Dimethylmethylene Blue Research Articles

A phosphate glass reinforced composite acrylamide gradient scaffold for osteochondral interface regeneration

The bone-cartilage interface is defined by a unique arrangement of cells and tissue matrix. Injury to the interface can contribute to the development of arthritic joint disease. Attempts to repair osteochondral damage through clinical trials have generated mixed outcomes. Tissue engineering offers the potential of integrated scaffold design with multiregional architecture to assist in tissue regeneration, such as the bone-cartilage interface. Challenges remain in joining distinct materials in a single scaffold mass while maintaining integrity and avoiding delamination. The aim of the current work is to examine the possibility of joining two closely related acrylamide derivatives such as, poly n-isopropyl acrylamide (pNIPAM) and poly n‑tert‑butyl acrylamide (pNTBAM). The target is to produce a single scaffold unit with distinct architectural regions in the favour of regenerating the osteochondral interface. Longitudinal phosphate glass fibres (PGFs) with the formula 50P2O5.30CaO.20Na2O were incorporated to provide additional bioactivity by degradation to release ions such as calcium and phosphate which are considered valuable to assist the mineralization process. Polymers were prepared via atom transfer radical polymerization (ATRP) and solutions cast to ensure the integration of polymers chains. Scaffold was characterized using scanning electron microscope (SEM) and Fourier transform infra-red (FTIR) techniques. The PGF mass degradation pattern was inspected using micro computed tomography (µCT). Biological assessment of primary human osteoblasts (hOBs) and primary human chondrocytes (hCHs) upon scaffolds was performed using alizarin red and colorimetric calcium assay for mineralization assessment; alcian blue staining and dimethyl-methylene blue (DMMB) assay for glycosaminoglycans (GAGs); immunostaining and enzyme-linked immunosorbent assay (ELISA) to detect functional proteins expression by cells such as collagen I, II, and annexin A2. FTIR analysis revealed an intact unit with gradual transformation from pNIPAM to pNTBAM. SEM images showed three distinct architectural regions with mean pore diameter of 54.5 µm (pNIPAM), 16.5 µm (pNTBAM) and 118 µm at the mixed interface. Osteogenic and mineralization potential by cells was observed upon the entire scaffold's regions. Chondrogenic activity was relevant on the pNTBAM side of the scaffold only with minimal evidence in the pNIPAM region. PGFs increased mineralization potential of both hOBs and hCHs, evidenced by elevated collagens I, X, and annexin A2 with reduction of collagen II in PGFs scaffolds. In conclusion, pNIPAM and pNTBAM integration created a multiregional scaffold with distinct architectural regions. Differential chondrogenic, osteogenic, and mineralized cell performance, in addition to the impact of PGF, suggests a potential role for phosphate glass-incorporated, acrylamide-derivative scaffolds in osteochondral interface regeneration.

Comparison of urine glycosaminoglycan excretion between children with autism spectrum disorder and typically developed children

BackgroundAbnormalities pertaining to glycosaminoglycan metabolism have been demonstrated in children with autism spectrum disorder. The aim of the present study was to compare urine excretion of sulfated glycosaminoglycans in children with autism spectrum disorder, with neurotypical controls and explore its association with co-occurring symptoms. MethodRandom urine samples were collected from children with autism spectrum disorder (n = 61) between the ages of 2 and 6 years, and age- and sex-matched neurotypical controls. Urine glycosaminoglycan levels were quantified by the dimethylmethylene blue (DMMB) dye-binding assay. ResultsMean glycosaminoglycan/creatinine ratio of children with autism spectrum disorder was 22.279 ± 13.044 mg/mmol while that of neurotypical controls was 19.121 ± 7.319 mg/mmol. Eight patients with autism spectrum disorder (13.11%) exhibited abnormally high glycosaminoglycan excretion. Unstandardized urine glycosaminoglycan levels are significantly higher (p = 0.019) in the autism spectrum disorder group when covariates such as age, urinary creatinine, and height are taken into consideration by ANCOVA. However, the outcome showed a trend towards significance when glycosaminoglycan/creatinine ratio was used in ANCOVA (p = 0.058). In neurotypical subjects, the urine glycosaminoglycan levels appear to decline with age, height, and weight while this trend was not apparent in subjects with autism spectrum disorder. Glycosaminoglycan excretion did not correlate with the presence of co-occurring symptoms of autism spectrum disorder; frequent gastrointestinal symptoms, self-injurious behaviors, food aversions, or parent-reported sleep problems. ConclusionsA subset of children with autism spectrum disorder exhibits higher urine glycosaminoglycan excretion. Further research is needed to explore the molecular basis of this finding.

Optimization of Automated Dimethylmethylene Blue Assay for Quantification of Pediatric Urine Samples.

The metachromatic dye dimethylmethylene blue is used to quantify total glycosaminoglycans in urine. Understanding the interaction of dimethylmethylene blue with glycosaminoglycans is pertinent to optimize the assay procedure depending on the type of sample and interpret the findings meaningfully. The present spectrophotometric study determined the optimum sample-to-dye ratio, primary wavelength for measuring absorbance, after studying the interaction of two different chondroitin sulfate species (unfractionated chondroitin sulfate from bovine trachea vs. chondroitin sulfate oligosaccharide with degree of polymerization of 12, from shark cartilage) with dimethylmethylene blue. Respective dye-glycosaminoglycan complexes of the two chondroitin sulfate species showed significantly different absorbance maxima, while that of the chondroitin sulfate oligosaccharide was closer to absorbance maxima of urine glycosaminoglycans. The chondroitin sulfate oligosaccharide showed relatively less stable absorbance readings at higher concentrations in the reaction volume. Furthermore, the chondroitin sulfate reference materials exhibited differences in the linearity of standard curves and hence parallelism. Based on the findings, the method was semiautomated on Beckman Coulter D✕C 700 biochemistry analyzer using the chondroitin sulfate oligosaccharide as the standard. The urine glycosaminoglycan concentration obtained was slightly lower but reasonably close to that obtained through the External Quality Assurance (EQA) scheme administrated by ERNDIM (European Research Network, Inherited Disorders of Metabolism). The findings of the present study can be used to guide the dimethylmethylene blue assay optimization, redevelopment efforts, and harmonization across laboratories. The chondroitin sulfate oligosaccharide is better than the unfractionated chondroitin sulfate from bovine trachea due to its absorbance maxima closer to urine glycosaminoglycans. On the other hand, unfractionated chondroitin sulfate exhibit poor parallelism leading to falsely lower urine glycosaminoglycan levels.

The potential of phenothiazinium dyes as cytotoxicity markers in cisplatin-treated cells

Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the strategies employed to determine cytotoxicity, MTT, neutral red, and resazurin are commonly used. Methylene blue (MB), a phenothiazinium salt, has several uses, such as dye, redox indicator, and even as treatment for human disease and health conditions, such as malaria and methemoglobinemia. However, MB has only been sparsely used as a cellular toxicity indicator. As a viability indicator, MB is mostly applied to fixed cultures at high concentrations, especially when compared to MTT or neutral red. Here we show that MB and its related compounds new methylene blue (NMB), toluidine blue O (TBO), and dimethylmethylene blue (DMMB) can be used as cytotoxicity indicators in live (non-fixed) cells treated for 72 h with DMSO and cisplatin. We compared dye uptake between phenothiazinium dyes and neutral red by analyzing supernatant and cell content via visible spectra scanning and microscopy. All dyes showed a similar ability to assess cell toxicity compared to either MTT or neutral red. Our method represents a cost-effective alternative to in vitro cytotoxicity assays using cisplatin or DMSO, indicating the potential of phenothiazinium dyes for the screening of candidate drugs and other applications.

Exogenous All-Trans Retinoic Acid Induces Myopia and Alters Scleral Biomechanics in Mice.

Ocular all-trans retinoic acid (atRA) levels are influenced by visual cues, and exogenous atRA has been shown to increase eye size in chickens and guinea pigs. However, it is not clear whether atRA induces myopic axial elongation via scleral changes. Here, we test the hypothesis that exogenous atRA will induce myopia and alter scleral biomechanics in the mouse. Male C57BL/6J mice were trained to voluntarily ingest atRA + vehicle (1% atRA in sugar, 25 mg/kg) (RA: n = 16 animals) or vehicle only (Ctrl: n = 14 animals). Refractive error (RE) and ocular biometry were measured at baseline and after 1 and 2 weeks of daily atRA treatment. Eyes were used in ex vivo assays to measure scleral biomechanics (unconfined compression: n = 18), total scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 23), and specific sGAGs (immunohistochemistry: n = 18). Exogenous atRA caused myopic RE and larger vitreous chamber depth (VCD) to develop by 1 week (RE: -3.7 ± 2.2 diopters [D], P < 0.001; VCD: +20.7 ± 15.1 µm, P < 0.001), becoming more severe by 2 weeks (RE: -5.7 ± 2.2 D, P < 0.001; VCD: +32.3 ± 25.8 µm, P < 0.001). The anterior eye biometry was unaffected. While scleral sGAG content was not measurably affected, scleral biomechanics were significantly altered (tensile stiffness: -30% ± 19.5%, P < 0.001; permeability: +60% ± 95.3%, P < 0.001). In mice, atRA treatment results in an axial myopia phenotype. Eyes developed myopic RE and larger VCD without the anterior eye being affected. The decrease in stiffness and increase in permeability of the sclera are consistent with the form-deprivation myopia phenotype.

Altered Structure and Function of Murine Sclera in Form-Deprivation Myopia.

The sclera is believed to biomechanically influence eye size, facilitating the excessive axial elongation that occurs during myopigenesis. Here, we test the hypothesis that the sclera will be remodeled and exhibit altered biomechanics in the mouse model of form-deprivation (FD) myopia, accompanied by altered retinoid concentrations, a potential signaling molecule involved in the process. Male C57 Bl/6J mice were subjected to unilateral FD (n = 44 eyes), leaving the contralateral eye untreated (contra; n = 44). Refractive error and ocular biometry were measured in vivo prior to and after 1 or 3 weeks of FD. Ex vivo measurements were made of scleral biomechanical properties (unconfined compression: n = 24), scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 18, and immunohistochemistry: n = 22), and ocular all-trans retinoic acid (atRA) concentrations (retina and RPE+choroid+sclera, n = 24). Age-matched naïve controls were included for some outcomes (n = 32 eyes). Significant myopia developed after 1 (-2.4 ± 1.1 diopters [D], P < 0.001) and 3 weeks of FD (-4.1 ± 0.7 D, P=0.025; mean ± standard deviation). Scleral tensile stiffness and permeability were significantly altered during myopigenesis (stiffness = -31.4 ± 12.7%, P < 0.001, and permeability = 224.4 ± 205.5%, P < 0.001). Total scleral sGAG content was not measurably altered; however, immunohistochemistry indicated a sustained decrease in chondroitin-4-sulfate and a slower decline in dermatan sulfate. The atRA increased in the retinas of eyes form-deprived for 1 week. We report that biomechanics and GAG content of the mouse sclera are altered during myopigenesis. All scleral outcomes generally follow the trends found in other species and support a retina-to-sclera signaling cascade underlying mouse myopigenesis.

The Dimethylmethylene Blue Assay (DMMB) for the Quantification of Sulfated Glycosaminoglycans.

The 1,9-dimethylmethylene blue (DMMB) assay enables the detection of sulfated glycosaminoglycans (sGAGs). This assay can be used to quickly quantify the sGAG content in a large number of samples using spectrophotometry. While this widespread assay appears straightforward, there are certain pitfalls that need to be considered.

Effect of Anti-Iduronate 2-Sulfatase Antibodies in Patients with Mucopolysaccharidosis Type II Treated with Enzyme Replacement Therapy

To assess the relationship between anti-Iduronate 2-sulfatase (IDS) antibodies, IDS genotypes, phenotypes and their impact in patients with enzyme replacement therapy (ERT)-treated Mucopolysaccharidosis type II. Dutch patients treated with ERT were analyzed in this observational cohort study. Antibody titers were determined by enzyme-linked immunosorbent assay. Neutralizing effects were measured in fibroblasts. Pharmacokinetic analysis of ERT was combined with immunoprecipitation. Urinary glycosaminoglycans were measured using mass spectrometry and dimethylmethylene blue. Eight of 17 patients (47%) developed anti-IDS antibodies. Three patients with the severe, neuronopathic phenotype, two of whom did not express IDS protein, showed sustained antibodies for up to 10years of ERT. Titers of 1:5120 or greater inhibited cellular IDS uptake and/or intracellular activity invitro. In 1 patient who was neuronopathic with a titer of 1:20 480, pharmacokinetic analysis showed that all plasma recombinant IDS was antibody bound. This finding was not the case in 2 patients who were not neuronopathic with a titer of 1:1280 or less. Patients with sustained antibody titers showed increased urinary glycosaminoglycan levels compared with patients with nonsustained or no-low titers. Patients with the neuronopathic form and lack of IDS protein expression were most at risk todevelop sustained anti-IDS antibody titers, which inhibited IDS uptake and/or activity invitro, and the efficacy of ERT in patients by lowering urinary glycosaminoglycan levels.

Histological evaluation of skin lesions induced by Leishmania braziliensis treated by PACT using Laser light and 1.9 dimethyl-methylene blue

This study aimed to perform a histological evaluation in skin lesions caused by Leishmania braziliensis after PACT treatment using Laser associated with 1.9. dimethyl methylene blue BALB/c mouse ear infection model was used. A total of 40 animals were assigned into two groups considering time intervals at 5 and 10 weeks and subdivided into four subgroups: Control, Photosensitizer, Laser and PACT. Two therapeutic interventions were performed after the 5th week of infection at 48 h intervals. 1.9 Dimethyl methylene blue was used as a photosensitizer at the concentration of 7 ng/mL, with a non-invasive topical administration method associated with Laser (λ = 660 nm, 40 mW, 12 J/cm2). Sample collection occurred 5 or 10 weeks after therapeutic interventions. The main histological findings were observed in the laser and PACT groups at the 10-week evaluation. The Laser group showed reduced lymphoplasmacytic inflammation and histiocytes (p = 0.0079). The PACT group showed reductions in lymphoplasmacytic inflammation at 5 and 10 weeks, discrete reduction of histiocytes and a higher percentage of tissue remodeling. PACT with non-invasive topical administration of the photosensitizer was able to reduce lymphoplasmacytic inflammation and increase tissue remodeling in leishmaniosis skin lesions. This protocol may be easily used in humans and clinical trial shall be carried out to confirm the animal's findings.

The influence of friedelin, resinone, tingenone and betulin of compounds on chondrogenic differentiation of porcine adipose-derived mesenchymal stem cells (pADMSCs)

The study investigated the influence of friedelin, resinone, tingenone and betulin plant-based secondary metabolite compounds on cellular proliferation, extracellular matrix (ECM) components synthesis, expression of chondrogenic markers and maturation of differentiated chondrocytes (cell proliferation and hypertrophy) in porcine adipose-derived mesenchymal stem cells (pADMSCs) undergoing chondrogenic differentiation. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Cyquant assays were used to determine cell proliferation, viability, and total cellular DNA, DMMB (Dimethyl methylene blue) was used for glycosaminoglycan (GAG) synthesis, RT-qPCR for gene expression and histology combined with immunohistochemistry for cartilage ECM proteoglycan deposition. The MTT results showed that friedelin at 37 μM, resinone at 36 μM and betulin at 18 μM with cell viability of above 100% compared to control. Tingenone at 37 μM showed cell viability of about 76%. These concentrations were considered the most effective with no toxicity effect on the cells and were further analysed with TGF-β3 (10 ng/mL) as a positive control. The results showed a high synthesis of DNA with friedelin on day 14. There was up-regulation of SOX 9, Col II and Col X with friedelin and resinone at day 14 with the significance of p < 0.01. Pellet from friedelin, resinone and tingenone showed more staining of the matrix for Safranin-O and Toluidine blue at day 14. Immunohistostaining of collagen type X (COL-10) showed more stain intensity at friedelin and resinone on day 21. These results provided new knowledge on the potential use of natural isolated secondary metabolites compounds as inducers for chondrogenic and bone differentiation.

Integration of a miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism

Defective biosynthesis or function of proteoglycans causes pathological conditions in a variety of tissue systems. Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by progressive cartilage destruction caused by imbalanced proteoglycan synthesis and degradation. Identifying agents that regulate proteoglycan metabolism may benefit the development of OA-modifying therapeutics. High-throughput screening (HTS) of chemical libraries has paved the way for achieving this goal. However, the implementation and adaptation of HTS assays based on proteoglycan measurement remain underexploited. Using primary porcine chondrocytes as a model, we report a miniaturized dimethyl-methylene blue (DMMB) assay, which is commonly used to quantitatively evaluate sulfated glycosaminoglycan (GAG) content, with an optimized detection range and reproducibility and its integration with HTS. Treatment with TGF-β1 and IL1-α, known as positive and negative proteoglycan regulators, respectively, supported the assay specificity. A pre-test of chemical screening of 960 compounds identified both stimulators (4.48%) and inhibitors (6.04%) of GAG production. Fluorophore-assisted carbohydrate electrophoresis validated the activity of selected hits on chondroitin sulfate expression in an alginate culture system. Our findings support the implementation of this simple colorimetric assay in HTS to discover modifiers of OA or other diseases related to dysregulated proteoglycan metabolism.

Phenothiazinium dyes for photodynamic treatment present lower environmental risk compared to a formulation of trifloxystrobin and tebuconazole.

The widespread use of conventional chemical antifungal agents has led to worldwide concern regarding the selection of resistant isolates. In this scenario, antimicrobial photodynamic treatment (APDT) has emerged as a promising alternative to overcome this issue. The technique is based on the use of a photosensitizer (PS) and light in the presence of molecular oxygen. Under these conditions, the PS generates reactive oxygen species which damage the biomolecules of the target organism leading to cell death. The great potential of APDT against plant-pathogenic fungi has already been reported both in vitro and in planta, indicating this control measure has the potential to be widely used in crop plants. However, there is a lack of studies on environmental risk with ecotoxicological assessment of PSs used in APDT. Therefore, this study aimed to evaluate the environmental toxicity of four phenothiazinium PSs: i) methylene blue (MB), ii) new methylene blue N (NMBN), iii) toluidine blue O (TBO), and iv) dimethylmethylene blue (DMMB) and also of the commercial antifungal NATIVO®, a mixture of trifloxystrobin and tebuconazole. The experiments were performed with Daphnia similis neonates and zebrafish embryos. Our results showed that the PSs tested had different levels of toxicity, with MB being the less toxic and DMMB being the most. Nonetheless, the environmental toxicity of these PSs were lower when compared to that of NATIVO®. Furthermore, estimates of bioconcentration and of biotransformation half-life indicated that the PSs are environmentally safer than NATIVO®. Taken together, our results show that the toxicity associated with phenothiazinium PSs would not constitute an impediment to their use in APDT. Therefore, APDT is a promising approach to control plant-pathogenic fungi with reduced risk for selecting resistant isolates and lower environmental impacts when compared to commonly used antifungal agents.

A human in vitro 3D neo-cartilage model to explore the response of OA risk genes to hyper-physiological mechanical stress

ObjectiveDue to the complexity and heterogeneity of osteoarthritis (OA) pathophysiology, studying the interaction between intrinsic molecular changes in chondrocytes after hyper-physiological mechanical stress (MS) and aberrant signalling of OA risk genes remains a challenge. In this study we set out to set up an in vitro 3D neo cartilage pellet model that enables us to explore the responses of OA risk genes to hyper-physiological MS. DesignHuman primary chondrocyte neo-cartilage pellets were exposed for 2 days to 2 ​× ​10 ​min of hyper-physiological dynamic MS attained by a 20% strain and a frequency of 5 ​Hz. In order to assess cartilage damage, sulphated glycosaminoglycan (sGAG) content in the neo-cartilage was quantified using Alcian blue staining and a dimethyl methylene blue (DMMB) assay, while cleavage of aggrecan was visualized by immunohistochemical staining of aggrecan neo-epitope NITEGE. In addition, changes in expression levels of catabolic, anabolic and hypertrophic genes, and of three OA risk genes; IL11, MGP and TGFA were determined. ResultsHyper-physiological MS induced cartilage damage, as reflected by decreased sGAG content. mRNA levels of aggrecanase ADAMTS5 were increased, while hypertrophic gene RUNX2 was downregulated. MS increased expression of pro-apoptotic marker NOXA. Furthermore, 20% MS led to increased expression of all three OA risk genes IL11, MGP and TGFA. ConclusionsWe established a human in vitro model in which hyper-physiological MS induced cartilage damage and catabolic signalling. Next, we demonstrated its usage to study OA risk genes and their response to the mechanical aspects of OA pathophysiology.

Activation of transient receptor potential vanilloid 4 protects articular cartilage against inflammatory responses via CaMKK/AMPK/NF-\u03baB signaling pathway

Transient receptor potential vanilloid 4 (TRPV4) plays an important role in chondrocytes via Ca2+ signaling. However, its role in the progression of osteoarthritis is unclear. This study aimed to evaluate the effects of TRPV4 activation on articular cartilage and chondrocytes stimulated with interleukin (IL)-1β. Bovine and human articular chondrocytes were stimulated with various agents, including IL-1β, GSK1016790A (GSK101; a TRPV4 agonist), Compound C (an AMP-activated protein kinase (AMPK) inhibitor), and STO-609 (a calmodulin-dependent protein kinase kinase (CaMKK) inhibitor), and were processed for Western blot analysis and real-time PCR. The dimethylmethylene blue (DMMB) assay and Safranin O staining were also performed. GSK101 reversed the IL-1β-induced increase in expression of matrix metalloproteinase (MMP)-13 and decrease in expression of aggrecan. GSK101 also decreased proteoglycan release in the DMMB assay and retained Safranin O staining of articular cartilage tissue. Furthermore, GSK101 increased AMPK phosphorylation and decreased IL-1β-induced nuclear factor kappa B (NF-κB) phosphorylation. Compound C and STO-609 reversed the suppressive effects of GSK101 on NF-κB activation and MMP-13 expression. In conclusion, TRPV4 activation had chondroprotective effects on articular cartilage stimulated with IL-1β by activating CaMKK/AMPK and suppressing the NF-κB pathway. TRPV4 activators may offer a promising therapeutic option for preventing the progression of osteoarthritis.

4 The effects of clodronate disodium on normal and inflamed equine cartilage in vitro

Bisphosphonates (BP) are a group of drugs which modulate osteoclastic resorption and alter bone turnover. BP are only approved for use in navicular syndrome in adult horses, however, these drugs are being used off-label for their potential anti-inflammatory and pain-relieving effects. When the BP, clodronate disodium (CLO), is administered intramuscularly at the labeled dose, synovial fluid concentrations are detectable within 2 h of administration. There are currently no published studies evaluating the effects of CLO on equine joint tissues. This study evaluated the effects of CLO on normal and inflamed equine cartilage explants in vitro. Cartilage explants were collected from the femorotibial joints of 3 horses which underwent euthanasia for reasons other than stifle osteoarthritis. Only visually normal cartilage was selected for biopsy. The harvested cartilage from each femorotibial joint was subdivided into 24-well plates. Cartilage explants were subjected to 6 different treatments with 8 replicates each, after a 24 h equilibration period: Control (Con), CLO 5 ng/ml (C/lo), CLO 50 ng/ml (C/hi), rEqIL-1β 10 ng/ml (IL), C/lo plus IL (ILC/lo) and C/hi concentration plus IL (ILC/hi). Explant culture media was collected every 24 h and stored at −20°C until analysis of sulfated glycosaminoglycan (GAG) content and nitric oxide (NO) content using a Dimethylmethylene Blue (DMMB) assay and a Greiss reaction, respectively. At the conclusion of the experiment (96 h), cartilage explants were papain-digested and GAG were quantified using a DMMB assay. Data were analyzed using PROC MIXED procedure (SAS 9.4), where horses were considered a random effect, and treatment and day were fixed effects. For all analyses, values of P < 0.05 were considered significant. At d 1, d 2, and d 3, the IL treatment group had a significantly higher concentration of GAG within the media compared with the Con group ( P < 0.05), indicating the expected catabolic effect of IL-1β on the explants. Treatments with clodronate (C/lo, C/hi) alone did not result in an increase in GAG or NO within the media compared with the control indicating CLO did not have a cytotoxic effect. However, the addition of CLO to the IL-1β treated explants (ILC/lo, ILC/hi) did not result in a significant decrease in GAG or NO release into the media when compared with rEqIL-1β (IL) alone. No differences were observed between treatments when GAG content of cartilage explants was measured following papain-digestion. Our in vitro study suggests that CLO does not result in cartilage toxicity. However, no anti-inflammatory or chondroprotective effects were observed.

Nano-Scale Stiffness and Collagen Fibril Deterioration: Probing the Cornea Following Enzymatic Degradation Using Peakforce-QNM AFM.

Under physiological conditions, the cornea is exposed to various enzymes, some of them have digestive actions, such as amylase and collagenase that may change the ultrastructure (collagen morphology) and sequentially change the mechanical response of the cornea and distort vision, such as in keratoconus. This study investigates the ultrastructure and nanomechanical properties of porcine cornea following incubation with α-amylase and collagenase. Atomic force microscopy (AFM) was used to capture nanoscale topographical details of stromal collagen fibrils (diameter and D-periodicity) and calculate their elastic modulus. Samples were incubated with varying concentrations of α-amylase and collagenase (crude and purified). Dimethylmethylene blue (DMMB) assay was utilised to detect depleted glycosaminoglycans (GAGs) following incubation with amylase. Collagen fibril diameters were decreased following incubation with amylase, but not D-periodicity. Elastic modulus was gradually decreased with enzyme concentration in amylase-treated samples. Elastic modulus, diameter, and D-periodicity were greatly reduced in collagenase-treated samples. The effect of crude collagenase on corneal samples was more pronounced than purified collagenase. Amylase was found to deplete GAGs from the samples. This enzymatic treatment may help in answering some questions related to keratoconus, and possibly be used to build an empirical animal model of keratoconic corneas with different progression levels.

Accuracy of the dimethylmethylene blue spectrophotometric assay in measuring the amount of encapsulated pentosan polysulfate into nanoparticles

Dimethylmethylene blue (DMMB) spectrophotometric assay was used to estimate the amount of pentosan polysulfate (PPS), a heparin-like glycosaminoglycan (GAG), encapsulated in chitosan-based nanoparticles (NPs) as an alternative method for capillary zone electrophoresis (CZE) that has been shown to lack sensitivity. The analytical performance of the DMMB method was evaluated, which revealed the response of PPS is linear in the investigated concentration range (R2 = 0.9996). The detection and quantification limits were < 0.22 µg/ml and < 0.75 µg/ml, respectively. The average recovery of the PPS was roughly 97.5%, with %DFA and %RSD < 5%. Based on indirect and direct estimation of the entrapment efficiency of the NPs, PPS encapsulated in NPs was 82.9 ± 0.084% and 84.1 ± 0.10%, respectively, with a %RSD of less than 1%. Comparison between the DMMB and CZE performances revealed that all the uncertainties related to results produced by the DMMB assay were at least 30-fold smaller than those produced by the CZE, revealing the higher precision and accuracy of the DMMB assay. The sensitivity of the DMMB was several times higher than the CZE. The DMMB also was found to have high stability and binding specificity for PPS. Overall, the DMMB was demonstrated to be more convenient than CZE in analytical terms and also in terms of practicality as a routine assay. Thus, the DMMB assay offers an alternative tool for the quantification of PPS, perhaps even in biological samples.

Study of Urinary Glycosaminoglycans among Essential Hypertensive Patients

Introduction: Essential hypertension is a systemic disease which affects endothelial basement membrane. Changes in Glycosaminoglycans (GAG) distribution pattern on glomerular basement membrane has been noted in hypertension. Hence it seems likely that an increase excretion of GAG levels may be an indicator of reduction in renal function. The qualitative or quantitative determination of urinary GAGs may be of value and could constitute a non invasive marker to assess the renal damage in essential hypertension. Aim: Differential analysis of urinary GAGs in essential hypertensive patients. Materials and Methods: This analytical case-control observational study which was conducted from November 2014 to June 2016 in Department of Biochemistry, SDM College of Medical Sciences and Hospital, Dharwad, Karnataka, India. The study group included 50 male patients with age group of 30-60 years, clinically diagnosed with essential hypertension. Control group included 50 healthy male individuals with age group of 30-60 years, blood donors visiting to hospital blood bank. Random urine sample was collected. Urine creatinine was estimated by Jaffe’s method, urine GAGs by Dimethyl methylene blue (DMMB) dye method and urine microalbumin by particle enhanced Turbidimetric inhibition immunoassay method. Correlation was tested with Spearman’s correlation coefficient. Level of significance was set for p-value &lt;0.05 with confidence interval of 95%. Results: In present study, urinary GAGs levels in essential hypertensive study group was 14.57±10.16 mg/dL and in control group was 10.09±6.04 mg/dL. Urinary GAGs levels in essential hypertensive group was significantly high (p-value=0.890) when compared with normotensive control group. But no statistical significant correlation was found between urine GAGs and urine microalbumin in hypertensive study group. Conclusion: Estimation of urine glycosaminoglycans in essential hypertensive patients is a simple, rapid and cost effective test which asseses the glomerular function. It can be used as one of the early marker for diagnosis of nephropathy before microalbuminuria sets in.

Performance of Cosmetic Ingredients Evaluated by Their Membrane Protection Efficiency

Plant extracts (PEs) are frequently used as ingredients in skin and hair care products to improve protection against the excess of sun exposure. PEs are usually selected based on their antioxidant capacity for reducing the damage in lipids and proteins, which are key molecules in the maintenance of skin homeostasis and hair integrity. Interestingly, biomolecules that do not have any antioxidant capacity can have a protective effect by physically adsorbing and inhibiting leakage of membranes. Our aims are: 1) to develop an experimental protocol that quantifies the level of membrane damage/protection; 2) to recommend an approach to compare the membrane protection efficiency of different PEs; 3) to briefly describe the mechanisms of membrane protection. Evaluation of membrane damage/protection is based on a phospholipid liposome system that signals the membrane rupture caused by photo-induced lipid oxidation. Carboxyfluorescein (CF) emission is monitored as a function of time (120 min) in a controlled experimental setup, in which a certain level of membrane damage is induced by Dimethyl-Methylene Blue (DMMB) photosensitized oxidation during red-light irradiation. Percentage levels of membrane damage/protection are calculated by comparing the CF fluorescence level of DMMB-liposomes in the presence of varying concentrations of membrane-protective ingredients with that of surfactant-dissolved DMMB-liposomes. Membrane protectants reduce the level of CF fluorescence during irradiation of DMMB-liposomes. Gallic acid (GA) and trehalose (Tre) were used as standard compounds to compute the levels of protection by the antioxidant and physical adsorption mechanisms, respectively. The percentage of membrane protection was shown to be linearly proportional to the concentration of the standard protectants. The performance of PEs was measured and compared in terms of GA or Tre membrane protection equivalents. All extracted also had their antioxidant capacities evaluate by the anti-radical DPPH assay. The membrane protection efficiency parallels the anti-radical efficiency for extracts that have antioxidant activity. However, even extracts that are mainly enriched in polysaccharides and that do not have any measurable anti-radical capacity were able to protect membranes. We propose that these extracts protect membranes by the physical adsorption mechanism, in comparison with the protection observed by Tre and also based on the known effect of carbohydrates that can protect against membrane micro-fissures. The membrane damage assay described here can be used to evaluate and compare the performance of cosmetic ingredients with claims of maintaining lipid membrane integrity of skin and hair products.

CircTMBIM6 promotes osteoarthritis-induced chondrocyte extracellular matrix degradation via miR-27a/MMP13 axis.

Osteoarthritis is a degenerative disease characterized by degeneration of articular cartilage, but the current mechanism is unclear. Circular RNA (circRNA) plays a significant role in a series of biological processes related to osteoarthritis, but its mechanism remains unclear. The purpose of this study was to investigate the role of the circTMBIM6/miR-27a/MMP13 axis in osteoarthritis. The expression levels of circTMBIM6, miR-27a and MMP13 in cartilage of osteoarthritis patients and normal human cartilage were detected by reverse transcription polymerase chain reaction (RT-PCR). Osteoarthritis cell model was induced by IL-1β and TNF-α, and the expression changes of circTMBIM6, miR-27a and MMP13 in the in vitro model were detected. In addition, the in vitro regulation of circTMBIM6 and miR-27a in osteoarthritis was verified by transfection of circTMBIM6 and miR-27a plasmids, and the regulation of miR-27a on MMP13 was also verified. The dimethylmethylene blue (DMMB) method was used to analyze the secretion and formation of soluble glycosaminoglycan sulfate (sGAG), and the effects of circTMBIM6 and miR-27a chondrocytes were evaluated. The expression levels of circTMBIM6 and MMP13 in cartilage tissue of patients with osteoarthritis were higher than that of normal group, while the expression level of miR-195 in cartilage tissue of patients with osteoarthritis was lower. After IL-1β and TNF-α treatment, the expression of circTMBIM6 and MMP13 in chondrocytes increased, while the expression of miR-27a decreased. CircTMBIM6 overexpression reduced miR-27a expression but increased MMP13 expression. The circTMBIM6 gene knockout showed the opposite effect. CircTMBIM6 promotes osteoarthritis-induced chondrocyte extracellular matrix degradation via miR-27a/MMP13 axis.