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International Journal of Medical Nano Research
Protease Biosensors Based on Peptide-Nanocellulose Conjugates: From Molecular Design to Dressing Interface
J Vincent Edwards1*, Krystal R Fontenot1, Nicolette T Prevost1, David Haldane2, Nicole Pircher3, Falk Liebner3, Alfred French1 and Brian D Condon1
1Cotton Chemistry and Utilization Research, United States Department of Agriculture, USA
2Innovatech Engineering Tallahassee, USA
3University of Natural Resources and Life Sciences, Vienna, Austria
*Corresponding author: J Vincent Edwards, Cotton Chemistry and Utilization Research, United States Department of Agriculture, 1100 Robert E. Lee Blvd, New Orleans, LA 70124, USA, E-mail: email@example.com
Int J Med Nano Res, IJMNR-3-018, (Volume 3, Issue 1), Research Article; ISSN: 2378-3664
Received: March 02, 2016 | Accepted: August 16, 2016 | Published: August 19, 2016
Citation: Edwards JV, Fontenot KR, Prevost NT, Haldane D, Pircher N, et al. (2016) Protease Biosensors Based on Peptide-Nanocellulose Conjugates: From Molecular Design to Dressing Interface. Int J Med Nano Res 3:018. 10.23937/2378-3664/1410018
Copyright: © 2016 Edwards JV, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The development of point of care diagnostic protease sensors applied to wound healing has received increased interest in chronic wound treatment as an interface for chronic wound dressings. Biosensor technology and the use of nanomaterials have grown exponentially in recent years. A biosensor is fundamentally a biomolecule (functioning as a transducer) attached to a transducer surface, which is activated by a biochemical property that prompts a detection signal specific to a target biomarker. For instance, nanomaterials are often derivatized with a biomolecule that provides selectivity and sensitivity for diagnostic biomarkers. Nanocellulose can be prepared as a transducer surface from an assortment of sources like wood and cotton in a variety of physical forms such as nanocrystals, nanocellulose composites, and nanocellulose aerogels that possess ideal properties including biodegradability, biocompatibility, functionality, and a high specific surface area. Interfacing nanocellulosic biosensors with a wound dressing having protease-lowering properties allows in situ sensor detection selectivity and sensitivity to monitor the effectiveness of the dressing and titer of protease removed from the wound. Here we discuss chronic wound dressing design and mechanism with an emphasis on protease-lowering dressings and chronic wound modalities and a discussion of a number of different types of nanocellulosic materials as interface materials for potential sensor-dressing application. As a specific model, we focus on nanocellulosic systems conjugated to the elastase substrate n-succinyl-Alanine-Alanine-Proline-Valine-7-amino-4-methylcoumarin and n-succinyl-Alanine-Proline-Alanine-7-amido-4-methyl-coumarin, and discuss comparative properties and molecular design, crystal structure, protease binding kinetics, specific surface area, permeability, surface charge, and sensitivity to proteases as relates to biosensor positioning in a dressing design.
Protease biosensors and chronic wound biomarkers
Advances in biosensor technology hold promise to revolutionize healthcare and diagnosis through assessment of health status, disease onset, and progression using non-invasive methods [1-4]. This is especially the case in the area of sensor and imaging for wound healing [5], which is critical for the management of wounds stalled in the inflammatory state i.e., a chronic wound [6].
Numerous inflammatory-based disease states are noted for secreting neutrophil and bacterial enzymes including, myeloperoxidase and gelatinases, and neutrophilic proteases. Two proteases that have been associated with chronic wound pathology are Matrix Metalloproteases (MMPs) [7] and Human Neutrophil Elastase (HNE) which have prolonged deleterious effects, from excessive degradation of extracellular matrix proteins [8-10], fibronectin [11,12], and growth factors [13]. It is also noteworthy that HNE and MMPs have been shown to work in a collaborative way to accelerate degradation [14].
Chronic wounds arise from an arrestment in the inflammatory stage of the natural flow of the wound healing phases of hemostasis, inflammation, proliferation, fibroplasia, and remodeling and are often found to have prolonged high titers of neutrophils which lead to high protease concentrations [15]. Although neutrophils play important roles in host defense and debridement of damaged tissue they contain free radical generating enzymes and proteases implicated in chronic inflammatory diseases. Thus, the stalled inflammatory state of chronic wounds [16,17] is often associated with the wound fluid containing high levels of harmful proteases (HNE and MMP) that have become increasingly important biomarkers for point-of-care diagnosis which is also important in appropriate wound dressing selection [18-20]. Thus, an increased interest in protease biosensors which may be designed to detect proteases of clinical interest has arisen giving rise to what has been referred to as a 'test and treat" strategy [21].
A biosensor's basic design may be defined as the interface of a molecular or receptor-recognition property with a cellular or biochemical activity, triggering a "bio-molecular switch" that in turn is connected with a detector signal [3,22]. Nanomaterials with high surface area and biocompatible properties make ideal transducer surfaces for biosensors. Nanocellulose, which we address in this paper, provides noteworthy advantages as a transducer surface, but has received less attention than other nanomaterials for sensor applications [18,23].
Chronic wound dressings
Chronic wound dressings used in the treatment of non-healing wounds may be organized into four groups including: passive, interactive, bioactive, and intelligent [24,25]. Passive dressings protect the wound bed from trauma and bacterial infection [24,26-28]. Interactive dressings maintain a moist wound and facilitate fibroblast proliferation and epithelialization [29,30] by way of a added functional property conferred by a material component of the dressing i.e. biomaterials like honey, collagen, hyaluronic acid, and chitosan have been associated with enabling mechanisms of wound healing. Current advancements in commercially available semiocclusive dressings include improvement on hydrocolloids, hydrogels, and semi permeable foam dressings that are designed to modulate fluid balance by retaining moisture or reducing exudate volume, and acting as a bacterial barrier while improving the range of their use i.e. to reduce pressure, friction, and shear for the patient for example [31].
Bioactive wound dressings, which include delivery systems, skin substitutes, and biological dressings, are being employed for their clinical efficacy to promote wound healing in challenging wounds by activating cellular roles in the proliferative or inflammatory stages of wound healing [24,32,33]. Bioactive dressings have been employed as a combination of keratinocytes, fibroblasts, and other cellular constituents as well as biopolymer composites designed to facilitate epidermal growth as are required in burn and full thickness wound patients [34,35]. It is noteworthy that a recent review by Dreifke, et al. has discussed FDA approved wound healing dressings based on collagen, silicon, chitosan, and hyaluronic acid in light of wound environment sensors for early detection of complications [36]. It is also important to note in the context of bioactive analogs the development of therapeutic growth factor and neuropeptide analogs, Advances in the understanding of wound healing pathophysiology have provided targets for bioactive topical therapies with an emphasis on peptide and growth factor therapies which have appeared promising for modulation of the proliferative phase of wound healing [37-39].
An intelligent dressing may perform a specific function, for example protease sequestration or low-level hydrogen peroxide generation. It may also have sensor capabilities. Sensor functions like color, pH, and temperature change, have been explored in dressing design where there is an underlying activation due to biochemical changes and cellular responses [31,40,41] in the wound. For example, biofilm detection and treatment is a clinically challenging area in 60 percent of chronic wounds; and Thet, et al. recently reported a prototype hydrogel wound dressing that is described as emitting a glow in response to bacterial biofilms present in a wound [42].
Wound fluids from chronic and acute wounds have different characteristic microenvironments and the composition of the fluid and the wound bed microenvironment undergoes basic changes in moisture, temperature, pH and oxygen. Thus, theranostic markers like pH useful in predicting whether the wound will heal may reflect the general condition of a wound in lieu of giving a more specific indication as a biomarker does [43-46]. In this regard it is notable that Rahimi, et al. evaluated a flexible pH (4-10) sensor fabricated on a polymer coated palette and paper substrate, consisting of cyto-compatible materials, which exhibited a sensitivity of -50 mV/pH unit in wound fluid i.e. a recent approach to point of care theranostic assessment [47]. The pH assessment is a beneficial indicator of wound healing considering acute and normal wounds have a slightly acidic pH (5.5-6.5) whereas chronic wounds tend to exhibit a higher pH ( > 7.4) due to alkaline byproducts from proliferating bacterial colonies [47]. The use of flexible polymer and paper substrates including silicone elastomers, parylene, polyimide, and cellulose paper as pH, oxygen, moisture, and temperature sensors also enable detection of changes in the wound microenvironment using materials that have conformability to the wound bed [45].
Some commercially available dressings now have the added functionality of indicating wound exudate saturation to signal dressing change [48]. However, current state of the art sensor nanoscience and wound dressing technology are poised to yield improved multifaceted sensor/dressings that will provide point of care diagnostics and theranostics by increasing sensitivity to detectable biomarkers while providing an improved opportunity to treat chronic wounds based on cellular and molecular specific pathologies.
Clearly, advanced dressings integrate more progressive characteristics and may overlap in their categorization. However, the combination of a bioactive, semiocclusive, or intelligent dressing with a point of care diagnostic or theranostic sensor is both an emerging paradigm and a reasonable extrapolation of the current technologies available for chronic wound care.
Protease sequestrant dressings
The amelioration of harmful levels of proteases in chronic wounds has been a topic of increasing interest and focus in the design and preparation of non-healing wound dressings over the last two decades. A protease sequestrant also termed protease-lowering or protease attenuation) dressing's design may be couched in a number of molecular motifs based on the structural features of the protease or target enzyme, which interferes with the healing process. The molecular features of the dressing's material may be targeted to the protease size, charge, active site, or conformation to enhance selective binding of the protein to the dressing material and remove proteases from the wound bed. Thus, the mechanism of action may also be through other cellular or biochemical events that modulate protease levels. Previously reported active wound dressings designed to redress the biochemical imbalance of the chronic wound in this manner are composed of collagen and oxidized regenerated cellulose [49], nanocrystalline silver-coated high density polyethylene [50], which also serves as an antimicrobial, deferioxamine-linked cellulose [51], electrophilic and ionically derivatized cotton [52], peptide [53] or carbohydrate-conjugates [54], and sulfonated ion exchange derivatives of hydrogel polymers [55]. Electrostatic uptake mechanisms have also been demonstrated with in situ fluorescence hydrogels that both detect and trap proteases [56]. In addition, phosphorylated cotton was shown to sequester both human neutrophil elastase and collagenase [57], and developed as an FDA approved dressing [58]. From this work it was shown that the best formulation for development was a treatment of sodium hexameta phosphate in urea that is attached through low cost conventional textile finishing approaches. More recently the role of polyphosphates when incorporated into a foam matrix have been shown to sequester metalloproteases (MMPs) derived from Pseudmonas aeruginosa [59]. Interestingly, when combined with silver in a hydrogel base polyphosphate was also found to give rise to anti-biofilm activity [60]. Also noteworthy is work reported by several authors that addresses the activity and mechanism of action of protease-lowering activity of phosphorylated analogs in terms of dressing functionalized active site inhibitors of elastase and matrix metalloprotease [61-63].
A veritable arsenal of different protease inhibitors has been developed for inflammatory diseases, and a variety of non-toxic protease inhibitors have been proposed and studied for controlled release into chronic wounds. Among these oleic acid, which is the principle component of olive oil has been demonstrated to be an elastase inhibitor [64], and more recently proposed as an inhibitor of matrix metalloprotease and plasmin [65] as well. Oleic acid has been demonstrated as an albumin-mediated elastase inhibitor when released from different dressing [62,66]. Interestingly olive oil has been studied for a healing-oriented mechanism of action [67] in chronic wounds as well as the subject of a recent clinical trial for chronic wound treatment [68]. Oleic acid has been shown to accelerate healing of acute wounds [69]. It is noteworthy that the judicious use of protease inhibitors should be considered for formulations where growth factor and peptide therapeutics are being applied to wounds to prevent the inactivation of proteinacesous drugs by inhibiting protein degradation in the high protease-containing wound environment. Although a number of groups have studied this approach and developed patents claiming formulations with growth factors and protease inhibitors, no products have yet been marketed using this approach [70].
MMP sensors
The progress toward clinically based MMP sensors for point of care diagnostics has been reviewed in recent years [7]. Biosensors of MMP have been reported based on fluorescence resonance energy transfer (FRET) and include fluorescent tripeptide substrates of proteases with the mechanism being linkage of a highly sensitive fluorescent response between donor and acceptor molecules [71]; quantum dots, gold nanoparticles or organic dyes have also been explored with a similar approach [72,73]. Graphene which has a very large specific surface area has also been employed as a transducer surface for an oxide-based sensor, a fluoresce in labeled peptide based on electrostatic interactions and as a fluoresce in isothiocyanate-labled peptide conjugate [74,75].
HNE sensors
A recent review of HNE biosensors has listed a variety of designs proposed and utilized with the corresponding protease sensitivity ranges [18]. Briefly, biosensor-based detectors of elastase have included a microchip (Henares, et al. 2006) [76], microdialysis probe [77], fluorometric supramolecular pore sensor [78], immobilization of peptides on quantum dots [79], immobilization of HNE on a biosensor chip for surface plasma resonance [80], ethoxyacrylate resin [81], using a colorimetric detection and fluorometric peptides coupled to paper [79] as well as cotton cellulose nanocrystals [18,82]. Schyrr, et al. recently reported fluorescent sensor film made from cellulose nanocrystals and polyvinyl alcohol has also been demonstrated as a protease sensor [83] with added functional porosity.
Other approaches and considerations with wound Sensors
A variety of imaging spectroscopy technologies are beginning to replace traditional visual cutaneous assessment of burn, diabetic and traumatic wounds [84], and recently an approach utilizing wound blotting of necrotic tissue for distribution analysis of marker proteins with nitrocellulose has been proposed as an approach to the prediction of healing progress in pressure ulcers [85]. Point-of-care protease detection has received increased attention and has been applied clinically throughout the world to enable chronic wound treatment decisions [19,86,87]. The pros and cons of a 'test and treat' strategy approach for elevated protease activity in venous ulcers was recently reviewed [21]. Moreover, peptide-based protease sensors are widely used in monitoring enzyme activity. They have a rich structure/function literature that can be utilized to design enzyme sensors and are central to a number of protease-based diseases and their therapy [88]. The clinical application of peptide-based biosensor approaches to protease biomarker disease assessment in chronic wounds is growing [19]. On the other hand, although there have been a few reports of prototypes, the combination of detection and treatment in a chronic wound dressing has not been adopted on a widespread clinical basis [18]. Thus, the ability to combine measurable protease detection with a dressing motif that removes proteases is an emerging research and development goal of wound healing biomaterial design [18,21,89].
There are many considerations in clinically evaluating a combined dressing-sensor system for protease detection. The use of an in situ detector should withstand scrutiny as to whether it be applied as an indicator to change the dressing and accurately detect the wound protease titer. It is also important to note that not all chronic wounds have abnormally high protease levels, and normal protease levels are important to wound remodeling [17]. In light of variations in wound protease levels a 'dipstick' approach for assessment of protease levels in chronic wounds [19,86] has been suggested before treatment options and dressing selection is made.
Nanocellulosic biosensors
Although the sophistication and sensitivity of detectors underlie much of the progress in the rapidly growing field of biosensors, the composition of the transducer surface material and its environment can also complicate and dampen the sensitivity of detection. Thus, biosensor materials should ideally be compatible with the complexity of the bio-system by being resistant to nonselective adsorption by lipids, proteins, polysaccharides, cellular debris, and breakdown of the transducer surface. In this regard, nanocellulose is both hydrophilic and structurally resilient, rendering it advantageous as a biomaterial.
The use of cellulose nanocrystals and nanofibrillated cellulose in biomaterials has been shown to have potential advantages in optics [90], nanocomposite materials [91], and has continued to show promise as an effective material in semiocclusive dressing design for chronic wound applications [92-94]. Nanocellulose is a crystalline biopolymer with hydrophilic properties and high surface area [91]; it possesses reactive hydroxyls that can be derivatized to covalently append a wide range of biologically active molecules. Its hydrophilic surface of nanocellulose improves wettability and abrogates binding by proteins and lipids.
The high specific surface area of the nanocellulose systems enables enhanced sensitivity of a peptide-based protease sensor by accommodating a higher peptide titer on the crystalline surface, and subsequently performing as a sensor more efficiently by way of increased binding of the protease on the sensor surface. For example, print cloth and filter paper, which are largely cellulosic when compared with and wood cellulose nanocrystals have a specific surface area of 0.016-0.020 m2g-1 and 261.2 m2g-1, respectively [95]. Upon attachment of peptides to these types of materials, it was found that the approximate number of peptides per 200 anhydroglucose cellulosic residues are print cloth (1), filter paper (2), and cellulose nanocrystals (12), and these ratios of peptide to cellulosic units correspond with a relative order of increasing specific surface area [95]. The relative activities also paralleled specific surface area of the cellulosic versus nanocellulosic materials and demonstrate the contrast in efficiency of cellulosic versus nanocellulosic sensors.
As introduced above elastase sensors provide a model for point of care biomarkers in inflammatory diseases and chronic wounds. It is understood that broad spectrum protease assessment would be beneficial in clinical point of care diagnosis. However, here we discuss elastase sensors as a model for broad spectrum protease sensors as a model for this approach. Here we evaluate fluorescent protease biosensors on nanocrystals, nanocellulose composites, and nanocellulose aerogels immobilized with the tetrapeptide substrate n-succinyl-Alanine-Alanine-Proline-Valine-7-amido-4-methyl-coumarin (Suc-Ala-Ala-Pro-Val-AMC) and the tripeptide substrate n-succinyl-Alanine-Proline-Alanine-7-amido-4-methyl-coumarin (Suc-Ala-Pro-Ala-AMC).
Material and Methods
Esterification of transducers
Standard fluorenylmethyloxycarbonyl (Fmoc) chemistry was used for esterification and immobilization of the peptide onto the transducer materials [96,97]. The esterification and peptide immobilization of the transducers is previously outlined by Edwards, et al. [82,95]. Briefly, the nanocellulosic transducers cotton cellulose nanocrystals (cCNC), wood nanocellulose composites 66/33 and 50/50 (wNCC), and nanocellulosic aerogel (NA) were esterified with Fmoc-Gly-OH with the respective coupling agents (Ethylcyanoglyoxylate-2-oxime (Oxyma Pure), Diisopropylcarbodiimide (DIC), and 4-dimethylaminopyridine (DMAP) in N,N-dimethylformamide (DMF)) and sonicated. The materials were washed thrice by centrifuging or filtration DMF, dichloromethane (DCM), and in select cases allowed to air dry. (Note: the NA-Gly-Fmoc was allowed to remain in DMF for storage and was not air dried). The transducers cCNC-Gly-Fmoc, wNCC-Gly-Fmoc, and NA-Gly-Fmoc were deprotected or stored at ~4-8°C.
A deblock solution (piperidine/DMF) was used to deprotect the Fmoc protecting group from the glycine esterified transducers. (Note: the NA-Gly-Fmoc was washed with DMF prior to soaking in the deblock solution). The materials were washed thrice by centrifuging or filtration with DMF, DCM, and in select cases allowed to air dry. (Note: the NA-Gly was washed thrice with DMF followed by preparation for peptide coupling or stored in DMF for further use).
Immobilization of the fluorescent peptide substrate
Peptide immobilization of cCNC-Gly, wNCC-Gly, and NA-Gly were achieved by activating the tripeptide (Suc-Ala-Pro-Ala-AMC) or tetrapeptide (Suc-Ala-Ala-Pro-Val-AMC) substrates with DMAP in DMF for twenty minutes, to which Oxyma Pure and DIC were added. The solutions were sonicated for 3 hours, placed in the refrigerator overnight, and purified by centrifuging or filtering thrice with DMF, DCM or methanol (MeOH), and in select cases allowed to air dry. (Note: the NA-Pep was washed thrice with DMF, thrice with PBS, and stored in PBS). The biosensors were stored at ~4-8°C until further use. Note "biosensor" will be defined as the glycine esterified transducer immobilized with the tripeptide or tetrapeptide substrate.
Upon the completion of drying, the peptide was cleaved from each biosensor by adding a mixture of TFA/water/triisopropylsilane to the biosensor for three hours. The solution for each biosensor was diluted (water/acetonitrile) and submitted for Electrospray ionization liquid chromatography mass spectrometry (ESI-LC/MS), which confirmed the intact sequence of the peptide component of the biosensor via its molecular weight.
Specific surface area (SSA)
The specific surface area (SSA), identifies the total surface area of a solid material per the unit mass of the material. The calculation of the cellulosic nanocrystals and the BET specific surface area of the wNCCs were previously outlined in by Edwards, et al. [95]. The Brunauer-Emmett-Teller (BET) specific surface area of the NA was determined using a Micrometrics ASAP 2020 instrument [98] with a nitrogen sorption source at 77 K detector. The NA (0.033 g) was degassed at 100°C for 4 hours prior to the analysis followed by N2 adsorption at 77.4 K.
Fluorogenic enzyme sensitivity assay
The sensitivity assay evaluates the limit of detection of which the biosensors are able to detect human neutrophil elastase (HNE). The sensitivity assay was previously outlined by Fontenot, et al. [99]. Briefly, duplicates of ~2 mg of each biosensor were placed into a 96 well plate and 100 μL of a phosphate buffer solution (PBS) was added. To start the reaction, 50 μL human neutrophil elastase (HNE) ranging from 2-0.0156 U/mL was added to the standard curve and to the biosensors to provide a total volume of 150 μL. Fluorescent measurements at 37°C were monitored for 1 hour at 1-minute intervals using a Biotech Synergy HT with tungsten halogen lamp and photomultiplier detection. The 96 well plates were shaken before each measurement for 3 seconds and the measurements were acquired at 360 nm excitation and 460 nm emission. The wound like fluid herein comprises of PBS and HNE.
Surface charge
The zeta potential (ζ) determines the surface charge properties of the peptide free transducers (cCNC, wNCC, NA), which are immersed in a conducting solvent and measured using a Malvern Zetasizer nano ZS90 equipped with a laser doppler and detected with a phase analysis light scattering. The wNCC and NA were ground into a powder using a Wig L Bug Ball Mill and pulsated for ~1.5 minutes. Stock solutions (5 mg/0.25 mL) of the ground transducers were prepared in DI H2O or PBS from which 1 mg/mL (cCNC and NA) and 0.5 mg/mL (wNCC) solutions were prepared. The respective solutions were sonicated for 2 hours prior to obtaining triplicate zeta potential measurements.
Moisture vapor transmission rate (MVTR)
The MVTR measures the rate at which moisture can permeate solid porous materials such as the wNCCs. Commercial Testing Company performed the water vapor transmission rate according to the ASTM E96-95 method with procedure B - water method at 23°C for the wNCC.
Results and Discussion
Cellulose nanocrystal-based sensors
Cotton cellulose nanocrystals (cCNC) and wood cellulose nanocrystals (wCNC) have a high surface area, mechanical robustness, a geometrical rod-like or whisker shape nanocrystal, and a negatively charged surface [18,100]. The cellulose nanocrystals have a high SSA of 186.2 m2/g-1 that allows for a greater number of peptide substrates to be anchored onto the surface. The fluorescence sensitivity of the nanocrystalline peptide biosensors was as low as 0.05 U/mL, which correlates to a higher concentration of peptide substrate loading and to a higher concentration of AMC-released fluorophore. Furthermore, as shown in table 1 the cellulose nanocrystals posses a negatively charged surface of -41 mV [82,101] that enable binding of elastase. The surface charge influences the interaction between the wound fluid and the wound dressing, which promotes uptake of positively charged proteases by negatively charged materials consistent with phosphorylation, sulfonation, and oxidation of cellulose. This electrostatic mechanism promotes increased binding to HNE by targeting positively charged amino acids in the proteins sequence [52,102]. Therefore, negatively charged cellulose nanocrystals bind the HNE proteases present both in the wound fluid and the dressing, which has been shown to accelerate the healing rate of the wound [52,102,103].
Table 1: List of each nanocellulosic biosensor with its respective peptide substrate, SSA, sensitivity, and surface charge values. View Table 1
Molecular design
Figure 1 shows a crystallite-tethered fluorescent tripeptide elastase substrate linked via the cellulose primary hydroxyls to a cotton cellulose nanocrystal surface. This depiction is representative of the molecular surface of a nanocellulose transducer with sensor molecules and constitutes the design components of a point-of-care protease sensor. Recently analogous structures have been discussed [99]. Modeling of the sensor molecule on the crystalline surface when accompanied with enzyme kinetic analysis is helpful in predicting the interaction of the protease with the sensor in terms of enzyme/substrate binding efficiency and crystal surface versus enzyme spatial considerations [104] i.e., of the enzyme on the transducer surface as it relates to peptide protease binding and kinetics. A peptide-nanocellulose analog characterized with a similar crystallite surface was shown to undergo a five-fold more efficient interaction with HNE than was observed of the analogous enzyme substrate in solution i.e. a higher Kcat/Km of the peptide-nanocrystalline material versus peptide in solution [104]. This improvement in the kcat/km is attributable in part to enhanced binding of the cationic serine protease to an anionic crystallite surface.
Figure 1: Model of the cotton cellulose nanocrystals biosensor with the tripeptide based on the cellulose I diffraction patterns with a width of 58.8Ǻ (109 chains and 15 layers) [82]. The tripeptide is substituted on the crystallite structure with a D.S. level of 0.044 and a SSA of 186.2 m2g-1. View Figure 1
Colorimetric sensor
Over the last ten years a variety of colorimetric protease sensors directed to point of care models have been reported. Edwards, et al. first reported a colorimetric approach for elastase detection with ethoxyacrylate resin as a transducer surface and demonstrated the relevant enzyme-substrate binding properties [105]. The incorporation of colorimetric detectors i.e. remazol brilliant blue into peptidoglycan and polymer motifs, with a goal of making them amendable to the wound environment, was reported with a variety of designs as a diagnostic device paradigm for wound infection relevant proteases [89,106]. Previously we proposed a colorimetric protease detection with dressings using cotton cellulose nanocrystals derivatized with both a tetrapeptide protease substrate (n-succinyl-Alanine-Alanine-Proline-Valine-p p-nitroanilide, Suc-AAPV-pNA) or tripeptide protease substrate (succinyl-Ala-Ala-Val-p-nitroanilide, Suc-AAV-pNA), which employs the visual amplification of a chromophore with a dye to enhance sensitivity [18] (Figure 2).
Figure 2: Shows the cleavage pathway of A) pNA with NEDH amplification; B) AMC from the tetrapeptide substrate with HNE [110]. View Figure 2
The colorimetric sensor-based dressing diagram shown in figure 3 comprises a matrix of layers functioning to: 1) absorb wound exudate and allow entry of the protease onto the surface of the sensor through a semipermeable absorptive layer and allow, 2) protease reaction with the protease sensor substrate where upon, 3) release of the chromophore onto a 10 kDa dialysis membrane permeated with amplifying dye, and 4) a barrier layer to prevent backwash of the released dye into the wound. The cellulose nanocrystal tripeptide conjugate with colorimetric amplifying agents was capable of detecting HNE at 0.05 U/mL [18]. The free particle nature of the cellulose nanocrystals allows efficient use of the nanoparticles properties as a sensor, which includes high SSA, versatility of application, and improved sensitivity to the protease biomarker. The benefit of interfacing a protease sensor with a dressing may be construed in its value as an indicator for dressing saturation of proteases, a detector to monitor the efficacy of the dressing and its interaction with the wound environment.
Figure 3: Diagram of the cotton cellulose nanocrystals conjugated to a chromophore derivatized (tripeptide) that in turn is interfaced with a semi permeable wound dressing. The contacting layer absorbs wound exudate and allows passage of wound exudate-containing proteases; the barrier layer prevents the para-nitroanilide (pNA) chromophore from entering the wound bed. The cellulose nanocrystal peptide conjugate is activated upon contact with protease when the dressing is saturated with protease. The pNA migrates through a 10 kD dialysis cellulose membrane to the color amplifying reagent layer where a color appears signaling the presence of proteases in the dressing. The semi permeable top layer protects the dressing. View Figure 3
Fluorescent-based sensors and dressing interface
Fluorescent imaging on the other hand has higher sensitivity limits toward visualization of molecular biomarkers at nano- or picomole levels of protease, but requires a fluorometric device for imaging. However it is notable that fluorescence imaging of wounds has shown potential to directly visualize the skin's natural fluorophores (collagen and elastin for example), and this type of approach has been demonstrated with nicotinamide adenine dinucleotide (NADH), which is associated with oxidative phosphorylation as a maker for cutaneous healing [107]. Direct exogenous fluorescent imaging, for dressing application, has also been applied thru the use of fluorescent dyes injected into the systemic system, and the dyes are visualized in the vasculature of burn wounds to assess wound depth [108]. Also noteworthy is an elegant approach to incorporation of fluorgenic esterase sensors demonstrating potential as an in situ sensor functionality that addresses challenging signal and diffusion issues and can be incorporated into various cellulosic dressing and substrate designs using a chemo-enzymatic approach [109].
A point of care diagnostic approach for chronic wound dressing selection is sensitive detection of the biomarker of interest [40]. Protease biomarkers that correlate to outcomes in wound healing include both MMPs and HNE as discussed above. Figure 4A, shows the interface of a fluorescent protease biosensor motif with a dressing. The dressing-sensor design directs uptake of wound exudate through a semipermeable contact layer onto the sensor surface where proteases react with the peptide substrate giving deposition of proteolytic reaction by-products on a barrier surface of the dressing tangential to the biosensor where it does not mix with wound fluid. Figure 4B portrays a series of images of the nanocrystals, nanocellulose composites, and nanocellulose aerogels, which offer a visual image of the disposition of properties benefitting sensor interface with a dressing as outlined below.
Figure 4: Illustration of an intelligent semiocclusive protease dressing designed with fluorescent sensor. Image 3A shows a contact layer that absorbs wound exudate secretion and allows passage of wound fluid. The surrounding layers allow protease capture and fluorophore release and entrapment. Each layer of the bandage contributes to maintaining moisture via its open semi permeable structure. Image 3B shows the images of the nanocellulose-based sensors used as a biosensor layer for interface with a semiocclusive dressing (the images were acquired using a Sony Full HD 1080 mega pixels). Each nanocellulosic material has a device specific advantage: 1) the cellulosic nanocrystals can be affixed to the barrier layer, 2) the nanocellulose composite film can be layered between the top layer and barrier layer, and 3) the nanocellulosic aerogel can be placed between the barrier layer and top layer or applied directly to the top layer of a dressing as a separate sensor. View Figure 4
Functional comparison of colorimetric and fluorescent protease sensors
The colorimetric and fluorescent tetrapeptide elastase substrates function as sensors through a respective chromophore (pNA) and fluorophore (AMC), which have different mechanism pathways. The para-nitroanilide (pNA) chromophore upon release absorbs at 405 nm, and when coupled with amplification by way of N-(1-Naphthyl) ethylenediamine (NEDH) elicits a colorimetric signal that gives enhanced sensitivity of detection, but notably slightly less than is emitted by the fluorescent signal found with AMC. On the other hand, AMC upon hydrolysis fluoresces by way of excitation at 360 and emission at 460 nm. Figure 2 shows the pathway of A) hydrolytic cleavage of pNA from the peptide substrate followed by reaction with NEDH amplification and B) hydrolysis to yield AMC from a tetrapeptide substrate with elastase. Comparatively, the AMC signal is more sensitive than the colorimetric signal when the transducer surface is cellulose nanocrystals. As noted previously [18] the sensitivity of the flurophore with the tetrapeptide substrate is approximately two-fold greater than the colorimetric response.
Figure 2A portrays the molecular sequence of events underlying colorimetric detection from hydrolytic cleavage of pNA from the tetrapeptide substrate via a two-step reaction. The sensor signal is prompted by the following: HNE hydrolyzes of the amide bond between valine and the COOH-terminal pNA whereupon an azide bond forms between pNA and NEDH. The detection signal of the pNA chromophore (405 nm) is amplified with formation of the azide bond between pNA and NEDH (545 nm), which results in a red shift in absorbance and increases the colorimetric signal [18,110].
Figure 2B demonstrates the one-step reaction that occurs upon HNE hydrolysis of the amide bond between valine and AMC subsequently resulting in a strong and sensitive fluorescence signal. It is notable that the one-step approach for the fluorescence signal as a detection method for a biosensor layer in a multilayered wound dressing is depicted in figure 5.
Figure 5: Depiction of nanocellulose aerogel peptide conjugates biosensor (diameter of 2.1 cm) with fluorescent protease activation interfaced in a multilayer bandage prototype with and without ultraviolet illumination. Image A demonstrates a gauze contact layer, barrier layer, and biosensor layer without ultraviolet activation. Image B depicts a barrier layer and gauze top layer above the biosensor layer with ultraviolet illumination. View Figure 5
Nanocellulosic composite-based sensors
Nanocellulosic composites (NCC) may be considered appropriate for wound dressings and are porous paper-like films that have good gas barrier and mechanical properties [111]. Generating nanocellulose composites from wood cellulose nanocrystals and microfibrillated cellulose into thin film nanocellulose composites in ratios of 66/33 and 5050, respectively [95] allowed for improved dressing construction while maintaining a porous structure that permits air, moisture, and gas permeation [111,112]. As shown in figure 6 the porous nature of the nanocomposites resulted in favorable water vapor permeability of ~553 gm-2d-2 and is commensurate with levels associated with gas exchange at the wound bed. Thin films typically have a low permeability MVTR of 400-800 gm-2d-2, a moderate permeability MVTR of 800-3000 gm-2d-2, and a high permeability MVTR ranging from 3000 gm-2d-2 or even higher [113]. The composition of the materials influences the physical structure including the porosity, which alters the MVTR values [114,115]. The MVTR properties of the commercial gauze (677 gm-2day-1), wNCC (553 gm-2day-1), and commercial films (394 gm-2day-1) [116], which shows the wNCCs are in range with the planar commercial gauze and films for wound dressings. The wNCCs MVTR results are promising for use as a second layer detector interface to a chronic wound dressing.
Figure 6: A bar graph depiction of the relative moisture vapor transmission rates (MVTR) for a nanocellulose composite film i.e. wNCC 50/50 (wood cellulose nanocrystals and microfibrillated cellulose) thickness ~300 micrometers, commercial gauze dressing material (36" wide folded to 4 ply (8"), 24/20 thread count, DeRoyal Textiles, Camden S.C., commercial film (Bioclusive, Johnson & Johnson, New Brunswick, N.J.) 0.077 mm thick. View Figure 6
It was observed that protease sensors made from the films had lower protease detection sensitivity of 0.25 and 0.125 U/mL, respectively when compared with the cellulose nanocrystal-based sensors [95]. The lower sensitivity is thought to be due to aggregation of the nanocellulose during the preparation process, which yields a lower specific surface area of 0.021-0.035 m2g-1. As shown in table 1 the incorporation of cellulose nanocrystals into the nanocomposite imparts a negatively charged surface [101] that functions to sequester excess positively charged proteases.
Nanocellulosic aerogel-based sensor
Nanocellulosic aerogels (NA) are lightweight solid materials with low densities, high SSA, and an interconnected open porous structure [117,118], which make them attractive for use in semiocclusive dressings. Nanocellulosic aerogels may be prepared from a variety of cellulose fiber sources including wood and cotton. The interconnected open porous structure enables gas exchange between the wound bed and environment [24], membrane permeability [119,33], and prevents wound dehydration by way of promoting a high water vapor transmission rate [24]. Furthermore, the wettability and swelling properties of the NA promotes a hydrophilic and absorbent structure conducive to absorbing wound fluid. In addition as shown in table 1, the NA is a potential sequestrant of HNE due to the NAs negative surface charge of ~-24 mV [52,102,103]. The high SSA, 162.9 m2/g-1, of the nanocellulosic aerogels enables a higher loading of the sensor molecules (elastase tripeptide substrate) and increases sensitivity of the sensor by increasing the transducer surface area for protease binding. Figure 5 shows the NA biosensor interfaced with a semiocclusive wound dressing. The sensitivity studies of the NA peptide conjugate show the biosensor detects HNE as low as 0.125 U/mL.
Evaluation of nanocellulosic biosensors
The nanocellulosic biosensors discussed here and depicted in figure 4B are viewed as advantageous to the dressing interface based on the relative structure, SSA, and sensitivity as a biosensor component of a multilayered dressing. The nanocrystals exist in a powder form that is not readily adaptable in a wound dressing prototype as are the planar nanocellulose composites and nanocellulose aerogel. On the other hand the free particle nature of the nanocrystals maximizes the interaction with the human neutrophil elastase enzyme by virtue of the higher SSA thereby yielding a higher sensitivity to detect HNE present in chronic wound fluid. On the other hand, the incorporation of the nanocrystals into the wNCCs to create films affords a planar structure but a 5000-8000 fold reduction in the SSA occurs due to the aggregation of the nanocrystals blended with microfibrillated cellulose. The reduction in the SSA parallels the lower level of sensitivity; however, the planar but porous structure of wNCCs permits the permeation of moisture vapor and gaseous molecules, making it suitable for dressing interface as a biosensor layer. Nevertheless, the NA structure is also a compatible component of a multilayered dressing and offers a SSA with only a 1.14 fold reduction compared to the nanocrystals. Contrary to the SSA value, a higher sensitivity of 0.125 U/mL is observed versus the lower 0.05 U/mL compared to the cCNC, which may potentially be attributed to the pore sizes restricting the HNE enzymes penetration into the NA structure in order to interact with peptide substrate.
Even though the sensitivity of the nanocomposite and aerogel biosensors are at borderline levels for previously reported titers of elastase in chronic wounds [120] they are viewed as beneficial biosensors capable of detecting and sequestering HNE, and can be applicable as a dressing that can monitor wound saturation of HNE versus a preliminary way to detect the onset of a developing chronic wound. The benefit to both applications can provide information as to how the wound is healing when a new method of treatment is selected.
Biosensor development is undergoing exponential growth in the field of health care. In the area of point of care diagnostics, wound care developments with in situ detection that is compatible with wound dressing structure and function promises to change the face of wound care and help address the rapidly rising number of chronic wound patients worldwide. Here we have explored, in the context of the area of point of care diagnostic technology and dressing development, the comparative properties of nanocellulosic-based protease sensors for their potential interface properties with chronic wound dressings. Three nanocellulosic materials (nanocrystals, nanocellulose composites, and nanocellulose aerogels) were selected and evaluated for their ability to serve as a biosensor component for a multilayered chronic wound dressing. The nanocellulosic materials surveyed offer ideal properties including SSA, permeability, and surface charge that confer a compatible function to protease sequestrant semiocclusive wound dressings in the removal and detection of harmful proteases in the chronic wound.
A tetrapeptide (cCNC and wNCCs) and tripeptide (NA) substrate were immobilized onto transducer surfaces to generate the biosensors examined here. Each nanocellulosic biosensor was assessed for its ability to detect HNE and for its level of sensitivity in the presence of HNE. The relevant molecular and enzyme kinetic attributes suggest that negatively charged nanocellulose promotes efficient uptake of positively charges proteases. All of the biosensors were effective at detecting HNE at levels comparable to those found in chronic wound fluid or as may be expected to be taken up in a dressing. Furthermore, the anionic surface charge of the cellulosic materials provides an uptake mechanism for cationic human neutrophil elastase and is compatible with protease lowering dressings in that regard. Therefore, a single dressing motif based on different nanocellulosic materials could both sequester harmful proteases while serving to detect their presence as well as signaling dressing saturation.
Each of the biosensors, discussed here, has physical properties similar to commercially available thin films, hydrogels, and hydrocolloid dressings. Therefore, interfacing these types of nanocellulose-based biosensors with a semiocclusive dressing can be construed as compatible with detection of other markers of clinical interest as well, through substitution of the biomolecule needed for the desired point of care diagnostics or theranostic interest. Future work will look to fine tuning the structure function relationships of sensor and dressing interface.
The biosensors presented herein are promising in view of nanocellulosic-based materials with a high SSA, permeability, hydrophilicity, absorbent structure, and anionic surface charge. The combination of these features in a single sensor will increase the sensitivity threshold and enhance uptake of the biomarker.
The U.S. Department of Agriculture financed this project. We thank Dr. Alfred D. French for the X-Ray Diffraction studies that yielded the nanocrystallite model.
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Wu P, Fisher AC, Foo PP, Queen D, Gaylor JD (1995) In vitro assessment of water vapour transmission of synthetic wound dressings. Biomaterials 16: 171-175.
Liebner F, Haimer E, Potthast A, Rosenau T (2012) Cellulosic aerogels. In: Habibi Y, Lucia LA Polysaccharide Building Blocks: A Sustainable Approach to the Development of Renewable Biomaterials. John Wiley & Sons, Inc., New Jersery, 51-103.
Liebner F, Potthast A, Rosenau T, Haimer E, Wendland M (2007) Ultralight-Weight Cellulose Aerogels from NBnMO-Stabilized Lyocell Dopes. Research Letters in Materials Science 2007: 4.
Kanani AG, Bahrami SH (2010) Review on Electrospun Nanofibers Scaffold and Biomedical Applications. Trends Biomater Artif Organs 24: 93-115.
Trengove NJ, Stacey MC, MacAuley S, Bennett N, Gibson J, et al. (1999) Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors. Wound Repair Regen 7: 442-452.


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2925 Dean Parkway Suite 300Minneapolis, MN 55416
United States
See map: Google Maps
612 281 8331
Contact Name:
Lara Derksen
Contact Email:
We are the leader in Early Intensive Behavioral Intervention, using all ABA-proven methods. We serve families whose goal is to recover their children from ASD. Our behavior analyst works with you for a full day each week to ensure that your behavior therapy is effective, supervising a full team of therapists in your home 6 days a week. Every parent is trained to be a behavior therapist for their own child, whose actual trajectory is re-evaluated every six months.

subset: commoncrawl


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- Our friend Rhonda has a cute, furry family~ Herself and three dogs! One of the nicest looking family pics we received. We always look forward to that picture.
- Three family Christmas letters told of tales from Northern Wisconsin for the July 4th holiday. Looks like this is "the" place to go once July rolls around.
- Looks like water parks, casinos, beaches, and Disney World are popular destinations this holiday season for several of our friends/family.
- And then there is the picture of my sister and her family~ I do wonder who that man is......When you have a picture taken in front of a monument in Washington D. C- note to self: Make sure there are no crazies in the photo making funny motions with their hands!
Seriously, it was fun to receive so many wonderful pictures (kids grow up way too fast) and to hear of all the wonderful activities and adventures your families have taken part in this past year.....I can't wait to see and hear what's in store for 2011 for all of you! After All, It's All In a Mom's Day, Right?


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Godzone Queenstown: Beautiful!/
FSX/P3D. Next up in the Godzone Photoscenery Subscription is Queenstown South Island. If I lived in New Zealand, Queenstown would be my choice to live followed by Nelson which oddly reminds me of Ireland. Godzone is showing off some beautiful screenshots of the area and if you are an owner of Orbx Queenstown, fear not, the developer cut a hole in the coverage which blends in perfectly with Orbx ZQN. This issue extends the Godzone Photoscenery Subscription over the entire Lake Wakatipu, so it will be possible to fly all the way from Mount Cook Airport to NZQN within the photoscenery area. More information available via the source link below.


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Affordable is great, but it is worth nothing without quality. When you shop with us, you will find some of the best flooring brand names on the market today. For instance, we carry a great line of Mohawk flooring products, including hardwood, carpet, and laminate flooring. We also carry a wonderful selection of Dixie Home flooring systems.
When you buy from us, you get some of the best quality flooring in Mount Vernon, WA, and that is a fact. Our flooring comes with some of the most powerful warranties, and that means you never have to take a risk when you purchase your floors from us. Our goal is to carry the best flooring in Mount Vernon, WA at a variety of price points. This means that virtually anyone who needs new flooring will be able to find what they want, need, and can afford with ease.
If you are looking for great selections on brand name flooring, and want to save money as well, come see us at Completely Floored in Everett, WA. We would love to show you why so many homeowners come to us when they want the best quality, affordable flooring in Mount Vernon, WA.


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Deutsche Bank AG's RREEF unit agreed to buy offices and apartments being built in Bucharest for 341 million euros ($537 million). / Source: BloombergÏîäðîáíåå
Asian Coast Development Ltd., a Toronto-based developer part owned by Harbinger Capital Partners, plans to invest $4.5 billion to build Vietnam's first casino resort. / Source: BloombergÏîäðîáíåå
SM Investments Corp., the holding company of Philippine billionaire Henry Sy, fell the most in a month in Manila trading, leading declines among consumer and real estate stocks on concerns record oil prices will curb spending. / Source: BloombergÏîäðîáíåå
Qatars US$40 billion property investment arm said on Tuesday that it had bought a French industrial firm for 1.5 billion euros, planned to develop a US$2 billion project in Hong Kong and had set up a US$3 billion global fund. / Source: LushhomemediaÏîäðîáíåå
Boston Properties Inc., Goldman Sachs Group Inc. and Middle Eastern investors are in talks to buy the General Motors Building and other New York skyscrapers from developer Harry Macklowe, two people familiar with the matter said. / Source: BloombergÏîäðîáíåå
Dubai World, the state-owned investment group managing more than $100 billion, agreed to buy Starwood Hotels & Resorts Worldwide Inc.'s Turnberry golf resort in Scotland to expand its Leisurecorp sports unit. / Source: BloombergÏîäðîáíåå
Global hotel acquisitions dropped more than 60 percent in the first three months of the year as credit tightened after losses and writedowns related to the U.S. subprime mortgage collapse, Jones Lang LaSalle Inc. said. / Source: BloombergÏîäðîáíåå
British Land Co. Plc, the biggest developer in London, reported its first annual loss in at least 20 years after writing down the value of office buildings and malls. / Source: BloombergÏîäðîáíåå
Central China Real Estate Ltd., part owned by Singapore's CapitaLand Ltd., may raise as much as HK$1.9 billion ($244 million) in a Hong Kong initial public offering, said two people familiar with the sale. / Source: BloombergÏîäðîáíåå
Metro AG, Germany's largest retailer, said it plans to invest as much as 300 million euros ($467 million) to open as many as 20 stores in Egypt, where the economic growth rate is almost three times that of Europe. / Source: BloombergÏîäðîáíåå
DTZ, the global commercial real estate and investment adviser was appointed a leasing agent of Naberezhnaya Tower, block C. The building affiliates the Moscow City project. The owner of the complex, ENKA, is one of the most professional and experienced developer on the Moscow real estate market. / Source: Arendator.ruÏîäðîáíåå
BSEL Infrastructure Realty Ltd., an Indian property developer, may invest 15 billion ringgit ($4.6 billion) over 12 years in Malaysia's so-called Iskandar Regional Development in the southern state of Johor. / Source: BloombergÏîäðîáíåå
Global real estate markets are frozen because buyers and sellers remain far apart on price and aren't ready to concede values won't return to levels achieved in 2007, Apollo Real Estate Advisors LP senior partner William Mack said. / Source: BloombergÏîäðîáíåå
Joint Stock Company Open Investments (OPIN or the Company), the Moscow-based real estate development and investment company, today announces the offer price of its rights offering. The price for the rights offering was approved by the Board of Directors of the Company, following the expiration of the statutory pre-emptive rights period, at its meeting today, 16 May 2008. / Source: Arendator.ruÏîäðîáíåå
YNH Property Bhd. is seeking as much as 1.2 billion ringgit ($370 million) by selling in advance half of a tower being designed by Norman Foster for Kuala Lumpur in the Malaysian capital's biggest commercial property transaction. / Source: BloombergÏîäðîáíåå
Agile Property Holdings Ltd., a Hong Kong-listed developer of real estate in China, scrapped a plan to sell $400 million of bonds, according to Moody's Investors Service. / Source: BloombergÏîäðîáíåå
Land Securities Group Plc, the U.K.'s largest real-estate investment trust, reported its first annual loss since at least 1990 after writing down the value of office buildings and malls. / Source: BloombergÏîäðîáíåå
Tesco Plc, the U.K.'s biggest supermarket company, agreed to buy South Korea's Homever discount chain for 958 million pounds ($1.9 billion), boosting sales by about a third in its most profitable overseas market. / Source: BloombergÏîäðîáíåå
Wal-Mart Stores Inc., the world's largest retailer and a benchmark for the U.S. economy, reported higher quarterly profit and said earnings may trail analysts' estimates after record gasoline prices buffeted consumers. / Source: BloombergÏîäðîáíåå
Hampton opened 11 properties in April 2008, representing 1,135 new rooms. The new openings include one Hampton Inn hotel, eight Hampton Inn & Suites properties and two Hampton Inn & Suites by Hilton properties. All openings are franchised, newly constructed hotels. / Source: HotelNewsResourceÏîäðîáíåå
Business Center
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Chairs & Seating
A distant cousin to some of our favorite midcentury shell seat designs, Heartwork's multipurpose chair is effortlessly egalitarian in its comfort. Added to the design's high-functioning form are contract-quality materials and future-forward details that befit both desk and stool-height designs. The durable, single-piece seat supports team members of varying sizes while responding to user movements with inherent flex. All that without the foul, off-gassing foam cushions of most office chairs.


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SAP has revealed its plans to launch a SaaS ERP SAP Version focussed on the mid market. Henning Kagermann SAP's CEO announced earlier this week but no specific dates on release.
Netsuite CEO Zach Nelson has welcomed the announcement and mentioned that this will validate on demand within the ERP space. "Larry Ellison once told me that the best thing that happened to Oracle in the early days was IBM's preannouncement of a relational database years before it was available. It gave credibility to the then-new idea of relational databases and created demand that IBM could not fulfil."
The thinking is SAP will use on demand as a loss leader and Netsuite is not too concerned (yeah right)and they have a eight year head start and that will give them space to keep ahead of SAP.


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[I'm feeling up to the 6 word story challenge this morning, so here goes….]
Creek's warble? Muffled by crickets' song.
And here is my amateur video I recorded early Saturday morning while strolling our backyard garden with the dogs. We are so fortunate to live beside this babbling creek (which is unassumingly quiet these days before the rain).
[In response to the Six Word Challenge: Muffled
The Rules:
(Submit your story by clicking here.)
- Write a story in six words or less inspired by the prompt.
- Write your story in a comment below to be entered. A link to a blog post is not an entry.
- A blog post and a pingback is not a requirement of the challenge, just your story in the comments below but feel free to add your own blog post if you wish. Remember to use the tag #stellarstorychallenge if you do and provide a link to your post.
- Read and 'like' at least one other story published on this week's prompt.
- The winner will be the story with the most likes at the end of the challenge. This week, the end of the challenge will be on Friday 30th September at 10pm GMT.
- I will announce the winner on this blog on Friday evening and include links to the winners' blogs.
- First, second and third place will have the honour of downloading and using one of my highly coveted awards on their blogs should they wish to do so.
- In the event of a tie for second and third places, I will choose a single story to take the placement.
- Only one entry per person please. Multiple entries will be deleted leaving the first submission only.
Good luck!]


text: When I turn on the R4 DS and select the games icon it will take me to 2 folders, games and music. Now obviously playing music in the games mode won't work so I just want to know how do you get rid of it? Also this is the same when I select the multimedia mode, the game folder also appears, how do I get rid of it? Was I meant to put the music folder in the moonshell section? Do I need to just delete music/games folders? I got the latest firmware update and moonshell Thanks


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TORONTO – Ginella Massa has made history to become the first television anchor to wear a hijab while reading the news at one of Canada's major broadcasters.
The 29-year-old, who became the country's first hijab-wearing television news reporter in 2015, was asked to fill in on the anchor desk at Toronto's CityNews during the 11pm broadcast last week.
She created a buzz after the broadcast ended and she tweeted: "That's a wrap! Tonight wasn't just important for me. I don't think a woman in hijab has ever anchored a newscast in Canada."
Massa first started reporting on television in 2015 in Kitchener, Ontario. She moved back to Toronto in February this year to take a reporting job at CityNews.
Although she recognized that moving onto the anchor desk was a step up in her career, she said it took her editor to point out the larger significance. "It wasn't until my editor said, "Hey, great job! Was that a first for Canada? A woman in a hijab?" And I said yes. And so I tweeted about it," she said.
Her achievement comes amid heightened concerns about anti-Muslim sentiment in the US and Europe. During the US presidential election campaign, Donald Trump called for a "total and complete shutdown of Muslims entering the United States until our country's representatives can figure out" what is going on.
Photo Courtesy: Biliana Gortchova
"I've talked to many women who are journalists in the US who work behind the scenes and they've told me that they face multiple challenges trying to get on air," said Massa. "They've been told because of their hijab, that's not going to happen. That makes me really sad because they're being held back by someone else's idea of what the public can or cannot handle."
Although the reaction to Massa's anchor stint and reporting role has been mostly positive in Canada, she said she has received a handful of negative comments and Tweets.
"But this is all the more reason in today's climate to see positive images of Muslim women," she said. "They are a symbol of Islam when they wear the hijab and that carries a powerful image. It's so important to see positive images of us in the media."
Photos by Biliana Gortchova.


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The Geosciences Young Alumni Award
Young Alumni are building a brighter future for the university through their dedication of time, talent, and energy. The Geosciences Young Alumni Award is presented annually to a former student of the College of Geosciences who has demonstrated outstanding professional achievement or endeavors, exemplifying the core values of Texas A&M University and the educational training from the College of Geosciences towards the advancement of the greater good.
The Geosciences Young Alumni Award is reserved for those who graduated in a College of Geosciences major less than 10 years prior, attending the university as an undergraduate or graduate student to the attainment of a degree and is living at the time of consideration.
- Outstanding Geosciences graduates within the last 10 years are eligible.
- Enrichment of some aspect of the College of Geosciences community through continuing dedication, advocacy and support.
- Demonstrable commitment to the ongoing vitality of the University.
- Advancement of the value and in their achievements within their profession.
- Significant voluntary contributions of energy, time, creativity, and skills for the advancement of community welfare, discovery, education, social or government service.
Guidelines for Consideration of Candidates:
Former students employed by the University are not eligible during their term of employment.Nomination Form


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Thanks for stopping by the blog!
Hello there friend! Thank you so much for stopping by- I so appreciate having you here but wanted to let you know that since the beginning of 2014 we have moved to www.imagodeiphotographyblog.com.
I hope that you will click HERE and and once you are on the new blogsite you would add it to your favorite browser. That way you can always find the new blog easily as we will no longer be updating this blog. Hope to see you on the new and improved blog!!
Tuesday, April 6, 2010
{Netarts and Oceanside Family Photographer}
7 comments:
- AnonymousApril 6, 2010 at 2:26 PM
I love your black and white work- it's very different!ReplyDelete
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Classical Hatha Yoga for all levels
in Yarmouth County, Nova Scotia
Letter from Shobhan Richard Faulds
Page 1 of 3
Deepening a yoga practice does not require you to perform evermore difficult postures, although some students may choose to do so. Practice deepens as you attune to the inner flow of energy and awareness. Guided from within, you move and stretch in ways that nurture the body, rebalance the emotions, and fine tune the mind. On some days you may challenge yourself physically to work the kinks out. On others you may move gently to soothe body and mind.
With regular practice, you discover that sensations grow distinct and the inner world of emotion becomes more vivid. Mental awareness is refined, helping you discriminate between surface thinking and deeper, creative streams of thought. An attitude of compassionate self-observation emerges that expands self-awareness and leads to periodic insights and occasional breakthroughs. It is this steady deepening of presence, both on the yoga mat and in your life, that is the true measure of practice.
With contemporary yoga so focused on the physical, and postures woven into glitzy media images, it is easy to forget that traditional yoga was designed to initiate a profound process of personal transformation. The transformative process is best understood as an amalgam of three components: healing, growth, and spiritual awakening.
Many of us come to yoga to address health concerns or find a refuge from exhausting and hectic lives. We are in need of healing, the process of returning to normal functioning after trauma or injury.Next Page


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This county was formed in 1786 from Washington, and lies amongst the dividing ridge -- Alleghany mountains to the north and Clinch mountain variety on the south. Tazewell county is around the east and Scott and Sensible within the west. The floor is mostly mountainous, and the lands In most cases uncultivated. There are a few very prosperous tracts from the valleys and alongside the rivers, which deliver substantial and wonderful crops, significantly exceeding the common in almost all of the other counties on the Point out.
The huge major floor is devoted entirely for the living/dining/kitchen region. Expansive excellent room showcasing limitless unobstructed ocean sights plus a grand piano! Tv set leisure procedure, gasoline fireplace (Nov 1 to Mar 31), spacious gourmet kitchen with Heart island and two sink areas, chrome steel appliances, granite counter tops, substantial dining place, fifty percent tub, front room with comfortable seating for everyone, significant sun deck overlooking ocean.
Accomack, together with North Hampton county in 1634, constitute one of many eight initial shires into which Virginia was divided, and what's often called the " Japanese Shore " of Virginia. In 1642 it modified its identify to Northampton, but in 1663 recurred its initial title, taken from hat of the tribe of Indians (Accawmacke), who first inhabited this section of your State. It can be bounded on he north via the Point out of Maryland, South by Northampton county, Va. east because of the Atlantic Ocean, and west through the Chesapeake Bay. It's a location of 388 1/two, sq. miles (248,605 acres), along with the land has a median assessed price of $13 for every acre. This county is indented on the east and rest by several Appears, inlets and maller h2o courses, that are more or less farmed by Oyster planters and being navigable just about their overall size, furnish implies of transportation to your marketplaces of the massive towns from the North and East. The New York. Philadelphia and Norfolk railroad considerably facilitates intercourse and organization interaction between the individuals of our have together with other States which portion, and make it Just about the most favored portions of Virginia. In addition to this wonderful actuary of trade the Japanese Shore Steamboat Company provides every day conversation with Baltimore from your Pocomoke River, as significantly down as Hungers Creek, in Northampton, although, in the course of the trucking period, steamers and sail vessels command the creeks bringing about the Ocean.
Under a calendar year afterwards, Dell planned to double its workforce to nearly three,000 staff and include a fresh building. These ideas ended up teak furniture jarvis creek reversed, as a consequence of a superior Canadian dollar that produced the Ottawa team somewhat costly, as well as as Component of Dell's turnaround, which included transferring these simply call-center Careers offshore to cut charges.[104]
Toronto is a big city, so all person listings must be moved to the right district article content, and this segment ought to consist of a short overview. Remember to assist to maneuver listings Should you be aware of this metropolis.
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The key streets in Toronto are laid out inside of a grid sample which makes it among the best cities to get all around in by car. Acquiring from issue to issue everywhere in town is often achieved with just a few turns.
Buses and streetcars are liable to get caught kitchen furniture jarvis creek in Toronto's notorious targeted visitors all through rush-hrs, although some streetcar traces have committed lanes.
Liberty, the county seat, within the Norfolk and Western railroad, may be the critical and flourishing centre of the fertile and beautiful nation. It's a click here now large number of tobacco manufacturing along with other concerns, In addition to two banking institutions and several mercantile properties of fine organization standing.
Fares to Locations outside the GTA Price additional. C£fifty three for the taxi to downtown. C$58 for a limousine to downtown. You will pay in funds or that has a credit history/debit card. Stop by the airport's Site for recent taxi and limo tariff maps. Any spot that is not shown about the map is going to be billed C$1.45/km for taxis, or C£1.fifty five/km for limos. You'll want to talk to the motive force to verify the fare from your tariff chart just before leaving the terminal.
The Toronto Islands. A short low-cost ferry trip within the foot of Bay St. and you permit the bustle of the city at the rear of. Visually, the views of the skyline with the islands is beautiful, and for cycling, going for walks, picnics or simply stress-free, the Toronto Islands are challenging to beat.
023 BMW's initial vertical showroom in North The united states and the largest of its variety in Canada consisted of retrofitting a six-storey Business making into a showroom dealership.
Dell located a possibility amid PC-savy people who favored the benefit of buying immediate, customizing their PC for their implies, and obtaining it delivered in times. In early 1997, Dell developed an interior sales and internet marketing my site group devoted to serving the home industry and released an item line made specifically for person customers.[29]
There are numerous water courses In this particular county, tributaries on the Rapidan river, which manage ample power for mills &c. A remarkable cascade on the headwaters with the South river, which falls about a precipice of one hundred sixty feet is rather grand and intimate. No operates of internal advancement are in Procedure in this county. The Virginia Midland Railroad at Barboursville is inside of a shorter length on the county line. The Rockingham turnpike (McAdamized) from Harrisonburg to Gordonsville passes straight via Standardsville and your complete county, and affords enough services to the farmers to acquire create to market. Copper ore is located in the mountain sections, and tiny shafts, each horizontal and perpendicular, have been sunk, but with what accomplishment just isn't generally recognised. It has an area of a hundred and sixty square miles (102,507 acres) and the standard assessed worth of its lands is $four.ninety per acre.
- 309 Visitors


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Should you injure one among your arms when pursuing your fitness goals, don't cease figuring out the opposite one. Research has found that people who only skilled one arm for 2 weeks were in a position to enhance their arm strength in the different arm by around ten p.c. It is because understanding one arm also prompts the fibers in the different arm.
When you've got a busy lifestyle, make train fit into your schedule as an alternative of blocking time for practising fitness. Make fitness a family activity in case you do not need enough time to spend along with your kin. For instance, turn workout routines into video games you'll be able to play together with your kids.
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The key to winning a dash is to extend the pace of your operating stride. When doing this, be sure that your front foot lands underneath you as opposed to in entrance of you. Use the toes of your back foot to push ahead. When you do that, your running speed will improve. Set … Read More