) 2001 American Institute of Physics.
Recent work by Rasanen and coworkers showed that photolysis of hydrides in rare-gas matrices results in part in formation of novel, rare-gas-containing molecules. Thus, photolysis of HCl in Xe and of H2O in Xe result respectively in formation of HXeCl and HXeOH in the Xe matrices. Ab initio calculations show that the compounds HRgY so formed are stable in isolation, and that by the strength and nature of the bonding these are molecules, very different from the corresponding weakly bound clusters Rg . . . HY. This paper presents a study of the formation mechanism of HRgY following the photolysis of HY in clusters Rg(n)(HY). Calculations are described for HXeCl, as a representative example. Potential energy surfaces that govern the formation of HXeCl in the photolysis of HCl in xenon clusters are obtained, and the dynamics on these surfaces is analyzed, partly with insight from trajectories of molecular dynamics simulations. The potential surfaces are obtained by a new variant of the DIM (diatomics in molecules) and DIIS (diatomics in ionic systems) models. Non-adiabatic couplings are also obtained. The main results are : (1) Properties of HXeCl predicted by the DIM-DIIS model are in reasonable accord with results of ab initio calculations. (2) The potential along the isomerization path HXeCl –> Xe . . . HCl predicted by DIM is in semiquantitative accord with the ab initio results. (3) Surface-hopping molecular dynamics simulations of the process in clusters, with ``on the fly'' calculations of the DIM-DIIS potentials and non-adiabatic couplings are computationally feasible. (4) Formation of HXeCl, following photolysis of HCl in Xe-54(HCl), requires cage-exit of the H atom as a precondition. The H atom and the Cl can then attack the same Xe atom on opposite sides, leading to charge transfer and production of the ionic HXeCl. (5) Non-adiabatic processes play an important role, both in the reagent configurations, and at the charge-transfer stage. The results open the way to predictions of the formation of new HRgY species.
It is sometimes stated that in order to be amenable to a simple statistical description. a system needs to have many coupled degrees of freedom. In this view, the statistical limit is to be understood from the dynamics. Here we discuss a complementary point of view where the role of `size' is to ensure that the probabilities of simple events, which are technically marginal probabilities, settle down to a canonical distribution, conditioned by additive constants of the motion. The requirement that the conditioning is on additive variables plays an essential technical role and it is not dear that this condition can be relaxed. In the view discussed here, the effective `size' is determined by the variance of the physical variables of interest. Implications for Monte Carlo sampling are also discussed, with examples. (C) 2001 Elsevier Science B.V. All rights reserved.
The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss-Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1-34) [hPTH(1-34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (microCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5-week and 9-week post-OVX, non-PTH-treated animals. PTH induced a approximately 35% recovery of this loss and a approximately 40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein-labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2- and 3-fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss-Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.
Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assessing the effectiveness of systems of gene therapy delivery. We applied a new light-detection cooled charged-coupled device (CCCD) camera for continuous in vivo assessment of commonly used gene therapy delivery systems (such as ex vivo manipulated cells, viral vectors, and naked DNA), without the need to kill animals. We examined a variety of criteria related to real-time monitoring of luciferase (luc) gene expression in tissues including bone, muscle, salivary glands, dermis, liver, peritoneum, testis, teeth, prostate, and bladder in living mice and rats. These criteria included determination of the efficiency of infection/transfection of various viral and nonviral delivery systems, promoter specificity, and visualization of luciferase activity, and of the ability of luciferin to reach various organs. The exposure time for detection of luc activity by the CCCD camera is relatively short (approximately 2 minutes) compared with the intensified CCD camera photon-counting method (approximately 15 minutes). Here we transduce a variety of vectors (such as viruses, transfected cells, and naked DNA) by various delivery methods, including electroporation, systemic injection of viruses, and tail-vein, high-velocity-high-volume administration of DNA plasmids. The location, intensity, and duration of luc expression in different organs were determined. The distribution of luciferin is most probably not a barrier for the detection of in vivo luciferase activity. We showed that the CCCD photon detection system is a simple, reproducible, and applicable method that enables the continuous monitoring of a gene delivery system in living animals.
Real time monitoring of organic monolayer self-assembly by molecular layer epitaxy (MLE) processes was studied by in situ spectroscopic ellipsometry techniques. For the MLE of imide-based organic heterostructures using chemisorption of 3,4,7,8-naphthalenetetracarboxylic dianhydride (NTCDA) and 1,6-diamino-n-hexane (DAH) on prefunctionalized surfaces, in situ ellipsometry reveals that the reaction kinetics can be best fitted to an S-shaped deposition curve with saturated coverage of about 20 min by the Langmuir−Hinshelwood model, with a slow initial phase, followed by a faster second phase. The rate of deposition at each moment is proportional to the number of empty sites multiplied by the number of occupied sites. Calculated deposition rate constants for every pulse are kT = 5.6 × 10-5 s-1 for first deposition of NTCDA on a template (T-layer), decreasing to kA = 1.5 × 10-5 s-1 for NTCDA and to kB = 7.2 × 10-6 s-1 for DAH assembly pulses correspondingly. A modified Rudzinski−Aharoni kinetic model for adsorption that correlates adsorption energy with valid numbers of reactive sites was used to estimate an equilibrium surface absorption energy of 16 kcal for a NTCDA layer and 29 kcal for a DAH layer.
An ink-jet ink compn. for piezoelec. in-jet printing comprises: (a) a hydrophobic dye; (b) an oil-in-water microemulsion including: (i) a volatile oil; (ii) water; (iii) a co-solvent; (iv) at least one surfactant; and (v) a binder, wherein upon application of the ink to a substrate surface, the volatile oil evaps. and nanoparticles are formed. The invention addnl. discloses a printing process using the ink compn. [on SciFinder(R)]
A therapeutic or cosmetic compn. for topical application, capable of stabilizing an active ingredient and delivering said ingredient, comprising a plurality of microcapsules having a core-shell structure and a diam. of approx. 0.1-100 μ. The core of each microcapsule includes at least one active ingredient, and is encapsulated within a microcapsular shell. The shell is comprised of at least one inorg. polymer obtained by a sol-gel process, and the shell protects the active ingredient before topical application and releases the ingredient after topical application. This compn. is useful to encapsulate active ingredients that are unstable in formulation, or are irritating to the skin. The present invention further discloses a process for the encapsulation of an active ingredient in the form of a dispersion within a hydrophobic phase. For example, combinations of erythromycin and benzoyl peroxide are useful in the treatment of acne but usually must be formulated as a two component system because of incompatibility of the two active ingredients. Thus, erythromycin was encapsulated in silica; 1.7 g of erythromycin was mixed with 14.9 g of octylmethoxy cinnamate, and 19.5 g of tetraethoxy silane (TEOS) was added. This oil phase was emulsified and the emulsion was poured into a basic soln. of pH 11.5. The mixt. was stirred at 50-240 rpm. Flocculation was induced by the addn. of MgSO4 at a final concn. of 0.1% by wt. The ppt. was collected by filtration and a product obtained was a paste with a particle size distribution of 1-12 μ (an av. size of 6.2 μ). Encapsulation of benzoyl peroxide (30 g of 7% soln. in diisopropyl sebacate) was carried out by mixing it with 20 g of TEOS. The org. phase was emulsified in 200 g of an aq. soln. contg. 1% CTAC under high shear. The emulsion obtained was poured into a reactor contg. 200 g NaOH aq. soln. at pH 10 and stirred. The final product was re-suspended in water to obtain a dispersion contg. a 3% benzoyl peroxide encapsulated in silica particles of 0.5015 μ. [on SciFinder(R)]
A review. This review describes the interfacial behavior of biomols., which were converted to more hydrophobic derivs. by covalent attachment of hydrophobic chains. The mols. presented are proteins (glucose oxidase, IgG, gelatin, ovalbumin) and polysaccharides (CM-cellulose, pullulan). In general, it was found that such hydrophobically modified biomols. have enhanced surface activity and ability to penetrate into phospholipid monolayers. In addn., it has been demonstrated, that such mols. can be used as efficient emulsifiers and foaming agents, and in unique biomedical application based on combining the surface activity and recognition ability. [on SciFinder(R)]
