Reactive Oxygen Species Induced Upregulation of TRPV1 in Dorsal Root Ganglia Results in Low Back Pain in Rats.

Background: Dorsal root ganglia (DRGs) contain sensory neurons that innervate intervertebral discs (IVDs) and may play a critical role in mediating low-back pain (LBP), but the potential pathophysiological mechanism needs to be clarified. Methods: A discogenic LBP model in rats was established by penetration of a lumbar IVD. The severity of LBP was evaluated through behavioral analysis, and the gene and protein expression levels of pro-algesic peptide substance P (SP) and calcitonin gene-related peptide (CGRP) in DRGs were quantified. The level of reactive oxygen species (ROS) in bilateral lumbar DRGs was also quantified using dihydroethidium staining. Subsequently, hydrogen peroxide solution or N-acetyl-L-cysteine was injected into DRGs to evaluate the change in LBP, and gene and protein expression levels of transient receptor potential vanilloid-1 (TRPV1) in DRGs were analyzed. Finally, an inhibitor or activator of TRPV1 was injected into DRGs to observe the change in LBP. Results: The rats had remarkable LBP after disc puncture, manifesting as mechanical and cold allodynia and increased expression of the pro-algesic peptides SP and CGRP in DRGs. Furthermore, there was significant overexpression of ROS in bilateral lumbar DRGs, while manipulation of the level of ROS in DRGs attenuated or aggravated LBP in rats. In addition, excessive ROS in DRGs stimulated upregulation of TRPV1 in DRGs. Finally, activation or inhibition of TRPV1 in DRGs resulted in a significant increase or decrease of discogenic LBP, respectively, suggesting that ROS-induced TRPV1 has a strong correlation with discogenic LBP. Conclusion: Increased ROS in DRGs play a primary pathological role in puncture-induced discogenic LBP, and excessive ROS-induced upregulation of TRPV1 in DRGs may be the underlying pathophysiological mechanism to cause nerve sensitization and discogenic LBP. Therapeutic targeting of ROS or TRPV1 in DRGs may provide a promising method for the treatment of discogenic LBP.

Characteristics of denitrifying bacteria in different habitats of the Yongding River wetland, China.

Nitrogen nutrient salts are considered the major environmental factors (RNH4+-N0.92, RTN0.85) affecting the structure and distribution of denitrogen bacteria. We aimed to investigate the mechanisms by which wetland bacteria adapt to environmental factors in different types of habitats. High-throughput sequencing technology was used to study the microbial community structure of sediments in three wetland habitats [fish ponds, surface flow wetlands (cattails and reeds), and ditches] of the Yongding River, China. The microbial community structure differed across different habitats. Species richness of nitrifying bacteria increased, while that of denitrifying bacteria decreased, with ammonium salt and total nitrogen concentrations increasing from surface flow wetland to ditch wetland. The characteristics of the three habitat types and their distribution in the Yongding River wetland are beneficial to the differential distribution of microbial communities across the wetland, and to the existence and denitrification of different dominant bacteria. Overall, these results help explain the natural filtering function of wetlands.

An antimicrobial impregnated urinary catheter that reduces mineral encrustation and prevents colonisation by multi-drug resistant organisms for up to 12 weeks.

Two major complications of indwelling urinary catheterisation include infection and mineral encrustation of the catheter. Our antimicrobial urinary catheter (AUC) impregnated with rifampicin, triclosan, and sparfloxacin has demonstrated long-term protective activity against major uropathogens. This study aimed to firstly assess the ability of the AUC to resist mineral encrustation in the presence and absence of bacteria. Secondly, it aimed to investigate the AUC's anti-biofilm activity against multi-drug resistant organisms. There was no difference in surface roughness between AUC and control segments. In a static and a perfusion model, phosphate deposition was significantly reduced on AUCs challenged with P. mirabilis. Furthermore, none of the AUCs blocked during the 28 day test period, unlike controls. The AUC prevented colonisation by methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, extended-spectrum beta-lactamase producing E. coli, and carbapenemase-producing E. coli for 12 consecutive weekly challenges. All three drugs impregnated into the catheter continued to exert protective activity throughout 12 weeks of constant perfusion. The drugs appear to migrate into the crystalline biofilm to continually protect against bacteria not it direct contact with the catheter surface. In conclusion, the AUC reduces mineral encrustation and may increase time to blockage in the presence of P. mirabilis, and does not predispose to mineral deposition under other conditions. It also offers 12 weeks of protection against multi-drug resistant bacteria. STATEMENT OF SIGNIFICANCE: Infection and associated mineral encrustation of urinary catheters are two serious complications of indwelling urinary catheters. Others have attempted to address this through various technologies such as coatings, dips, and surface modifications to prevent infection and/or encrustation. However, all current 'anti-infective' urinary catheter technologies are limited to short-term use. Some patients with spinal injuries, multiple sclerosis, stroke survivors and others use long-term catheters for 4-12 weeks at a time with multiple catheterisation possibly throughout the rest of their life. We present a urinary catheter for long-term use that is impregnated with three antimicrobials by a patient-protected process to prevent infection and encrustation for up to 12 weeks, the maximum lifetime of a long-term catheter before it is changed.

Simulation of the Effect of Artificial Water Transfer on Carbon Stock of Phragmites australis in the Baiyangdian Wetland, China.

How to explain the effect of seasonal water transfer on the carbon stocks of Baiyangdian wetland is studied. The ecological model of the relationship between the carbon stocks and water depth fluctuation of the reed was established by using STELLA software. For the first time the Michaelis-Menten equation (1) introduced the relation function between the water depth and reed environmental carrying capacity, (2) introduced the concept of suitable growth water depth, and (3) simulated the variation rules of water and reed carbon stocks of artificial adjustment. The model could be used to carry out the research on the optimization design of the ecological service function of the damaged wetland.

Cobalt-Catalyzed Decarboxylative 2-Benzoylation of Oxazoles and Thiazoles with α-Oxocarboxylic Acids.

Cobalt-catalyzed decarboxylative cross-coupling of oxazoles and thiazoles with α-oxocarboxylic acids was developed through an sp(2) C-H bond functionalization process. This work represents the first example of cobalt-catalyzed decarboxylative C-H bond functionalization and provides an efficient means of building some important bioactive heteroaryl ketone derivatives.

Biomaterial modification of urinary catheters with antimicrobials to give long-term broadspectrum antibiofilm activity.

Catheter-associated urinary tract infection (CAUTI) is the commonest hospital-acquired infection, accounting for over 100,000 hospital admissions within the USA annually. Biomaterials and processes intended to reduce the risk of bacterial colonization of the catheters for long-term users have not been successful, mainly because of the need for long duration of activity in flow conditions. Here we report the results of impregnation of urinary catheters with a combination of rifampicin, sparfloxacin and triclosan. In flow experiments, the antimicrobial catheters were able to prevent colonization by common uropathogens Proteus mirabilis, Staphylococcus aureus and Escherichia coli for 7 to 12weeks in vitro compared with 1-3days for other, commercially available antimicrobial catheters currently used clinically. Resistance development was minimized by careful choice of antimicrobial combinations. Drug release profiles and distribution in the polymer, and surface analysis were also carried out and the process had no deleterious effect on the mechanical performance of the catheter or its balloon. The antimicrobial catheter therefore offers for the first time a means of reducing infection and its complications in long-term urinary catheter users.

Nickel-catalyzed and benzoic acid-promoted direct sulfenylation of unactivated arenes.

A nickel-catalyzed and benzoic acid-promoted direct sulfenylation of unactivated arenes using removable 2-(pyridine-2-yl)-isopropylamine as a directing group is described. This strategy provides an efficient access to valuable aryl sulfides with ample substrate scope and a high degree of functional group tolerance.

Combination of (M)DSC and surface analysis to study the phase behaviour and drug distribution of ternary solid dispersions.

PURPOSE: Miscibility of the different compounds that make up a solid dispersion based formulation play a crucial role in the drug release profile and physical stability of the solid dispersion as it defines the phase behaviour of the dispersion. The standard technique to obtain information on phase behaviour of a sample is (modulated) differential scanning calorimetry ((M)DSC). However, for ternary mixtures (M)DSC alone is not sufficient to characterize their phase behaviour and to gain insight into the distribution of the active pharmaceutical ingredient (API) in a two-phased polymeric matrix. METHODS: MDSC was combined with complementary surface analysis techniques, specifically time-of-flight secondary ion mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM). Three spray-dried model formulations with varying API/PLGA/PVP ratios were analyzed. RESULTS: MDSC, TOF-SIMS and AFM provided insights into differences in drug distribution via the observed surface coverage for 3 differently composed ternary solid dispersions. CONCLUSIONS: Combining MDSC and surface analysis rendered additional insights in the composition of mixed phases in complex systems, like ternary solid dispersions.

Understanding the interfacial properties of nanostructured liquid crystalline materials for surface-specific delivery applications.

Nonlamellar liquid crystalline dispersions such as cubosomes and hexosomes have great potential as novel surface-targeted active delivery systems. In this study, the influence of internal nanostructure, chemical composition, and the presence of Pluronic F127 as a stabilizer, on the surface and interfacial properties of different liquid crystalline particles and surfaces, was investigated. The interfacial properties of the bulk liquid crystalline systems with coexisting excess water were dependent on the internal liquid crystalline nanostructure. In particular, the surfaces of the inverse cubic systems were more hydrophilic than that of the inverse hexagonal phase. The interaction between F127 and the bulk liquid crystalline systems depended on the internal liquid crystalline structure and chemical composition. For example, F127 adsorbed to the surface of the bulk phytantriol cubic phase, while for monoolein cubic phase, F127 was integrated into the liquid crystalline structure. Last, the interfacial adsorption behavior of the dispersed liquid crystalline particles also depended on both the internal nanostructure and the chemical composition, despite the dispersions all being stabilized using F127. The findings highlight the need to understand the specific surface characteristics and the nature of the interaction with colloidal stabilizer for understanding and optimizing the behavior of nonlamellar liquid crystalline systems in surface delivery applications.

Nanoparticle-induced morphology and hydrophilicity of structured surfaces.

Nanoparticles have been applied into the construction of micro- and nanoscaled surface structures with extreme wettability over the past few years. However, the details of processing and employing colloidal nanosuspensions for this purpose have not yet been fully investigated. In this work, we study the surface structures formed via nanosuspensions, in which nanoparticles of solid phase are presented, and the caused surface wettability. We disperse silica nanoparticles with different sizes into pure ethanol to prepare nanosuspensions with a series of concentrations. The suspensions are ultrasonically processed to prompt uniform distribution of nanoparticles before application. The deposited nanosuspensions are thermally treated to assist the regulation of surface patterns based on nanoparticles. Hence, the investigation explores a variety of experimental conditions that will lead to distinctive surface structures and wettabilities. Accordingly, the wettability of the induced surfaces is investigated using contact angle measurement, and the structures of those surfaces are mainly revealed by atomic force microscopy (AFM). Superhydrophilicity is observed on many of such formed surfaces, and the pattern of surface structures in micro- and nanoscale is closely related to the processing conditions and the size of nanoparticles. Thus, we report the characteristics of the surface patterns based on nanoparticles and the formed wettability.

Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: an atomic force microscopy study.

The knowledge of mechanical properties of root cell walls is vital to understand how these properties interact with relevant genetic and physiological processes to bring about growth. Expansion of cell walls is an essential component of growth, and the regulation of cell wall expansion is one of the ways in which the mechanics of growth is controlled, managed and directed. In this study, the inherent surface mechanical properties of living Arabidopsis thaliana whole-root epidermal cells were studied at the nanoscale using the technique of atomic force microscopy (AFM). A novel methodology was successfully developed to adapt AFM to live plant roots. Force-Indentation (F-I) experiments were conducted to investigate the mechanical properties along the length of the root. F-I curves for epidermal cells of roots were also generated by varying turgor pressure. The F-I curves displayed a variety of features due to the heterogeneity of the surface. Hysteresis is observed. Application of conventional models to living biological systems such as cell walls in nanometer regimes tends to increase error margins to a large extent. Hence information from the F-I curves were used in a preliminary semiquantitative analysis to infer material properties and calculate two parameters. The work done in the loading and unloading phases (hysteresis) of the force measurements were determined separately and were expressed in terms of "Index of Plasticity" (η), which characterized the elasticity properties of roots as a viscoelastic response. Scaling approaches were used to find the ratio of hardness to reduced modulus (H/E(*)).

Nanoscale surface characterization and miscibility study of a spray-dried injectable polymeric matrix consisting of poly(lactic-co-glycolic acid) and polyvinylpyrrolidone.

Injectable controlled-release formulations are of increasing interest for the treatment of chronic diseases. This study aims to develop and characterize a polymeric matrix for intramuscular or subcutaneous injection, consisting of two biocompatible polymers, particularly suitable for formulating poorly soluble drugs. For this matrix, the water-insoluble polymer poly(lactic-co-glycolic acid) (PLGA) is combined with the water-soluble polymer polyvinylpyrrolidone (PVP). Microparticles of these two polymers were prepared by spray drying. The phase behavior of the samples was studied by means of modulated differential scanning calorimetry and the results showed that phase separation occurred in the bulk sample through evidence of two mixed amorphous phases, namely, a PLGA-rich phase and a PVP-rich phase. Characterization of the samples by scanning electron microscopy demonstrated that the spray-dried particles were hollow with a thin shell. Because of the importance in relation to stability and drug release, information about the surface of the microparticles was collected by different complementary surface analysis techniques. Atomic force microscopy gathered information about the morphology and phase behavior of the microparticle surface. Time-of-flight secondary ion mass spectrometry analysis of the particles revealed that the surface consisted mainly of the PLGA-rich phase. This was confirmed by X-ray photoelectron spectroscopy at an increased sampling depth (≈ 10 nm). Nanothermal analysis proved to be an innovative way to thermally detect the presence of the PLGA-dominated surface layer and the underlying PVP phase. Taken together, this information provides a rational basis for predicting the likely drug release behavior this formulation will display.

Surface-templated fibril growth of peptide fragments from the shaft domain of the adenovirus fibre protein.

Here we present a study of five analogues of a fragment from the shaft domain of the adenovirus fibre protein that readily form fibrils under a range of conditions. Using atomic force microscopy the fibrillisation of these peptides at the liquid/solid interface utilizing ordered crystalline substrates has been investigated. Our results demonstrate that the assembly pathway at the liquid/solid interface enables only the formation of truncated fibrillar structures, which align along the substrate's underlying atomic lattice during growth. Furthermore, that the concentration and volume of solution applied can be used to directly control the density of fibrillar coverage at the surface.

Polymers with hydro-responsive topography identified using high throughput AFM of an acrylate microarray.

Atomic force microscopy has been applied to an acrylate polymer microarray to achieve a full topographic characterisation. This process discovered a small number of hydro-responsive materials created from monomers with disparate hydrophilicities that show reversibility between pitted and protruding nanoscale topographies.

Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA.

The Escherichia coli curved DNA-binding protein A (CbpA) is a nucleoid-associated DNA-binding factor and chaperone that is expressed at high levels as cells enter stationary phase. Using a combination of genetics, biochemistry, structural modelling and single-molecule atomic force microscopy we have examined dimerization of, and DNA binding by, CbpA. Our data show that CbpA dimerization is driven by a hydrophobic surface comprising amino acid side chains W287 and L290 located on the same side of an α helix close to the C-terminus of CbpA. Derivatives of CbpA that are unable to dimerize are also unable to bind DNA. Free in solution, CbpA can exist as either a monomer or dimer. However, when bound to DNA, CbpA forms large aggregates that can protect DNA from degradation by nucleases. These CbpA-DNA aggregates are similar in morphology to protein-DNA complexes formed by the DNA-binding protein from starved cells (Dps), the only other stationary phase-specific nucleoid protein. Conversely, protein-DNA complexes formed by Fis, the major growth phase nucleoid protein, have a markedly different appearance.

Extracellular matrix-mediated osteogenic differentiation of murine embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent and have the ability to differentiate into mineralising cells in vitro. The use of pluripotent cells in engineered bone substitutes will benefit from the development of bioactive scaffolds which encourage cell differentiation and tissue development. Extracellular matrix (ECM) may be a suitable candidate for use in such scaffolds since it plays an active role in cellular differentiation. Here, we test the hypothesis that tissue-specific ECM influences the differentiation of murine ESCs. We induced murine ESCs to differentiate by embryoid body formation, followed by dissociation and culture on ECM prepared by decellularisation of either osteogenic cell (MC3T3-E1) or non-osteogenic cell (A549) cultures, or on defined collagen type I matrix. We assessed osteogenic differentiation by formation of mineralised tissue and osteogenic gene expression, and found it to be significantly greater on MC3T3-E1 matrices than on any other matrix. The osteogenic effect of MC3T3-E1 matrix was reduced by heat treatment and abolished by trypsin, suggesting a bioactive proteinaceous component. These results demonstrate that decellularised bone-specific ECM promotes the osteogenic differentiation of ESCs. Our results are of fundamental interest and may help in tailoring scaffolds for tissue engineering applications which both incorporate tissue-specific ECM signals and stimulate stem-cell differentiation.

Substrate stiffness affects early differentiation events in embryonic stem cells.

Embryonic stem cells (ESC) are both a potential source of cells for tissue replacement therapies and an accessible tool to model early embryonic development. Chemical factors such as soluble growth factors and insoluble components of the extracellular matrix are known to affect the differentiation of murine ESCs. However, there is also evidence to suggest that undifferentiated cells can both sense the mechanical properties of their environment and differentiate accordingly. By growing ESCs on flexible polydimethylsiloxane substrates with varying stiffness, we tested the hypothesis that substrate stiffness can influence ESC differentiation. While cell attachment was unaffected by the stiffness of the growth substrate, cell spreading and cell growth were all increased as a function of substrate stiffness. Similarly, several genes expressed in the primitive streak during gastrulation and implicated in early mesendoderm differentiation, such as Brachyury, Mixl1 and Eomes, were upregulated in cell cultures on stiffer compared to softer substrates. Finally, we demonstrated that osteogenic differentiation of ESCs was enhanced on stiff substrates compared to soft substrates, illustrating that the mechanical environment can play a role in both early and terminal ESC differentiation. Our results suggest a fundamental role for mechanosensing in mammalian development and illustrate that the mechanical environment should be taken into consideration when engineering implantable scaffolds or when producing therapeutically relevant cell populations in vitro.

Nanoscale thermal analysis of pharmaceutical solid dispersions.

Formation of a solid solution of a drug in a water-soluble polymer is one of the primary techniques used to improve the dissolution rate and thus bioavailability of a poorly water-soluble drug. Understanding and detecting the state of the drug inside such a polymer matrix is critically important since issues such as drug stability, safety and efficacy can be greatly affected. In this study, two model formulations were prepared containing low and high levels of drug content. The heterogeneity of the formulations has been investigated using a novel nanothermal analysis technique. This technique has demonstrated a promising capability for imaging and quantitatively characterising the nanoscale properties of solid dispersion formulations.

Dynamic equilibria in solvent-mediated anion, cation and ligand exchange in transition-metal coordination polymers: solid-state transfer or recrystallisation?

The solution properties of a series of transition-metal-ligand coordination polymers [ML(X)(n)](infinity) [M=Ag(I), Zn(II), Hg(II) and Cd(II); L=4,4'-bipyridine (4,4'-bipy), pyrazine (pyz), 3,4'-bipyridine (3,4'-bipy), 4-(10-(pyridin-4-yl)anthracen-9-yl)pyridine (anbp); X=NO(3) (-), CH(3)COO(-), CF(3)SO(3) (-), Cl(-), BF(4) (-); n=1 or 2] in the presence of competing anions, metal cations and ligands have been investigated systematically. Providing that the solubility of the starting complex is sufficiently high, all the components of the coordination polymer, namely the anion, the cation and the ligand, can be exchanged on contact with a solution phase of a competing component. The solubility of coordination polymers is a key factor in the analysis of their reactivity and this solubility depends strongly on the physical properties of the solvent and on its ability to bind metal cations constituting the backbone of the coordination polymer. The degree of reversibility of these solvent-induced anion-exchange transformations is determined by the ratio of the solubility product constants for the starting and resultant complexes, which in turn depend upon the choice of solvent and the temperature. The extent of anion exchange is controlled effectively by the ratio of the concentrations of incoming ions to outgoing ions in the liquid phase and the solvation of various constituent components comprising the coordination polymer. These observations can be rationalised in terms of a dynamic equilibrium of ion exchange reactions coupled with Ostwald ripening of crystalline products. The single-crystal X-ray structures of [Ag(pyz)ClO(4)](infinity) (1), {[Ag(4,4'-bipy)(CF(3)SO(3))]CH(3)CN}(infinity) (2), {[Ag(4,4'-bipy)(CH(3)CN)]ClO(4) 0.5 CH(3)CN}(infinity) (3), metal-free anbp (4), [Ag(anbp)NO(3)(H(2)O)](infinity) (5), {[Cd(4,4'-bipy)(2)(H(2)O)(2)](NO(3))(2)4 H(2)O}(infinity) (6) and {[Zn(4,4'-bipy)SO(4)(H(2)O)(3)] 2 H(2)O}(infinity) (7) are reported.

A quantitative assessment of inhaled drug particle-pulmonary surfactant interaction by atomic force microscopy.

To date limited consideration has been given to the physical interaction between inhaled drug particles and pulmonary surfactant (PS). This study combines atomic force microscopy (AFM) with a Langmuir-Blodgett (LB) approach to quantify the force of adhesion between micronised budesonide particles and simulated PS monolayers. A LB approach was used to prepare Survanta monolayers at pre-determined surface pressures and AFM was employed to facilitate their visualisation. Adhesion measurements between drug particles and PS monolayers were executed via AFM. Contact angle measurements were performed to probe material wetting characteristics, the data confirmed that budesonide is hydrophobic and Survanta films at increasing surface pressure exhibit a rising hydrophobic character. AFM revealed that PS properties were governed by applied surface pressure and that the degree of interaction of budesonide was greater at higher surface pressure, where packing of the lipid film was increased; consistent with the point of exhalation. This correlates well with the accepted inhaler technique. The increasing hydrophobicity of the PS film, on increased pressure, was believed to be the primary reason for increased interaction with the hydrophobic budesonide. Surface chemistries of the drug particles and PS interface are considered to be important for inhaled drug delivery.