Dye-binding Assay Research Articles

Evaluation of a point-of-care portable analyzer for measurement of plasma immunoglobulin G, total protein, and albumin concentrations in ill neonatal foals

To compare the diagnostic performance of a point-of-care (POC) analyzer with that of established methods for the measurement of plasma IgG, total protein, and albumin concentrations in neonatal foals. Evaluation study. 100 neonatal foals < 7 days of age. Procedures-Plasma IgG, total protein, and albumin concentrations were measured with a POC analyzer via an immunoturbidimetric method. Corresponding measurements of plasma IgG, total protein, and albumin concentrations were measured by means of automated biochemical analyzers via automated immunoturbidimetric, biuret, and bromocresol green dye-binding assays, respectively (standard laboratory methods). The sensitivity and specificity of the POC analyzer for detection of failure of passive transfer of immunity (FPTI) in foals were 80.7% and 100%, respectively, when FPTI was defined as a plasma IgG concentration < 400 mg/dL and were 75.9% and 100%, respectively, when FPTI was defined as a plasma IgG concentration < 800 mg/dL. The POC analyzer overestimated plasma albumin concentrations and, to a lesser extent, plasma total protein concentrations, compared with values determined with the standard laboratory methods. Results suggested the POC analyzer was acceptable for determination of plasma IgG and total protein concentrations in ill foals. The POC analyzer overestimated plasma albumin concentration such that its use was clinically unacceptable for the determination of that concentration. The POC analyzer provided timely measurements of plasma IgG concentrations, which is necessary information for the assessment of passive transfer of maternal antibodies to neonatal foals.

Chemical and Thermal Unfolding of Calreticulin

Calreticulin is a soluble endoplasmic reticulum chaperone, which has a relatively low melting point due to its remarkable structure with a relatively high content of flexible structural elements. Using far ultraviolet circular dichroism (CD) spectroscopy and a fluorescent dye binding thermal shift assay, we have investigated the chemical and thermal stability of calreticulin. When the chemical stability of calreticulin was assessed, a midpoint for calreticulin unfolding was calculated to 3.0M urea using CD data at 222 nm. Using the fluorescent dye binding thermal shift assay, calreticulin was found to obtain a molten structure in urea concentrations between 1-1.5 M urea, and to unfold/aggregate at high and low pH values. The results demonstrated that the fluorescent dye binding assay could measure the thermal stability of calreticulin in aqueous buffers with results comparable to melting points obtained by other techniques.

Aza-BODIPY: Improved synthesis and interaction with soluble Aβ1–42 oligomers

Dye-binding assays that are used to evaluate anti-aggregation ability of small molecule inhibitors towards amyloids are known to be prone to false-positive effects due to spectral overlaps between the dye and the inhibitor. Aza-BODIPY dye, which has both excitation and emission maxima above 600nm, exhibits a significant increase in its fluorescence intensity in the presence of soluble oligomers of Aβ1–42. These results indicate that aza-BODIPY could serve as a near-IR probe for detecting conformational changes of Aβ1–42 soluble oligomers in vitro, and it should eliminate false-positive effects that are associated with currently utilized thioflavin T-based dyes. In addition, a facile synthesis of aza-BODIPY has been developed, which might further expand the applications of this dye.

Urinary Excretion of Glycosaminoglycans in Patients with Urolithiasis

The interaction between tubular epithelial cells and calcium oxalate crystals or oxalate ions is a very precarious event in the lithogenesis. Urine contains ions, glycoproteins and glycosaminoglycans that inhi bit the crystallization process and may protect the kidney against lithogenesis. Hundred patients (24 women and 76 men) with a history of stone disease and hundred controls (25 women and 75 men) were evaluated for urinary GAG concentration. By using a new dye-binding assay, the total GAG concentration in the urine was measured and corrected to urinary creatinine levels (milligram of GAG per gram creatinine). There was a preponderance of urinary stones in males ; the highest incidence being in Group 2. Excretion of GAGs in 24-hour urine sample was significantly lower in patients compared to controls, for males in all age groups and for females, however, there was no statistically significant difference in the urinary GAGs value between males and females in a given age group for either controls or patients. The urinary GAGsexcretion idecreased with age in controls, but the levels in patients were lower. Conclusion: This study shows that low urinary GAGsareassociated with urinary stones in patients.Our studylead us to propose that urinary GAG may play an important role in

Dye-Binding Assays for Evaluation of the Effects of Small Molecule Inhibitors on Amyloid (Aβ) Self-Assembly

Dye-binding assays, such as those utilizing Congo red and thioflavin T, are among the most widely used tools to probe the aggregation of amyloidogenic biomolecules and for the evaluation of small molecule inhibitors of amyloid aggregation and fibrillization. A number of recent reports have indicated that these dye-binding assays could be prone to false positive effects when assessing inhibitors' potential toward Aβ peptides, species involved in Alzheimer's disease. Specifically, this review focuses on the application of thioflavin T for determining the efficiency of small molecule inhibitors of Aβ aggregation and addresses potential reasons that might be associated with the false positive effects in an effort to increase reliability of dye-binding assays.

Biomarkers in dogs surgically treated for ruptured cranial cruciate ligaments

Thirty-one dogs with cranial cruciate ligament rupture (CCLR) were randomly treated with tibial plateau levelling osteotomy (TPLO; TPLO group) or proximal tibial osteotomy (PTO; PTO group). Synovial fluid was collected...

Effect of Caffeic Acid on Nasal Polyp Derived Fibroblasts

Objectives: Caffeic acids are known to have anti-oxidant, anti-inflammatory, immunomodulatory, and tissue reparative effects. The purposes of this study were to determine the effect of caffeic acid on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and collagen production, and to determine whether caffeic acid is involved in the antioxidant effect. Method: Nasal polyp-derived fibroblasts (NPDFs) were pre-treated with caffeic acid (5-25 µM) for 2 hours and stimulated with TGF-β1 (5 ng/mL) for 24 hours. The expression of α-SMA, collagen types I and III, and Nox4 mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein was determined by immunofluorescence microscopy. The amount of total soluble collagen production was analyzed by the Sircol collagen dye-binding assay. The reactive oxygen species (ROS) generated by NPDFs were determined using 2′,7′-dichlorfluorescein-diacetate. siNox4 was used to determine the effect of Nox4. Results: The expression of α-SMA and production of collagen were significantly increased following TGF-β1 treatment. In contrast, the level of expression of α-SMA and the level of production of collagen were decreased by pre-treatment with caffeic acid. The activation of Nox4 and the subsequent production of ROS were also reduced by pre-treatment with caffeic acid. The expression of α-SMA was prevented by inhibition of ROS generation with siNox4. Conclusion: Caffeic acid may inhibit TGF-β1-induced differentiation of fibroblasts into myofibroblasts and collagen production by regulating NADPH oxidase-derived ROS.

The Diagnostic Accuracy of a Urine Albumin-Creatinine Ratio Point-of-Care Test for Detection of Albuminuria in Primary Care

Albuminuria is an important sign of chronic kidney disease and is detected routinely by measurement of urinary albumin-creatinine ratio (ACR). A Siemens CLINITEK test designed for use at the point of care is available that can semiquantitatively measure ACR. Diagnostic accuracy study evaluating a urinary ACR point-of-care test. The semiquantitative ACR test was evaluated at the point of care in a representative primary care population (those with or at increased risk of chronic kidney disease) of 642 patients under standard operational conditions and compared with the reference standard of ACR measurement in the clinical laboratory. The point-of-care CLINITEK semiquantitative ACR test. This test uses dye-binding and catalytic assays for albumin and creatinine, respectively, on a Microalbumin 9 strip, which is read by the CLINITEK Status Analyzer, and ACR is calculated automatically. Laboratory measurement of albumin and creatinine on an Abbott Architect analyzer by immunoturbidimetric and enzymatic assays, respectively, and calculation of ACR. The prevalence of albuminuria (laboratory ACR≥30 mg/g) in the study population was 20.2%. Sensitivity and specificity of the point-of-care test for detecting albuminuria were 83.2% and 80.0%, respectively. Positive and negative predictive values were 51.2% and 95.0%, respectively; positive and negative likelihood ratios were 4.16 and 0.21, respectively. Twenty-three (3.6%) samples measured at the point of care were not analyzed in the central laboratory for a variety of reasons, including laboratory reception data entry errors. Our sensitivity calculation is accurate to an approximately 8% CI. The instrument-read reagent strip test was a poor rule-in test for albuminuria at the point of care, as evidenced by the low positive predictive value, but was a reasonable rule-out test. Observed sensitivity was lower than reported in earlier laboratory-based studies. This decreased diagnostic accuracy needs to be balanced against the potential advantages of a point-of-care testing approach.

Effect of [6]-gingerol on myofibroblast differentiation in transforming growth factor beta 1-induced nasal polyp-derived fibroblasts.

[6]-Gingerol is one of the major pungent principles of ginger and has diverse effects, including anti-inflammatory, and antioxidative effects. Reactive oxygen species (ROS) are released during the phenotypic transformation of fibroblasts to myofibroblasts, a process that is involved in the growth of nasal polyps by inducing extracellular matrix (ECM) accumulation. The purpose of this study was to determine the effect of [6]-gingerol on myofibroblast differentiation and collagen production of nasal polyp-derived fibroblasts (NPDFs) and to determine if the effect of [6]-gingerol is linked to an antioxidant effect. NPDFs were incubated and treated with transforming growth factor (TGF) beta 1. The ROS generated by NPDFs were determined using 2″,7″-dichlorfluorescein-diacetate. The fluorescence was captured by a fluorescent microscope and measured using a fluorometer. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein and pSmad2/3 was determined by immunofluorescence microscopy and or Western blotting. The amount of total soluble collagen production was analyzed by the SirCol collagen dye-binding assay. TGF-beta 1 stimulation increased ROS production by NPDFs. [6]-Gingerol decreased the production of ROS in TGF-beta 1-induced NPDFs. Myofibroblast differentiation, collagen production, and phosphorylation of Smad2/3 were prevented by [6]-gingerol and inhibition of ROS generation with antioxidant such as diphenyliodonium, N-acetylcysteine, and ebselen. These results suggest the possibility that [6]-gingerol may play an important role in inhibiting the production of the ECM in the development of nasal polyps through an antioxidant effect.

SDS Can Be Utilized as an Amyloid Inducer: A Case Study on Diverse Proteins

Sodium dodecyl sulphate (SDS), an anionic surfactant that mimics some characteristics of biological membrane has also been found to induce aggregation in proteins. The present study was carried out on 25 diverse proteins using circular dichroism, fluorescence spectroscopy, dye binding assay and electron microscopy. It was found that an appropriate molar ratio of protein to SDS readily induced amyloid formation in all proteins at a pH below two units of their respective isoelectric points (pI) while no aggregation was observed at a pH above two units of pI. We also observed that electrostatic interactions play a leading role in the induction of amyloid. This study can be used to design or hypothesize a molecule or drug, which may counter act the factor responsible for amyloid formation.

NON-REDUCING ALKALINE SOLUBILIZATION AND RAPID ON-COLUMN REFOLDING OF RECOMBINANT PRION PROTEIN

Mature prion protein (PrP) is a 208-residue polypeptide that contains a single disulfide bond. We report an alternative method to purify recombinant mouse PrP produced in Escherichia coli. Bacterial inclusion bodies were solubilized in a buffer containing 2 M urea at pH 12.5. The solubilized protein was rapidly purified on a nickel affinity column without a chaotrope gradient, followed by ion-exchange chromatography. The yield and purity of PrP produced by this alternative approach was similar to that obtained using a conventional solubilization and on-column refolding protocol. Recombinant PrP produced using the non-reducing purification protocol is properly folded, as determined by circular dichroism, and a competent substrate for amyloid fibril formation, as determined by Thoflavin-T dye binding assays. In summary, this report describes a rapid method for producing properly folded recombinant PrP without reducing agents or a chaotrope gradient.

COMPARISON OF ADSORPTION BEHAVIOR OF BOVINE SERUM ALBUMIN AND OSTEOPONTIN ON HYDROXYAPATITE AND ALUMINA

Adsorption behavior of bovine serum albumin (BSA) and osteopontin (OPN) on osteoconductive hydroxyapatite (HA) and non-osteoconductive alpha-type alumina (-Al2O3) was studied as function of protein concentration and pH, using Bradford dye binding assay. BSA showed a much larger binding capacity on -Al2O3 than that on HA irrespective of BSA solution concentration and pH. Furthermore, OPN is likely to show the same adsorption characteristic as BSA. Definite correlation was not observed between the albumin or OPN adsorption capacity and the osteoconductivity of materials, suggesting that other factors (e.g., orientation, arrangement, etc. of albumin and/or OPN) likely govern expression of the osteoconductivity. (Received February 2, 2012; Accepted March 30, 2012)

Phosphatidate Phosphatase Plays Role in Zinc-mediated Regulation of Phospholipid Synthesis in Yeast

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated by mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G. S. (2007) Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion. Biochim. Biophys. Acta 1771, 322-330; Eide, D. J. (2009) Homeostatic and adaptive responses to zinc deficiency in Saccharomyces cerevisiae. J. Biol. Chem. 284, 18565-18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative upstream activating sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme) and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme) but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway.

Effect of Simvastatin on Transforming Growth Factor Beta-1–Induced Myofibroblast Differentiation and Collagen Production in Nasal Polyp-Derived Fibroblasts

Statins are the most commonly prescribed drugs for the treatment of hypercholesterolemia. Statins exert not only lipid-lowering but also other cellular effects, including antifibrotic properties. The purpose of this study was to determine the effect of simvastatin on transforming growth factor (TGF)-beta-1-induced myofibroblast differentiation and collagen production in nasal polyp-derived fibroblasts (NPDFs) and to verify the mechanism of the effect of simvastatin in TGF-beta-1-induced myofibroblast differentiation in NPDFs. NPDFs were pretreated with simvastatin with or without mevalonate or Y-27643 for 2 hours before induction by TGF-beta-1. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of alpha-SMA protein was determined by immunofluorescent cytochemical staining. Total soluble collagen production was analyzed by the SirCol collagen dye-binding assay (Biocolor, Belfast, U.K.). Phosphorylation of Smad 2/3 was evaluated by Western blot analysis. In TGF-beta-1-induced NPDFs, simvastatin significantly inhibited the expressions of α-SMA and collagen type IV mRNA and reduced alpha-SMA and collagen protein levels. Pretreatment with mevalonate reversed the effect of simvastatin. The expression of alpha-SMA mRNA and protein was significantly decreased by pretreatment with Y-27632. The TGF-beta-1-induced expression of pSmad 2/3 protein was notably decreased by pretreatment with simvastatin. We showed that simvastatin inhibits TGF-beta-1-induced myofibroblast differentiation (expression of alpha-SMA) and collagen production in NPDFs and Rho/Rock and TGF-β/Smad signaling is involved as an underlying mechanism. The results of our study suggest that simvastatin is a possible candidate for the suppression of nasal polyp formation.

Chondrogenic differentiation of canine myoblasts induced by cartilage-derived morphogenetic protein-2 and transforming growth factor-β1 in vitro

The meniscus has limited ability to repair itself following injury. However, tissue engineering provides new means of meniscus repair. Myoblasts, which possess the potential of multi-directional differentiation, may be ideal seed cells in meniscus tissue engineering. Myoblasts from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated myoblasts from canines and induced chondrogenesis in order to establish a new experimental model of seed cells. Myoblasts were isolated and harvested from Beagle canines. To induce chondrogenesis, cartilage-derived morphogenetic protein-2 (CDMP-2) at different concentrations (10, 20, 50 and 100 ng/ml), transforming growth factor-β1 (TGF-β1; 10, 20, 30 and 50 ng/ml), and different concentrations of CDMP-2 (10, 20, 50 and 100 ng/ml) together with TGF-β1 (20 ng/ml) were added to the cultured pellets. After 21 days of in vitro culture, chondrogenic differentiation was evaluated by histological and immunohistochemical techniques. The degree of gene expression was measured by quantitative RT-PCR. Based on the histological staining of glycosaminoglycan, using the toluidine blue dye-binding method, we found that CDMP-2 initiated chondrogenic differentiation of myoblasts, as did TGF-β1. Furthermore, CDMP-2 conferred a stronger stimulatory effect than TGF-β1. The combination of CDMP-2 and TGF-β1 synergistically induced chondrogenesis of myoblasts. This synergistic chondrogenic effect of CDMP-2 together with TGF-β1 was further confirmed by quantification of glycosaminoglycan using dimethylmethylene blue dye-binding assay and immunohistochemical analysis of the expression of cartilage-specific proteins collagen I and II. Canine myoblasts can be induced into chondrocytes by CDMP-2 and TGF-β1 in vitro, suggesting that myoblasts are suitable as seed cells for meniscus tissue engineering.

Synthesis and Transfection Efficiency of Cationic Oligopeptide Lipids: Role of Linker

In the design of new cationic lipids for gene transfection, the chemistry of linkers is widely investigated from the viewpoint of biodegradation and less from their contribution to the biophysical properties. We synthesized two dodecyl lipids with glutamide as the backbone and two lysines to provide the cationic headgroup. Lipid 1 differs from Lipid 2 by the presence of an amide linkage instead of an ester linkage that characterizes Lipid 2. The transfection efficiency of lipoplexes with cholesterol as colipid was found to be very high with Lipid 1 on Chinese Hamster Ovary (CHO) and HepG2 cell lines, whereas Lipid 2 has shown partial transfection efficiency on HepG2 cells. Lipid 1 was found to be stable in the presence of serum when tested in HepG2 and CHO cells albeit with lower activity. Fluorescence-based dye-binding and agarose gel-based assays indicated that Lipid 1 binds to DNA more efficiently than Lipid 2 at charge ratios of >1:1. The uptake of oligonucleotides with Lipid 1 was higher than Lipid 2 as revealed by confocal microscopy. Transmission electron microscopy (TEM) images reveal distinct formation of liposomes and lipoplexes with Lipid 1 but fragmented and unordered structures with Lipid 2. Fusion of Lipids 1 and 2 with anionic vesicles, with composition similar to plasma membrane, suggests that fusion of Lipid 2 was very rapid and unlike a fusion event, whereas the fusion kinetics of Lipid 1 vesicles was more defined. Differential scanning calorimetry (DSC) revealed a high T(m) for Lipid 1 (65.4 °C) while Lipid 2 had a T(m) of 23.5 °C. Surface area-pressure isotherms of Lipid 1 was less compressible compared to Lipid 2. However, microviscosity measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed identical values for vesicles made with either of the lipids. The presence of amide linker apparently resulted in stable vesicle formation, higher melting temperature, and low compressibility, while retaining the membrane fluid properties suggesting that the intermolecular hydrogen bonds of Lipid 1 yielded stable lipoplexes of high transfection efficiency.

477 NEW ALGINATE-CHITOSAN HYDROGEL BEADS WITH ANTI-INFLAMMATORY AND ANABOLIC EFFECTS ON HUMAN CHONDROCYTES

Purpose: Proper selection of cell source is a persisting problem for cartilage tissue engineering because it is difficult to attain sufficient number of chondrocytes from sparsely cellular cartilage tissue. Chondrocytes can be increased in number by cell culture however they readily dedifferentiate towards fibroblast like and lose their chondrocytic phenotype. Previously we have shown that co-culture of these cultured passaged dedifferentiated chondrocytes with small numbers of primary chondrocytes induces redifferentiation in dedifferentiated cells. Here we show that the age affects ability of primary chondrocytes to induce redifferentiation in passaged cells. Methods: Cell preparation: Human fetal (17 weeks), human adult articular (18–60 yrs.) and bovine articular cartilage (young 6–9 months, old 24–36 months) were harvested and chondrocytes were isolated by sequential enzymatic digestion. Co-culture: the passaged dedifferentiated cells were mixed with primary chondrocytes at ratio of 4:1 and cocultured on MillicellTM filter inserts which allow for 3D culture in 20% FBS containing DMEM-HG. Analysis: After 4 weeks the newly formed tissue was characterized and compared with bovine and human primary chondrocyte cultures. Histological qualitative assessment was done by Toluidine Blue staining. Proteoglycan accumulation was quantitatively determined by dimethylmethylene blue dye-binding assay. Values were normalized to total DNA content determined by Hoechst 33258 dye binding assay. Gene expression was quantified by qPCR. Statistics: Results were expressed as mean ± SD of 3 independent experiments and significance was determined by one-way ANOVA. Results: To determine the effect of age on the ability of chondrocytes to accumulate proteoglycans and induce redifferentiation chondrocytes from older and younger cows were compared with each other after 4 weeks of in vitro culture (Fig. 1). After 4 weeks when visualized under microscope after Toluidine blue staining more young cells appeared to be alive and surrounded by newly deposited cartilaginous matrix. Dimethylmethylene blue assay showed that the proteoglycan content of tissue formed by co-cultured of young primary chondrocytes and old passaged cells at 4 weeks was significantly higher than tissue formed by the co-culture with old primary chondrocytes. qPCR showed higher gene expression of type II collagen, aggrecan, and SOX9. passged cells alone failed to produce cartilaginous matrix regardless of age or species difference. Conclusion: We conclude that young primary chondrocytes produce factors which affect the cartilaginous matrix deposition capabilities of dedifferentiated cells. Further studies on human fetal chondrocytes are in progress. Acknowledgements: Supported by CIHR.

459 THE TENSION OF ALIGNED NANOFIBROUS POLYURETHANE SCAFFOLDS INFLUENCES ANNULUS FIBROSUS CELL FUNCTION

Purpose: Degeneration of the intervertebral disc (IVD) causes loss of disc function and is associated with back pain. Tissue engineering an IVD replacement is complicated by the complex orientation of annulus fibrosus (AF), one of the tissues of the IVD. The AF is under tension in vivo and loss of this strain has been implicated in the pathogenesis of AF degeneration. Aligned nanofibrous polycarbonate urethane (PU) scaffolds have been used to tissue engineer AF tissue. This study examined whether the tension of PU scaffolds modulate AF cell shape and function. Methods: Scaffolds: Biodegradable PU was synthesized from poly(1,6hexyl 1,2-ethyl carbonate)diol, 1,6-hexane diisocyanate and 1,4butanediol in N, N-dimethylacetamide. Aligned scaffolds were formed by electrospinning and placed under tension in a specially designed holding apparatus. Cell culture: AF cells were isolated from bovine caudal discs by sequential enzymatic digestion and seeded onto strained PU scaffolds. 24 hours later cultures were divided into two groups – (1) strain maintained; or (2) strain released (relaxed) and were grown in vitro for up to 7 days. Tissue Analysis: Either H-thymidine to quantify cell proliferation or Hproline to quantify newly synthesized collagen was added to the culture during the last 24 hours of culture. The tissue was harvested, papain digested and the amount of isotope incorporated determined. DNA content was quantified by Hoechst 33258 dye binding assay. Morphology: Cultures were processed for scanning electron microscopy (SEM) to visualize cell shape. Other samples were fixed with 4% paraformaldehyde and stained with phalloidin (actin), DAPI (nuclei) or antibody reactive with collagen type I and examined by confocal microscopy. Scaffold analysis: The elastic modulus of the scaffold was evaluated by a mechanical testing device to the breaking point (Instron) using a tensile strain of 10mm/min. Statistics: Results were expressed as mean ± SEM of 3 independent experiments and significance was determined by one-way ANOVA. Results: SEM images of the continuously strained and relaxed scaffolds show that the fibres remained aligned under both conditions. The applied strain in the direction of the oriented fibres resulted in a change in scaffold length of 20.1±3% (mean±SEM), and this decreased by 11.1±2% after releasing the tension on the scaffold. Cells on relaxed scaffolds were significantly more proliferative and synthesized more collagen type I when compared to cells on the continuously strained scaffolds. The strained scaffolds displayed cells which were elongated and oriented parallel to each other as well as to the type I collagen fibres, both of which were aligned parallel to the scaffold fibers. In contrast cells on relaxed scaffolds were more polygonal in shape and did not show collagen alignment. The cell response to the change in strain was delayed by 3 to 5 days after tension release and this correlated with the time at which changes in scaffold length were detected. By 7 days the elastic modulus was significantly greater in the continuously strained scaffolds as compared to the relaxed scaffolds. Conclusions: AF cell shape and matrix synthesis and orientation were influenced by changes in PU substrate tension. Understanding how scaffold tension regulates AF cell function will help facilitate the engineering of AF tissue with an organized extracellular matrix, a critical feature for creating functional AF tissue. Acknowledgements: Supported by CIHR.

Abstract 2540: Evaluation of novel bis-amidine osmium complex as anti-proliferative agents

Abstract Introduction. Despite improvement in current treatment strategies, including surgery, chemotherapy (CT) and radiotherapy (RT), survival of cancer patients in advanced stages has not improved significantly over the last decade. Organometallic complexes of platinum (cisplatin, carboplatin and oxaliplatin) are in use as chemotherapeutic agents worldwide. However, acquired drug resistance to these platinum complexes has necessitated the search for novel strategies to overcome this limitation, which includes combination therapies and the development of novel platinum or non-platinum complexes as drugs. Methods. Anti-proliferative effects of a bis-amidine complex (Os-Sh1) was determined by measuring the inhibition of cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry-based cell-cycle analysis, annexin V dye binding assay and TUNEL assay in endometrial cancer (Hec1A and KLE) and cervical cancer (HeLa) cells. Results: Cell viability (MTT) assays revealed inhibition of cell proliferation on treatment with the bis-amidine osmium complex (Os-Sh1) in HEC1A, KLE and HeLa cell lines in a dose- (1μM – 50μM) and time-dependent manner (24h – 96h). Cell-cycle analysis showed increased cell death, i.e. the sub-G0 fraction, with G0/G1-arrest and reduction in the G2/M phase of cell cycle on treatment with the Os-Sh1 in these cell lines. Results of annexin V assay suggested apoptosis as the mechanisms of Os-complex induced cell death. Apoptosis was further confirmed by TUNEL assay using flow cytometry analysis. Western blot analysis showed cleavage of caspase 9, caspase 3 and poly-ADP ribose polymerase (PARP), suggesting activation of the intrinsic mitochondrial pathway of apoptosis. Interestingly, Os-Sh1 was more effective in inhibiting cell proliferation at a lower dose and shorter period of time (LD50=20 μM, 48h) in comparison to cisplatin, carboplatin and oxaliplatin in HEC1A, KLE and HeLa cell lines. Conclusions: Our in vitro data clearly demonstrated anti-proliferative effects of a novel bis-amidine osmium complex in endometrial and cervical cancer cells at a lower dose and in shorter period of time in comparison to platinum drugs. Based on these results, we hypothesize that such osmium complex has the potential as effective cytotoxic agents wherein cancer cells acquire resistance to commonly used platinum drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2540. doi:10.1158/1538-7445.AM2011-2540

7,3′,4′-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses Ultraviolet B-induced Skin Cancer by Targeting Cot and MKK4

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3',4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3',4'-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3',4'-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3',4'-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3',4'-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3',4'-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3',4'-THIF, a potential skin cancer chemopreventive agent.