Soft tissue regeneration in animal models using grafts from adipose mesenchymal stem cells and peripheral blood fibrin gel.

OBJECTIVE: Our study aimed to evaluate the effect of soft tissue regeneration in nude mice using grafts made from the combination of adipocytes from fat tissue mesenchymal stem cells and fibrin gel from peripheral blood. MATERIALS AND METHODS: Mesenchymal stem cells were isolated from adipose tissue and identified according to ISCT criteria. The scaffold used was fibrin obtained from peripheral blood. The grafts in this study were generated by transferring mesenchymal stem cells onto a fibrin scaffold. Two types of grafts, the research sample (fibrin scaffold containing adipocytes differentiated from mesenchymal stem cells) and the control sample (fibrin scaffold only), were grafted under the dorsal skin of the same mouse. After each research period, samples were collected and evaluated by histological methods to observe the existence and growth of cells inside the grafts. RESULTS: The results showed that the study group's graft integrated better within the tissue when compared with the control group. In addition, the grafts in the study group showed the presence of cells with characteristic morphology of adipocytes one week after transplantation. In contrast, control samples showed dimorphous shapes and features mainly composed of non-homogenous fragments. CONCLUSIONS: These initial conclusions might be considered a first step in generating safe bio-compatible engineered grafts specifically usable in post-traumatic tissue regeneration procedures.

Diagnostic performance of MRI perfusion and spectroscopy for brainstem glioma grading.

OBJECTIVE: This study investigated the roles of dynamic susceptibility contrast (DSC) perfusion and multivoxel magnetic resonance spectroscopy (MRS) in grading brainstem glioma (BSG). PATIENTS AND METHODS: Our retrospective study comprised 12 patients, including 6 with pathology verified low-grade BSGs and 6 with high-grade BSGs. We examined differences in age, relative cerebral blood volume (rCBV), regional cerebral blood flow (rCBF), and the metabolite ratios of choline (Cho)/N-acetyl aspartate (NAA) and Cho/creatine (Cr) between these two groups using the Mann-Whitney U test and Chi-square test. Receiver operating characteristic (ROC) curve analysis was used to establish cutoff values and assess their usefulness in grading BSG. RESULTS: The Cho/NAA metabolite ratio had the strongest preoperative predictive performance for identifying the correct histological grade among BSGs, with an area under the ROC curve (AUC) value of 0.944 (cutoff: 3.88, sensitivity [Se]: 83.3%; specificity [Sp]: 100%), followed by the Cho/Cr ratio (cutoff: 3.08; AUC: 0.917; Se: 83.3%; Sp: 100%), rCBF (cutoff: 3.56, AUC: 0.917; Se: 83.3%; Sp: 100%), rCBV (cutoff: 3.16, AUC: 0.889; Se: 100%; Sp: 66.7%), and age (cutoff: 9.5 years, AUC: 0.889; Se: 100%; Sp: 83.3%). CONCLUSIONS: rCBF and rCBV values comparing solid tumors with the normal brain parenchyma and the metabolite ratios for Cho/NAA and Cho/Cre may serve as useful indices for establishing BSG grading and provide important information when determining treatment planning and prognosis in patients with BSG.

Diagnostic performance of quantitative signal intensity measurements on magnetic resonance imaging for distinguishing cerebellopontine angle meningioma from acoustic schwannoma.

OBJECTIVE: Our study investigated magnetic resonance imaging measurements for differentiating cerebellopontine angle (CPA) meningioma from vestibular schwannoma (VS). PATIENTS AND METHODS: This retrospective study compared 36 meningioma and 36 VS patients. The tumor volume (Vtumor) and peritumor edema index (EI) relationship was analyzed. T2-weighted three-dimensional gradient-echo image signal intensity (T23D) and apparent diffusion coefficient (ADC) differentiation cutoff values were defined. Mann-Whitney U test, independent-samples t-test, receiver operating characteristic curve, and Spearman's correlation analyses were applied. RESULTS: Meningioma had higher Vtumor (p=0.009) and EI (p=0.031) values than VS. Meningioma had significantly (p<0.001) lower values than VS for mean ADC (ADCmean: 0.841±0.083×10-3 vs.1.173±0.190×10-3 mm2/s), minimum ADC (ADCmin: 0.716±0.078×10-3 vs.1.045±0.178×10-3 mm2/s), tumor:white matter ADC ratio (rADC: 1.198±0.19 vs. 1.59±0.30), mean T23D (T23Dmean: 142.91±19.9 vs. 218.72±84.73), and tumor:adipose T23D ratio (rT23d: 0.19±0.06 vs. 0.30±0.28) Cutoff, sensitivity (Se), and specificity (Sp) values were ADCmin, 0.856×10-3 mm2/s (Se: 96.6%, Sp: 100%); ADCmean, 0.963×10-3 mm2/s (Se: 96.6%, Sp: 95.5%); rADC, 1.3189 (Se: 93.1%, Sp: 81.8%), T23Dmean (Se: 96.6%, Sp: 100%); rT23D, 0.1951 (Se: 89.7%, Sp: 100%), Vtumor, 14828.65 mm3 (Se: 75.0%, Sp: 66.7%), and EI, 1.1025 (Se: 47.2%, Sp: 100%). CONCLUSIONS: ADCmin, ADCmean, rADC, T23Dmean, rT23D, Vtumor, and EI, effectively discriminated meningioma from VS.

Synthesis of primary N-arylthioglyoxamides from anilines, elemental sulfur and primary C-H bonds in acetophenones.

A simple method for coupling of anilines, acetophenones, and elemental sulfur to afford N-arylthioglyoxamides has been developed. Reactions proceeded in the presence of Na2SO3 and DMSO, thus eliminating the need for transition metals and external oxidants. Functionalities such as halogen, ester, methylthio, and heterocycle groups were compatible with the conditions. Electron-poor acetophenones sometimes gave isosteric glyoxamides.

Intrinsic correlation between ß-relaxation and spatial heterogeneity in a metallic glass.

ß-relaxation has long been attributed to localized motion of constituent molecules or atoms confined to isolated regions in glasses. However, direct experimental evidence to support this spatially heterogeneous scenario is still missing. Here we report the evolution of nanoscale structural heterogeneity in a metallic glass during ß-relaxation by utilizing amplitude-modulation dynamic atomic force microscopy. The successive degeneration of heterogeneity during ß-relaxation can be well described by the Kohlrausch-Williams-Watts equation. The characteristic relaxation time and activation energy of the heterogeneity evolution are in accord with those of excess enthalpy release by ß-relaxation. Our study correlates ß-relaxation with nanoscale spatial heterogeneity and provides direct evidence on the structural origins of ß-relaxation in metallic glasses.

Stochastic and deterministic models for agricultural production networks.

An approach to modeling the impact of disturbances in an agricultural production network is presented. A stochastic model and its approximate deterministic model for averages over sample paths of the stochastic system are developed. Simulations, sensitivity and generalized sensitivity analyses are given. Finally, it is shown how diseases may be introduced into the network and corresponding simulations are discussed.

Sensitivity of dynamical systems to parameters in a convex subset of a topological vector space.

We develop a theory for sensitivity with respect to parameters in a convex subset of a topological vector space of dynamical systems in a Banach space. Specific motivating examples for probability measure dependent differential, partial differential and delay differential equations are given. Schemes that approximate the measures in the Prohorov sense are illustrated with numerical simulations for distributed delay differential equations.

A dynamic model for induced reactivation of latent virus.

We develop a deterministic mathematical model to describe reactivation of latent virus by chemical inducers. This model is applied to the reactivation of latent KSHV in BCBL-1 cell cultures with butyrate as the inducing agent. Parameters for the model are first estimated from known properties of the exponentially growing, uninduced cell cultures. Additional parameters that are necessary to describe induction are determined from fits to experimental data from the literature. Our initial model provides good agreement with two independent sets of experimental data, but also points to the need for a new class of experiments which are required for further understanding of the underlying mechanisms.

Directed evolution and Phenomics screening of new biocatalysts.

Biodiversity screening and directed evolution are two fruitful complementary approaches for the discovery and design of novel biocatalysts. A new technology for directed evolution, L-Shuffling, has been designed and patented by Proteus. L-Shuffling technology offers several competitive advantages over other technologies including (i) directed evolution of large genes: L-Shuffling" means "Large-Shuffling"; (ii) high fidelity recombination and (iii) Control over location and frequency of recombination. The thousands of new recombinants generated by L-Shuffling can be further screened for their biochemical characteristics using Phenomics. Phenomics is a proprietary functional HTS technology designed and patented by Proteus for the screening of natural biodiversity as well as biodiversity generated by combinatorial biology. Phenomics is a function to gene structure approach which provides an alternative to genomics and proteomics. The traditional limits of expression libraries are thereby circumvented especially those related to cytotoxic products in usual or specific surrogate hosts. The quality of the answer given by the screening is directed dependent on the quality of the question asked. Thanks to a new substrates synthesis technology named CLIPS-O, the company can design highly specific molecules simulating the chemical structure and energetic state of the industrial substrates. The whole process of novel biocatalysts discovery has been automated using commercially available high throughput robotics.

Minimising the secondary structure of DNA targets by incorporation of a modified deoxynucleoside: implications for nucleic acid analysis by hybridisation.

Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N:4-ethyldeoxycytidine (d(4Et)C), which hybridises specifically with natural dG to give a G:(4Et)C base pair with reduced stability compared to the natural G:C base pair. Substitution by d(4Et)C greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.

Block and graft copolymers and NanoGel copolymer networks for DNA delivery into cell.

Self-assembling complexes from nucleic acids and synthetic polymers are evaluated for plasmid and oligonucleotide (oligo) delivery. Polycations having linear, branched, dendritic. block- or graft copolymer architectures are used in these studies. All these molecules bind to nucleic acids due to formation of cooperative systems of salt bonds between the cationic groups of the polycation and phosphate groups of the DNA. To improve solubility of the DNA/polycation complexes, cationic block and graft copolymers containing segments from polycations and non-ionic soluble polymers, for example, poly(ethylene oxide) (PEO) were developed. Binding of these copolymers with short DNA chains, such as oligos, results in formation of species containing hydrophobic sites from neutralized DNA polycation complex and hydrophilic sites from PEO. These species spontaneously associate into polyion complex micelles with a hydrophobic core from neutralized polyions and a hydrophilic shell from PEO. Such complexes are very small (10-40 nm) and stable in solution despite complete neutralization of charge. They reveal significant activity with oligos in vitro and in vivo. Binding of cationic copolymers to plasmid DNA forms larger (70-200 nm) complexes. which are practically inactive in cell transfection studies. It is likely that PEO prevents binding of these complexes with the cell membranes ("stealth effect"). However attaching specific ligands to the PEO-corona can produce complexes, which are both stable in solution and bind to target cells. The most efficient complexes were obtained when PEO in the cationic copolymer was replaced with membrane-active PEO-b-poly(propylene oxide)-b-PEO molecules (Pluronic 123). Such complexes exhibited elevated levels of transgene expression in liver following systemic administration in mice. To increase stability of the complexes, NanoGel carriers were developed that represent small hydrogel particles synthesized by cross-linking of PEI with double end activated PEO using an emulsification/solvent evaporation technique. Oligos are immobilized by mixing with NanoGel suspension, which results in the formation of small particles (80 nm). Oligos incorporated in NanoGel are able to reach targets within the cell and suppress gene expression in a sequence-specific fashion. Further. loaded NanoGel particles cross-polarized monolayers of intestinal cells (Caco-2) suggesting potential usefulness of these systems for oral administration of oligos. In conclusion the approaches using polycations for gene delivery for the design of gene transfer complexes that exhibit a very broad range of physicochemical and biological properties, which is essential for design of a new generation of more effective non-viral gene delivery systems.

Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents.

Cationic copolymers consisting of polycations linked to non-ionic polymers are evaluated as non-viral gene delivery systems. These copolymers are known to produce soluble complexes with DNA, but only a few studies have characterized the transfection activity of these complexes. This work reports the synthesis and characterization of a series of cationic copolymers obtained by grafting the polyethyleneimine (PEI) with non-ionic polyethers, poly (ethylene oxide) (PEO) or Pluronic 123 (P123). The PEO-PEI conjugates differ in the molecular mass of PEI (2 kDa and 25 kDa) and the degree of modification of PEI with PEO. All of these conjugates form complexes upon mixing with plasmids, which are stable in aqueous dispersion for several days. The sizes of the particles formed in these systems vary from 70 to 200 nm depending on the composition of the complex. However, transfection activity of these systems is much lower than that of PEI (25 kDa) or Superfect as assessed in in vitro transfection experiments utilizing a luciferase reporter expression in Cos-7 cells as a model system. In contrast, conjugate of P123 with PEI (2 kDa) mixed with free P123 (9:1(wt)) forms small and stable complexes with DNA (110 nm) that exhibit high transfection activity in vitro. Furthermore, gene expression is observed in spleen, heart, lungs and liver 24 h after i.v. injection of this complex in mice. Compared to 1,2-bis(oleoyloxy)-(trimethylammonio) propane:cholesterol (DOTAP:Chol) and PEI (25 kDa) transfection systems, the P123-PEI system reveals a more uniform distribution of gene expression between these organs, allowing a significant improvement of gene expression in liver.

Smoothing of the thermal stability of DNA duplexes by using modified nucleosides and chaotropic agents.

The effect of alkyltrimethylammonium ions on the thermostability of natural and modified DNA duplexes has been investigated. We have shown that the use of tetramethylammonium ions TMA+along with the chemical modification of duplexes allow the fine adjustment of T m and the possibility of obtaining several duplex systems with varied isostabilizedtemperatures, some of which show greater stability than those of natural DNA. This approach could be very useful for DNA sequencing by hybridization.

The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content. Implication for DNA sequencing by hybridization.

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G4EtC base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51 degrees C). The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G4EtC base pairs is mainly due to the sequence effect and not to their AT/G4EtC ratio content. Thus N -4-ethyl-2'-deoxycytidine (d4EtC) hybridizes specifically with natural deoxyguanosine leading to a G4EtC base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d4EtC can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d4EtCTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.

Modification of DNA duplexes to smooth their thermal stability independently of their base content for DNA sequencing by hybridization.

The possibility of equalizing DNA duplex stability is essential for the application of sequencing by hybridization. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their base content. Modified *C bases have been developed and incorporated into oligonucleotides. The influence of these modifications on duplex stability has been studied by absorption spectroscopy, thus allowing selection of N -4-ethyl-2'-deoxycytidine (d4EtC), which hybridizes specifically with natural dG to give a G4EtC base pair whose stability is very close to that of natural AT base pairs. Duplexes built with AT and/or G4EtC base pairs exhibit thermal stabilities independent of their base content in a classical buffer solution, thus enabling control of the stability of DNA hybrids as a function of their length only.

Evaluation of the implantable force transducer for chronic tendon-force recordings.

In vivo force measurements from tendons and ligaments have become an important technique to determine internal forces, joint loading, and mechanisms of neuromotor control. The most frequently used transducers for such force recordings were placed external to the target tissue. Because of space restrictions and the associated impingement artifacts, these external transducers cannot be used for force measurements in all tendons and ligaments. In these situations, transducers placed inside the target tissue have been used recently; however, the suitability and performance characteristics of these internal transducers have not been assessed systematically. The purpose of this study was to assess the suitability and performance characteristics of an internally placed force transducer which has been used previously. The results indicated that small angular displacements of the transducer within the target tissue, as well as small relative rotations of the corresponding bones, resulted in substantially changed transducer output for given externally applied loads. Also, the transducer output was found to depend on the rate of load application. It was concluded that, although the internal force transducer gave reliable signals within a given experiment, and thus, could be used to assess relative changes in tissue forces pre- and post-interventions, it would be difficult to use the transducer for the accurate determination of the actual tissue forces during unrestrained animal locomotion.

Synthesis and biological activity of 7-alkylidenecephems.

Several 7-alkylidenecephalosporins were synthesized and biologically evaluated as beta-lactamase inhibitors. The three beta-lactamase enzymes used in this study included two type C beta-lactamases, derived from Enterobacter cloacae P99 and E. cloacae SC12368, and one type A beta-lactamase, derived from Escherichia coli WC3310. Of the cephalosporins prepared, compound 7e, the sodium salt of 7-[(Z)-(2'-pyridyl)methylene]cephalosporanic acid sulfone, was found to have excellent inhibitory properties against both type C enzymes. Also, compound 7f, the sodium salt of 7-[(Z)-(tert-butoxycarbonyl)methylene]cephalosporanic acid sulfone showed high activity as an inhibitor of the type A enzyme. The inhibition kinetics of 7e were further explored. The IC50 value of 7e indicated that this compound was approximately 20-fold more active than tazobactam against the enzyme derived from E. cloacae P99 and 167-fold more active than tazobactam against the enzyme derived from E. cloacae SC12368. A plot of enzymatic activity vs incubation time with stoichiometric amounts of inhibitor reveals a rapid deactivation of the enzyme followed by an extremely slow reactivation. 7e exhibited a second-order rate constant of k3' = 5.3 x 10(6) L/mol.min, and a partition ratio of approximately 20:1 inhibitor:enzyme was determined for this inhibitor. After separation of excess inhibitor with Sephadex filtration, a rate constant of enzyme reactivation was measured at kreactiv = 1.0 x 10(-3) s-1. Following 24 h of incubation of enzyme with a large excess of inhibitor and sephadex filtration to remove excess inhibitor, the enzyme was able to recover only 43% of its original activity, indicating an irreversible component to the inhibition. Potential mechanisms of inhibition for both 7e and 7f are suggested.

Efficacy of cefixime in the treatment of acute otitis media in children.

OBJECTIVE: To compare the efficacy of cefixime with amoxicillin in the treatment of acute otitis media in children. DESIGN: Randomized, nonblinded study. SETTING: General pediatric clinic at a university hospital in Texas. PARTICIPANTS: A volunteer sample of 201 children, aged 2 months through 6 years. INTERVENTIONS: A 10-day oral course of cefixime (8 mg/kg per day administered once daily) or amoxicillin (40 mg/kg per day administered in three divided doses [every 8 hours]). MEASUREMENTS/MAIN RESULTS: Tympanocentesis for bacterial culture was performed on all affected ears on enrollment and after 4 to 6 days of therapy. The patients were evaluated clinically 4 to 6 days after starting therapy, at the end of therapy, and 3 to 4 weeks after therapy was completed. Using Fisher's Exact Test, no significant difference was found between the two treatment groups for rate of clinical improvement or rate of eradication of Haemophilus influenzae and Streptococcus pneumoniae. However, combining the results from this study and two previously reported studies, cefixime was found to be more effective in eradication of H influenzae and less effective in eradication of S pneumoniae.

Ca2+-calmodulin regulates the induction and expression of macrophage cytotoxicity.

Ca2+-activated calmodulin (CAM) is known to regulate cellular responses of diverse cell types to external stimuli. To examine the effects of Ca2+ influx and CAM on macrophage (MP) cytotoxicity, we used 51Cr-labeled cells as targets and in vivo or in vitro activated MPs from C57 BL/6 or BALB/c mice as effectors in a 16-h cytotoxicity assay. MPs activated in vivo by administration of vaccinia virus or in vitro with lymphokine (LK) were cytotoxic for a variety of tumor cell lines, including SV3T3, but not for normal 3T3 cells. Addition of verapamil (0.1 mM), a Ca2+ channel blocker, to MPs activated in vivo by vaccinia virus markedly reduced their cytotoxicity for SV3T3 cells. This correlated with an inhibition of Ca2+ uptake by MPs, as measured by 45Ca influx. Chlorpromazine (20 microM), trifluoperazine (20 microM), and W13 (75 microM), inhibitors of CAM activity, also suppressed MP cytotoxicity for SV3T3 cells when added to the assay, suggesting that Ca2+-activated CAM is an integral component in expression of MP cytotoxicity. To further explore the mechanism of MP cytotoxicity, supernatants from activated MPs treated with various pharmacological agents were examined for cytotoxicity. Vaccinia-activated MPs released a soluble factor(s) that was cytotoxic for SV3T3 cells. Resident MPs cultured under the same condition produced no significant cytotoxic activity. As observed with direct MP cytotoxicity, addition of a Ca2+ channel blocker or CAM inhibitors to cultured activated MPs markedly reduced the cytotoxic activity of the supernatants, suggesting an active secretory process. The requirement for Ca2+ and CAM in the expression of MP cytotoxicity was confirmed in an in vitro MP activation system. Resident cultured MPs activated by an LK preparation were reduced in their tumoricidal capability when verapamil or CAM inhibitors were added to the cytotoxicity assay. This correlated with a reduction in lytic activity of the MP culture supernatants. Further, addition of verapamil or CAM inhibitors to resident MP cultures markedly reduced the induction of tumoricidal MPs by LK, suggesting that Ca2+ and CAM are necessary for both the induction and the expression of MP cytotoxicity. Suppression of cytotoxicity of in vitro activated MP cell line B6MP102 with verapamil and CAM inhibitors confirmed that the MP was the cytotoxic cell being modulated by Ca2+-activated CAM.(ABSTRACT TRUNCATED AT 400 WORDS)