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1
Silva, Abdênego R., Fernanda V. Cabral, Camila R. Silva, Daniela F. T. Silva, Anderson Z. Freitas, Adriana Fontes, and Martha S. Ribeiro. "New Insights in Phenothiazinium-Mediated Photodynamic Inactivation of Candida Auris." Journal of Fungi 9, no. 7 (June 30, 2023): 717. http://dx.doi.org/10.3390/jof9070717.
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In recent years, Candida auris has emerged as a hazardous hospital-acquired pathogen. Its resistance to antifungal treatments makes it challenging, requiring new approaches to manage it effectively. Herein, we aimed to assess the impact of photodynamic inactivation mediated by methylene blue (MB-PDI) or 1,9-dimethyl MB (DMMB-PDI) combined with a red LED against C. auris. To evaluate the photoinactivation of yeasts, we quantified colony-forming units and monitored ROS production. To gain some insights into the differences between MB and DMMB, we assessed lipid peroxidation (LPO) and mitochondrial membrane potential (ΔΨm). After, we verified the effectiveness of DMMB against biofilms by measuring metabolic activity and biomass, and the structures were analyzed through scanning electron microscopy and optical coherence tomography. We also evaluated the cytotoxicity in mammalian cells. DMMB-PDI successfully eradicated C. auris yeasts at 3 μM regardless of the light dose. In contrast, MB (100 μM) killed cells only when exposed to the highest dose of light. DMMB-PDI promoted higher ROS, LPO and ΔΨm levels than those of MB. Furthermore, DMMB-PDI was able to inhibit biofilm formation and destroy mature biofilms, with no observed toxicity in fibroblasts. We conclude that DMMB-PDI holds great potential to combat the global threat posed by C. auris.
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Shtivelman, E., and J. M. Bishop. "The PVT gene frequently amplifies with MYC in tumor cells." Molecular and Cellular Biology 9, no. 3 (March 1989): 1148–54. http://dx.doi.org/10.1128/mcb.9.3.1148-1154.1989.
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The line of human colon carcinoma cells known as COLO320-DM contains an amplified and abnormal allele of the proto-oncogene MYC (DMMYC). Exon 1 and most of intron 1 of MYC have been displaced from DMMYC by a rearrangement of DNA. The RNA transcribed from DMMYC is a chimera that begins with an ectopic sequence of 176 nucleotides and then continues with exons 2 and 3 of MYC. The template for the ectopic sequence represents exon 1 of a gene known as PVT, which lies 50 kilobase pairs downstream of MYC. We encountered three abnormal configurations of MYC and PVT in the cell lines analyzed here: (i) amplification of the genes, accompanied by insertion of exon 1 and an undetermined additional portion of PVT within intron 1 of MYC to create DMMYC; (ii) selective deletion of exon 1 of PVT from amplified DNA that contains downstream portions of PVT and an intact allele of MYC; and (iii) coamplification of MYC and exon 1 of PVT, but not of downstream portions of PVT. We conclude that part or all of PVT is frequently amplified with MYC and that intron 1 of PVT represents a preferred boundary for amplification affecting MYC.
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Shtivelman, E., and J. M. Bishop. "The PVT gene frequently amplifies with MYC in tumor cells." Molecular and Cellular Biology 9, no. 3 (March 1989): 1148–54. http://dx.doi.org/10.1128/mcb.9.3.1148.
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The line of human colon carcinoma cells known as COLO320-DM contains an amplified and abnormal allele of the proto-oncogene MYC (DMMYC). Exon 1 and most of intron 1 of MYC have been displaced from DMMYC by a rearrangement of DNA. The RNA transcribed from DMMYC is a chimera that begins with an ectopic sequence of 176 nucleotides and then continues with exons 2 and 3 of MYC. The template for the ectopic sequence represents exon 1 of a gene known as PVT, which lies 50 kilobase pairs downstream of MYC. We encountered three abnormal configurations of MYC and PVT in the cell lines analyzed here: (i) amplification of the genes, accompanied by insertion of exon 1 and an undetermined additional portion of PVT within intron 1 of MYC to create DMMYC; (ii) selective deletion of exon 1 of PVT from amplified DNA that contains downstream portions of PVT and an intact allele of MYC; and (iii) coamplification of MYC and exon 1 of PVT, but not of downstream portions of PVT. We conclude that part or all of PVT is frequently amplified with MYC and that intron 1 of PVT represents a preferred boundary for amplification affecting MYC.
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Lee, Ah-Chiou, Fang-Shin Liao, and Hsiao-Feng Lo. "Temperature, Daylength, and Cultivar Interact to Affect the Growth and Yield of Lettuce Grown in High Tunnels in Subtropical Regions." HortScience 50, no. 10 (October 2015): 1412–18. http://dx.doi.org/10.21273/hortsci.50.10.1412.
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The production of lettuce, a cool-season leafy vegetable, in high tunnels the year around is a challenge for growers in subtropical regions. The aims of this research were to characterize the growth of locally grown lettuce cultivars, develop a new high-yielding cultivar by crossing romaine-type lettuce ‘Jhih Li Wo’ and Batavia type lettuce ‘Fu San’, and determine the relationships between climatic variables, temperature, and daylength, and days to harvest for maximum marketable yield (DMMY) in individual cultivars in high tunnels. Nine cultivars were grown in high tunnels in the spring and winter of 2008 and summer of 2009 to evaluate growth and maximum marketable yield (MMY), the latter being defined as the aboveground fresh weight of 5 ± 0.7 cm of plant stem. Romaine lettuce ‘Jhih Li Wo’ had a higher growth rate during the initiation of plant growth in the spring of 2008. ‘Jhih Li Wo’ and Batavia lettuce ‘Fu San’ also showed higher growth rate before harvest for the MMY (GRBHD) and exhibited higher MMY and DMMY than butterhead lettuce and leaf lettuce cultivars under summer and winter regimes. However, landraces of leaf lettuce are the main lettuces grown in high tunnels in summer rather than ‘Fu San’ and ‘Jhih Li Wo’ due to their needing fewer DMMY and having a more upright growth form. Among nine cultivars studied, Batavia lettuce ‘Fu San’, romaine lettuce ‘Jhih Li Wo’, and landrace ‘Bai Yeh Wo’ were found to be more adaptable to summer weather. Genotypes with superior growth and yield traits are essential for not only production but also breeding. A new cultivar, Taoyuan No.3, was developed by introducing the high growth rate trait during the initial period of plant growth from romaine lettuce ‘Jhih Li Wo’ into high-yielding Batavia lettuce ‘Fu San’. Another experiment was performed over eight successive seasons to analyze the correlation of temperature and daylength on DMMY for each cultivar using multiple regression analysis from 2008 to 2009. This showed that the proposed models expressed as coefficients of multiple determinants (R2) accounted for 72% to 91% of the total variation in DMMY in each cultivar. Temperature affected DMMY the most and the relative contributions of temperature and daylength to DMMY differed with cultivar. These results provide information about production practices for growers in subtropical regions to use in choosing suitable lettuce cultivars.
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Hosoi, Junichi, Masahiro Tanida, and Toru Tsuchiya. "Regulation of Plasma Substance P and Skin Mast Cells by Odorants." Journal of Cutaneous Medicine and Surgery 7, no. 4 (July 2003): 287–91. http://dx.doi.org/10.1007/s10227-002-0129-y.
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Background: Mast cells stimulate inflammation and itch sensation in the skin by releasing various mediators when they are activated. Stress exacerbates some skin diseases. We have reported that inhalation of certain odorants modulates immune reactions in the skin. Objective: The possible usage of odorants in the regulation of skin inflammation and itch sensation was to be examined. Methods: Female volunteers were subjected to interview stress with or without odorant inhalation. Mice were immobilized while inhaling odorants. Toluidene blue-stained sections were analyzed for activated mast cells. Plasma substance P level was determined by enzyme-linked immunoassay. Results: Interview stress induced plasma substance P only in volunteers who did not inhale odorants containing 2% 1,3-dimethoxy-5-methyl benzene (DMMB). Immobilization stress induced mast cell activation in mice and the activation was blocked by exposure to DMMB. Conclusions: Stress causes mast cell activation via an increase in substance P. The effect of stress is suppressed by inhalation of DMMB.
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Yeo, Tsin W., J. Brice Weinberg, Daniel A. Lampah, Enny Kenangalem, Peggy Bush, Youwei Chen, Richard N. Price, et al. "Glycocalyx Breakdown Is Associated With Severe Disease and Fatal Outcome in Plasmodium falciparum Malaria." Clinical Infectious Diseases 69, no. 10 (February 12, 2019): 1712–20. http://dx.doi.org/10.1093/cid/ciz038.
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AbstractBackgroundInteractions between the endothelium and infected erythrocytes play a major role in the pathogenesis of falciparum malaria, with microvascular dysfunction and parasite sequestration associated with worsening outcomes. The glycocalyx is a carbohydrate-rich layer that lines the endothelium, with multiple roles in vascular homeostasis. The role of the glycocalyx in falciparum malaria and the association with disease severity has not been investigated.MethodsWe prospectively enrolled Indonesian inpatients (aged ≥18 years) with severe (SM) or moderately severe (MSM) falciparum malaria, as defined by World Health Organization criteria, and healthy controls (HCs). On enrollment, blood and urine samples were collected concurrently with measurements of vascular nitric oxide (NO) bioavailability. Urine was assayed for glycocalyx breakdown products (glycosaminoglycans) using a dimethylmethylene blue (GAG-DMMB) and liquid chromatography-tandem mass spectrometry (GAG-MS) assay.ResultsA total of 129 patients (SM = 43, MSM = 57, HC=29) were recruited. GAG-DMMB and GAG-MS (g/mol creatinine) were increased in SM (mean, 95% confidence interval: 3.98, 2.44–5.53 and 6.82, 5.19–8.44) compared to MSM patients (1.78, 1.27–2.29 and 4.87, 4.27–5.46) and HCs (0.22, 0.06–0.37 and 1.24, 0.89–1.59; P < 0.001). In SM patients, GAG-DMMB and GAG-MS were increased in those with a fatal outcome (n = 3; median, interquartile range: 6.72, 3.80–27.87 and 12.15, 7.88–17.20) compared to survivors (n = 39; 3.10, 0.46–4.5 and 4.64, 2.02–15.20; P = 0.03). Glycocalyx degradation was significantly associated with parasite biomass in both MSM (r = 0.48, GAG-DMMB and r = 0.43, GAG-MS; P < 0.001) and SM patients (r = 0.47, P = 0.002 and r = 0.33, P = 0.04) and inversely associated with endothelial NO bioavailability.ConclusionsIncreased endothelial glycocalyx breakdown is associated with severe disease and a fatal outcome in adults with falciparum malaria.
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Zeng, Wen-Wen, Tsan-Chi Chen, Cheng-Huan Liu, Sheng-Yang Wang, Jei-Fu Shaw, and Yu-Ting Chen. "Identification and Isolation of an Intermediate Metabolite with Dual Antioxidant and Anti-Proliferative Activity Present in the Fungus Antrodia cinnamomea Cultured on an Alternative Medium with Cinnamomum kanehirai Leaf Extract." Plants 10, no. 4 (April 9, 2021): 737. http://dx.doi.org/10.3390/plants10040737.
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The fungus Antrodia cinnamomea has been used as a folk medicine for various diseases, especially cancer. When A. cinnamomea is cultured on the original host, an endangered woody plant Cinnamomum kanehirai Hayata, the fungus produces more active ingredients, but its growth is slow. Here, C. kanehirai leaf ethanol extract (KLEE) was used as a substitute for C. kanehirai wood to culture A. cinnamomea on solid medium to shorten the culture period and produce active metabolites en masse. The antioxidant activities of methanol extracts from A. cinnamomea cultured on KLEE (MEAC-KLEE) were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, reducing power, and ferrous ion-chelating effect, and the effective concentration (EC50) values were 0.27, 0.74, and 0.37 mg mL−1, respectively. MEAC-KLEE exhibited specific anti-proliferative activity against a non-small-cell lung cancer cell line (A549) by Annexin V assay. A secondary metabolite (2,4-dimethoxy-6-methylbenzene-1,3-diol, DMMB) present in the extract (MEAC-KLEE) was purified by high-performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR) spectra. DMMB exhibited moderate antioxidant activity against DPPH radicals and reducing power, with EC50 values of 12.97 and 25.59 μg mL−1, respectively, and also induced apoptosis in A549 cells. Our results provide valuable insight into the development of DMMB for nutraceutical biotechnology.
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Jiang, Shijie, Pifeng Chen, Yang Zhan, and Chunyu Zhao. "Theoretical and Computational Analysis on the Melt Flow Behavior of Polylactic Acid in Material Extrusion Additive Manufacturing under Vibration Field." Applied Sciences 10, no. 11 (May 29, 2020): 3801. http://dx.doi.org/10.3390/app10113801.
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Material extrusion (ME), an extrusion-based rapid prototyping technique, has been extensively studied to manufacture final functional products, whose forming quality is significantly influenced by the melt flow behavior (MFB) inside the extrusion liquefier. Applied vibration has a great potential to improve the MFB, and thereby promote the forming quality of the built product. To reveal the mechanism, a dynamic model of the melt flow behavior (DMMFB) is established based on fluid dynamics, Tanner nonlinear constitutive equation and Newton’s power law equation. The MFB, i.e., pressure drop, shear stress and apparent viscosity, is investigated without and with different vibration applied. The corresponding finite element analysis (FEA) is then carried out. From the comparison between DMMFB and FEA results, it is concluded that the proposed model is reliable. When vibration is applied onto the extrusion liquefier, the time-domain MFB will change periodically. Its effective value decreases significantly, and further decreases with the increase of vibration frequency or amplitude. This paper provides the theoretical basis to improve the MFB by applied vibration, and thereby to enhance the forming quality of ME products.
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Lee, W. M., M. Schwab, D. Westaway, and H. E. Varmus. "Augmented expression of normal c-myc is sufficient for cotransformation of rat embryo cells with a mutant ras gene." Molecular and Cellular Biology 5, no. 12 (December 1985): 3345–56. http://dx.doi.org/10.1128/mcb.5.12.3345-3356.1985.
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We studied the effect of altered c-myc structure and expression upon the ability of c-myc to promote the transformation of normal rat embryo cells when it was supplemented by EJras (the mutant c-H-ras1 gene from EJ/T24 bladder carcinoma cells). We tested several c-myc alleles cloned from normal and tumor tissues of chicken and human origin and found that only LL4myc (derived from a bursal lymphoma in which an avian leukosis virus long terminal repeat resides within the first c-myc intron in the same transcriptional orientation) had cotransforming activity. No activity was observed with normal chicken and human c-myc alleles, two other bursal lymphoma c-myc alleles (LL3myc and LL6myc), and two human c-myc genes (HSRmyc and DMmyc) from human neuroectodermal tumor cell line COLO320, in which c-myc is amplified. Some of these inactive alleles had the following alterations that are frequently found in tumor-derived c-myc: point mutations affecting the encoded protein (LL3myc); a truncated structure with loss of the first, noncoding exon (LL3myc and DMmyc); and proviral integration within or near the myc locus (LL3myc and LL6myc). The following two experimental approaches indicated that cotransforming activity was directly related to the transcriptional activity of the alleles in cultured rat cells: when cotransfected into Rat-2 cells, LL4myc was more highly expressed than the other (inactive) alleles; and augmented expression of HSRmyc, DMmyc, or normal human or normal chicken c-myc placed under the transcriptional control of retroviral long terminal repeats or increased expression of normal human c-myc under the influence of a retroviral enhancer element was accompanied by cotransformation activity. We concluded that augmented expression of even a normal c-myc gene is sufficient for cotransforming activity and that additional structural alterations frequently found in tumor-derived alleles are neither necessary nor sufficient for the gene to acquire rat embryo cell cotransforming properties.
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Lee, W. M., M. Schwab, D. Westaway, and H. E. Varmus. "Augmented expression of normal c-myc is sufficient for cotransformation of rat embryo cells with a mutant ras gene." Molecular and Cellular Biology 5, no. 12 (December 1985): 3345–56. http://dx.doi.org/10.1128/mcb.5.12.3345.
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We studied the effect of altered c-myc structure and expression upon the ability of c-myc to promote the transformation of normal rat embryo cells when it was supplemented by EJras (the mutant c-H-ras1 gene from EJ/T24 bladder carcinoma cells). We tested several c-myc alleles cloned from normal and tumor tissues of chicken and human origin and found that only LL4myc (derived from a bursal lymphoma in which an avian leukosis virus long terminal repeat resides within the first c-myc intron in the same transcriptional orientation) had cotransforming activity. No activity was observed with normal chicken and human c-myc alleles, two other bursal lymphoma c-myc alleles (LL3myc and LL6myc), and two human c-myc genes (HSRmyc and DMmyc) from human neuroectodermal tumor cell line COLO320, in which c-myc is amplified. Some of these inactive alleles had the following alterations that are frequently found in tumor-derived c-myc: point mutations affecting the encoded protein (LL3myc); a truncated structure with loss of the first, noncoding exon (LL3myc and DMmyc); and proviral integration within or near the myc locus (LL3myc and LL6myc). The following two experimental approaches indicated that cotransforming activity was directly related to the transcriptional activity of the alleles in cultured rat cells: when cotransfected into Rat-2 cells, LL4myc was more highly expressed than the other (inactive) alleles; and augmented expression of HSRmyc, DMmyc, or normal human or normal chicken c-myc placed under the transcriptional control of retroviral long terminal repeats or increased expression of normal human c-myc under the influence of a retroviral enhancer element was accompanied by cotransformation activity. We concluded that augmented expression of even a normal c-myc gene is sufficient for cotransforming activity and that additional structural alterations frequently found in tumor-derived alleles are neither necessary nor sufficient for the gene to acquire rat embryo cell cotransforming properties.
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Rovas, Alexandros, Julia Katharina Neumann, Carolin Christina Drost, Richard Vollenberg, Gerold Thölking, Manfred Fobker, Martin Witzenrath, and Philipp Kümpers. "Analysis of Urinary Glycosaminoglycans to Predict Outcome in COVID-19 and Community-Acquired Pneumonia—A Proof-of-Concept Study." Journal of Clinical Medicine 12, no. 16 (August 13, 2023): 5269. http://dx.doi.org/10.3390/jcm12165269.
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Although coronavirus disease 2019 (COVID-19) is considered a systemic disease associated with vascular inflammation and eventual destruction of the protective endothelial glycocalyx (eGC), biomarkers of eGC damage are not yet available in the clinic. The most prominent components of eGC are sulphated glycosaminoglycans (sGAGs) attached to core proteoglycans. We hypothesised that the amount of sGAG fragments shed in urine (as a surrogate for systemic eGC damage) would correlate with disease severity and outcome. Total urinary sGAG concentration was measured using an in-house optimised 1,9-dimethylmethylene blue (DMMB) assay, which is highly accurate and insensitive to interferences. The median urinary sGAG concentration was significantly higher in 67 hospitalised patients with COVID-19 compared to 72 hospitalised patients with community-acquired pneumonia (CAP). In both groups, urinary sGAG concentrations predicted a combined endpoint (including intubation and death) with an area under the receiver operator characteristic curve of 0.72 (95% CI 0.55–0.88, p = 0.01) and 0.70 (95% CI 0.57–0.83, p = 0.007), respectively. In conclusion, the inexpensive and easy-to-perform DMMB assay provides a surrogate parameter for eGC damage that may be useful for risk stratification of patients with COVID-19 and CAP.
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Murakami Junior, Mario Minor, Yollanda E. Moreira Franco, Maurício Da Silva Baptista, and Suely Kazue Nagahashi Marie. "Analysis of the use of photosensitization in human glioblastoma multiforme to induce cell death." Revista de Medicina 98, Suppl (October 4, 2019): 25. http://dx.doi.org/10.11606/issn.1679-9836.v98isupplp23-23.
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Introduction: The most frequent primary tumor of the central nervous system is the malignant glioma, being the glioblastoma (GBM), grade IV astrocytoma, the most aggressive and lethal glioma. Malignant astrocytomas are responsive for therapy targeting autophagy as temozolomide, the standard adjuvant treatment which induces autophagic cell death. Autophagy is a homeostatic intracellular process that eliminate old proteins and recycle cellular components. Mitophagy is a subtype of autophagy that regulates the removal of damaged, dysfunctional or redundant mitochondria. Parallel damage against lysosomes and mitochondria membranes using photosensitized oxidations and strong redox stress leads to activation of mitophagy and malfunction of autophagy. This mechanism of photosensitization, ultimately, causes cell death. Challenging cells with a low concentration of a photosensitizer as 1,9-dimethyl methylene blue (DMMB) combined with light- irradiation of 12 joules/cm2 have induced mitochondrial damage with activation of mitophagy and concomitant lysosome damage, in skin-derived cell lines. This experimental design was applied to U87MG GBM cells to verify if tumor cell death may be obtained with combined mitochondrial and lysosomal damages to open new therapeutic strategies for GBM and to better understand the mechanisms of mitophagy.Objectives: Our primary objective is to analyze the impact of challenging GBM cells with a low concentration of 1,9-dimethyl methylene blue (DMMB) with combined light- irradiation of 12 joules/cm2.Methodology: U87MG a human GBM cell line was used. The photodamage was performed using DMMB photosensitized by a LED with maximum emission wavelength at 630 nm providing 12 J/cm2. Cell proliferation and viability assays were performed using MTT to assess whether there was proliferation inhibition and/or alteration of cell viability after photosensitization. Quantification of cells in different stages of apoptosis, and in the various phases of the cell cycle were analyzed using flow cytometry after photosensitization. Acridine orange assay was used to assess lysosome damage. RT-PCR and Western Blotting were performed to evaluate the expression levels of the main autophagy and mitophagy genes and proteins.Partial Results: Cell proliferation and viability assays demonstrated that the concentration of DMMB to cause 50% inhibition of biological activity of cells (IC50) was 10 nM after 48h. The apoptosis and cell cycle experiments were performed in this concentration. Increase in apoptosis was observed after 24hs of photosensitization. Currently, the cell cycle flow cytometry assay has been performed, followed by the quantification of lysosomes damage by Acridine Orange assay. The genes and proteins involved in the mechanisms of autophagy and mitophagy will be determined by expression analysis through RT-PCR and Western blot assays.Discussion and Conclusion: For a future perspective, if this prove of concept is achieved, i.e. death of tumor cells by the combined approach of photosensitizer with irradiation, a new therapeutic strategy of light-activated drugs may be offered to cancer patients.
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Monteiro, Juliana S. C., Emília E. Rangel, Susana C. P. S. de Oliveira, Pedro J. L. Crugeira, Iago P. F. Nunes, Sandra R. C. de A. Fagnani, Fernando J. P. Sampaio, Paulo F. de Almeida, and Antônio L. B. Pinheiro. "Enhancement of photodynamic inactivation of planktonic cultures of Staphylococcus aureus by DMMB-AuNPs." Photodiagnosis and Photodynamic Therapy 31 (September 2020): 101930. http://dx.doi.org/10.1016/j.pdpdt.2020.101930.
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Jafaryazdi, Rokhsareh, Sedigheh Shams, Aria Setoodeh, Reza Shervin Badv, Vahid Ziaee, Farzaneh Abbasi, Mohammad Taghi Haghi Ashtiani, Fatemeh Mozafari, and Lila Shafeghat. "Evaluation of Patients Referred to Children's Medical Center Laboratory for Diagnosis of Mucopolysaccharidoses: Eight Years' Experience from Iran." Journal of Child Science 11, no. 01 (January 2021): e299-e305. http://dx.doi.org/10.1055/s-0041-1740059.
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AbstractMucopolysaccharidoses (MPSs) are rare lysosomal storage diseases, resulting from deficiencies of enzymes responsible for Glycosaminoglycans (GAGs) degradation. This leads to accumulation of GAGs in tissues and their excretion in urine, with a wide variety of manifestations. Early diagnosis of MPSs is strictly recommended due to available therapy that can slow down disease progression during the early ages. This study aimed to evaluate patients with suspected MPS referred to Children's Medical Center laboratory over eight years. We also evaluated the usefulness of urine GAG as a screening test for identification of such patients. A total of 1414 patients (40% female, 60% male, with mean age 3.1 ± 4.1years) have participated in this study. The urinary GAG analysis (uGAG) was performed by 1, 9-dimethyl-methylene blue (DMMB) and Berry spot test (BST). All patients with positive and mild positive results or with disease-related symptoms were evaluated in terms of definitive diagnosis, received treatments, morbidity, and mortality rate. In 407 (36.5%) patients uGAG were positive or mild positive, of which 26.3% suffered from one of the types of MPSs, 28.5% suffered from other diseases, 32.9% were undiagnosed, 12.3% were apparently healthy, and 19 died. The negative predictive value of uGAG test in our study was 100%. About 21% of MPSs patients received enzyme replacement therapy, while four patients underwent stem cell transplants. The rest received supportive care. We concluded that a combination of DMMB and BST methods has acceptable sensitivity for screening suspicious MPS patients.
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Scaria, George S., Gary Ramsay, and Alisa L. Katzen. "Two components of the Myb complex, DMyb and Mip130, are specifically associated with euchromatin and degraded during prometaphase throughout development." Mechanisms of Development 125, no. 7 (July 2008): 646–61. http://dx.doi.org/10.1016/j.mod.2008.02.005.
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Rabel, R. A. C., L. Osterbur, A. Maki, J. Lewis, and M. B. W. Wheeler. "193 HYALURONIC ACID-GLYCIDYL METHACRYLATE HYDROGELS SUPPORT IN VITRO CHONDROGENIC DIFFERENTIATION OF PORCINE ADIPOSE-DERIVED STEM CELLS." Reproduction, Fertility and Development 26, no. 1 (2014): 211. http://dx.doi.org/10.1071/rdv26n1ab193.
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There is a great need for bioengineered cartilage because of the lack of medical or surgical therapies to improve articular cartilage healing. We hypothesised that porcine adipose-derived stem cells (pASC) can be induced to undergo chondrogenic differentiation within hyaluronic acid (HA) hydrogels. The objective of this study was to develop UV-curable pASC-laden HA hydrogels aimed at application in cartilage tissue engineering. HA was treated with glycidyl methacrylate (GM) to allow chemical gelation of the polymer upon exposure to UV light. 2% HAGM hydrogel was obtained by mixing HAGM with chondrogenic medium consisting of TGFβ, ascorbic acid, ITS+ premix (insulin, transferrin, selenous acid; Cat. No. 354352, BD Biosciences, Franklin Lakes, NJ), sodium pyruvate, and dexamethasone. Passage three-pASC were resuspended in 2% HAGM hydrogel with 2 × 107 cells mL–1. Twelve-and-one-half (12.5)-μL droplets (micromasses) of this suspension containing 250 000 pASC were placed in 24-well culture plates and incubated for 2 h at 37°C and 5% CO2 to allow for cell attachment. Subsequently, the cell-laden hydrogels were cured with ~10 mW cm–2 365-nm UV light for 10 min, covered with 500 μL of chondrogenic medium, and cultured for up to 11 days at 37°C and 5% CO2. Additionally, pASC micromasses were cultured in chondrogenic medium without loading on 2% HAGM hydrogels as positive controls, and in non-chondrogenic DMEM as negative controls. Samples were collected at 4, 7, and 11 days in to culture for cryopreservation (for immunohistochemistry; IHC) and dimethylmethylene blue (DMMB) assay. IHC on day 11 of culture demonstrated the expression of cartilage specific proteins type-II collagen and aggrecan. On the basis of data from the DMMB assay, chondrogenic differentiation of pASC-laden micromasses in positive controls and 2% HAGM treatments were not different (P > 0.05). This indicates that ASC can produce cartilage equally well under both conditions, supporting the idea that HAGM may be used as a matrix for cartilage formation in vitro and possibly in vivo. In conclusion, using a micromass cell culture system, we demonstrated that 2% HAGM hydrogels support proliferation and chondrogenic differentiation of pASC. Further experiments testing different concentrations of HAGM and UV exposure levels, and larger sample numbers are warranted to further improve this procedure.
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Hallé, Jean-Pierre, Danielle Landry, Alain Fournier, Michèle Beaudry, and Francois A. Leblond. "Method for the Quantification of Alginate in Microcapsules." Cell Transplantation 2, no. 5 (September 1993): 429–36. http://dx.doi.org/10.1177/096368979300200511.
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Alginate is a key reagent in the preparation of microcapsules for cell transplantation. To address the question of the intracapsular alginate concentration, a sensitive assay has been developed to quantify the alginate content of microcapsules. The method is based on the metachromatic change induced by alginate binding to the dye, 1,9-dimethyl methylene blue (DMMB). The assay has a high sensitivity and precision. It covers a wide concentration range enabling the measurement of alginate in dilute supernatants as well as in microcapsules. For the latter, the membrane is initially dissolved by incubating the microcapsules in an alkaline medium. The effect of potentially interfering substances (poly-l-lysine (PLL), citrate, chloride, sodium) and of pH has been studied. Poly-l-lysine interfered with the assay at pH 6.5 but not at pH 13. Interference by sodium augmented with increasing sodium concentration and reached a plateau at 200 mM. This problem was overcome by routinely adjusting all samples to 500 mM sodium. The other substances tested had a negligible effect on the assay. The reliable measurement of alginate with this new assay will allow the optimization of the intracapsular alginate concentration.
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Santos, Darcy de A., Pedro Jorge L. Crugeira, Iago P. F. Nunes, Paulo Fernando de Almeida, and Antônio Luiz B. Pinheiro. "A novel technique of antimicrobial photodynamic therapy – aPDT using 1,9-dimethyl-methylene blue zinc chloride double salt-DMMB and polarized light on Staphylococcus aureus." Journal of Photochemistry and Photobiology B: Biology 200 (November 2019): 111646. http://dx.doi.org/10.1016/j.jphotobiol.2019.111646.
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Kazaili, Ahmed, Hayder Abdul-Amir Al-Hindy, Jillian Madine, and Riaz Akhtar. "Nano-Scale Stiffness and Collagen Fibril Deterioration: Probing the Cornea Following Enzymatic Degradation Using Peakforce-QNM AFM." Sensors 21, no. 5 (February 26, 2021): 1629. http://dx.doi.org/10.3390/s21051629.
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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.
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Schröder, Agnes, Ute Nazet, Dominique Muschter, Susanne Grässel, Peter Proff, and Christian Kirschneck. "Impact of Mechanical Load on the Expression Profile of Synovial Fibroblasts from Patients with and without Osteoarthritis." International Journal of Molecular Sciences 20, no. 3 (January 30, 2019): 585. http://dx.doi.org/10.3390/ijms20030585.
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Osteoarthritis (OA) affects the integrity of the entire joint including the synovium. The most abundant cells in the synovium are fibroblasts (SF). Excessive mechanical loading might contribute to OA pathogenesis. Here, we investigate the effects of mechanical loading on SF derived from non-OA (N-SF) and OA patients (OA-SF). We treated N-SF and OA-SF with or without mechanical loading for 48h after 24h of preincubation. Then we assessed gene and protein expression of proinflammatory factors (TNFα, COX-2, PG-E2, IL-6), extracellular matrix (ECM) components (COL1, FN1) and glycosaminoglycans (GAGs) via RT-qPCR, ELISA, DMMB assay and HPLC. Mechanical loading significantly increased TNFα and PG-E2 secretion by N-SF and OA-SF, whereas in OA-SF IL-6 secretion was reduced. COL1 and FN1 secretion were downregulated in N-SF during loading. OA-SF secreted less COL1 compared to N-SF under control conditions. In contrast, OA-SF in general expressed more FN1. GAG synthesis was upregulated in N-SF, but not in OA-SF during loading with OA-SF displaying a higher charge density than N-SF. Mechanical loading enhanced proinflammatory factor expression and GAG synthesis and decreased secretion of ECM components in N-SFs, indicating a contributing role of SF to OA development.
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Passos, Marcele F., Nayara M. S. Carvalho, Ana Amélia Rodrigues, Vanessa P. Bavaresco, André L. Jardini, Maria Regina W. Maciel, and Rubens Maciel Filho. "PHEMA Hydrogels Obtained by Infrared Radiation for Cartilage Tissue Engineering." International Journal of Chemical Engineering 2019 (January 31, 2019): 1–9. http://dx.doi.org/10.1155/2019/4249581.
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Although the exposure of polymeric materials to radiation is a well-established process, little is known about the relationship between structure and property and the biological behavior of biomaterials obtained by thermal phenomena at 1070 nm wavelength. This study includes results concerning the use of a novel infrared radiation source (ytterbium laser fiber) for the synthesis of poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel in order to produce medical devices. The materials were obtained by means of free radical polymerization mechanism and evaluated regarding its cross-linking degree, polymer chain mobility, thermal, and mechanical properties. Their potential use as a biomaterial toward cartilage tissue was investigated through incubation with chondrocytes cells culture by dimethylmethylene blue (DMMB) dye and DNA quantification. Differential scanning calorimetry (DSC) results showed that glass transition temperature (Tg) was in the range 103°C–119°C, the maximum degree of swelling was 70.8%, and indentation fluency test presented a strain of 56%–85%. A significant increase of glycosaminoglycans (GAGs) concentration and DNA content in cells cultured with 40 wt% 2-hydroxyethyl methacrylate was observed. Our results showed the suitability of infrared laser fiber in the free radicals formation and in the rapid polymer chain growth, and further cross-linking. The porous material obtained showed improvements concerning cartilage tissue regeneration.
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Wosek, J., I. Kuźmicz, R. Wiśniewska, J. Nazaruk, and A. Galicka. "Effect of selected flavonoids on glycosaminoglycans in human skin fibroblasts." Progress in Health Sciences 6, no. 2 (December 1, 2016): 59–63. http://dx.doi.org/10.5604/01.3001.0009.5049.
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Purpose: Glycosaminoglycans (GAGs) and proteoglycans (PG) in addition to collagen are the main components of extracellular matrix (ECM). They play an important role in intercellular communication and interactions between cells and ECM. The biological changes in ECM that occur during aging are induced by decrease in GAG biosynthesis. The purpose of this study was to evaluate the effect of selected flavonoids isolated from Cirsium palustre (L.) Scop. on GAG content in human skin fibroblasts. Materials and methods: Human skin fibroblasts were treated with eriodictyol 7-O-glucoside (C1), 6-hydroxyluteolin 7-O-glucoside (C2), scutellarein 7-O-glucoside (C3) and pedalitin (C4) at 1, 20 and 40 μM for 24 h. Concentration of GAGs in the medium was assayed using method based on their ability to bind the cationic dye 1,9- dimethylmethylene blue (DMMB). Results: C1, C2 and C4 at concentration of 20 and 40 µM significantly increased content of sulphated GAGs in the medium. In contrast, treatment of cells with compound C3 did not have a statistically significant impact on GAG level. Ascorbic acid used as a positive control at 50 µM showed no effect on GAG concentration and increased their content at 100 µM but to a much lower extent than flavonoids. Conclusion: Flavonoids C1, C2 and C4 showed greater than ascorbic acid stimulatory impact on GAGs in healthy human skin fibroblasts, demonstrating their therapeutic potential in the aging.
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Bedini, Emiliano, Alfonso Iadonisi, Chiara Schiraldi, Laura Colombo, Diego Albani, Paola Petrini, Carmen Giordano, and Marta Tunesi. "Microbiological-Chemical Sourced Chondroitin Sulfates Protect Neuroblastoma SH-SY5Y Cells against Oxidative Stress and Are Suitable for Hydrogel-Based Controlled Release." Antioxidants 10, no. 11 (November 16, 2021): 1816. http://dx.doi.org/10.3390/antiox10111816.
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Chondroitin sulfates (CS) are a class of sulfated glycosaminoglycans involved in many biological processes. Several studies reported their protective effect against neurodegenerative conditions like Alzheimer’s disease. CS are commonly derived from animal sources, but ethical concerns, the risk of contamination with animal proteins, and the difficulty in controlling the sulfation pattern have prompted research towards non-animal sources. Here we exploited two microbiological-chemical sourced CS (i.e., CS-A,C and CS-A,C,K,L) and Carbopol 974P NF/agarose semi-interpenetrating polymer networks (i.e., P.NaOH.0 and P.Ethanol.0) to set up a release system, and tested the neuroprotective role of released CS against H2O2-induced oxidative stress. After assessing that our CS (1–100 µM) require a 3 h pre-treatment for neuroprotection with SH-SY5Y cells, we evaluated whether the autoclave type (i.e., N- or B-type) affects hydrogel viscoelastic properties. We selected B-type autoclaves and repeated the study after loading CS (1 or 0.1 mg CS/0.5 mL gel). After loading 1 mg CS/0.5 mL gel, we evaluated CS release up to 7 days by 1,9-dimethylmethylene blue (DMMB) assay and verified the neuroprotective role of CS-A,C (1 µM) in the supernatants. We observed that CS-A,C exhibits a broader neuroprotective effect than CS-A,C,K,L. Moreover, sulfation pattern affects not only neuroprotection, but also drug release.
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Chou, Cheng-Hung, Tzong-Fu Kuo, Chien-Cheng Lin, Jui-Che Tsai, and Feng-Huei Lin. "GLYCOSAMINOGLYCAN SYNTHESIS OF CHONDROCYTES IN FIBRIN GLUE WITH GHC6S PARTICLES." Biomedical Engineering: Applications, Basis and Communications 20, no. 05 (October 2008): 329–35. http://dx.doi.org/10.4015/s1016237208000945.
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Articular cartilage provides functions of lubrication to shear stress and protection from compressive force, but it has poor ability to repair itself after suffering damage. The advanced method of tissue engineering is developed and used to maintain cell functions for tissue regeneration. In order to improve the ECM synthesis for the regeneration, many materials have been examined on chondrocytes or other cell sources. In this study, fibrinogen was concentrated from plasma cryoprecipitation and then polymerized by thrombin into fibrin. Gelatin/hyaluronic acid/chondroitin-6-sulfate (GHC6S) was prepared by the cross-linking reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and ground in liquid nitrogen to particles. The GHC6S particles were mixed with fibrin glue as the tissue engineering scaffold. Porcine articular cartilage chondrocytes were expanded and seeded into the scaffolds. The engineered constructs were cultured and harvested after cultured for 1 and 2 weeks. Morphology of GHC6S particle was examined by scanning electron microscopy (SEM). Total glycosaminoglycans (GAGs) and sulfated GAGs were quantified by p-dimethylaminobenzaldehyde reaction and 1,9-dimethymethylene blue (DMMB) assay, respectively. The results demonstrated that the total GAGs, especially the content of nonsulfated GAGs, hyaluronic acid, were increased with time in chondrocytes growing in fibrin glue with GHC6S particles. It suggested that the GHC6S in fibrin glue chondrocyte kept the GAGs synthesis, which could help resist the compressive force. Therefore, the GHC6S particles mixed within fibrin glue can be used as a promising scaffold for articular tissue engineering.
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Li, Yu-Fei, Shu-Hua Li, Yong Liu, and Ya-Tong Luo. "Long Noncoding RNA CIR Promotes Chondrocyte Extracellular Matrix Degradation in Osteoarthritis by Acting as a Sponge For Mir-27b." Cellular Physiology and Biochemistry 43, no. 2 (2017): 602–10. http://dx.doi.org/10.1159/000480532.
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Background/Aims: Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation. The degradation of the extracellular matrix (ECM) of chondrocyte is closely associated with the destruction of joints in OA patients. lncRNAs are non-coding segments of RNA that possess important regulatory functions at the cellular level and in a variety of pathophysiological processes. The present study was conducted to investigate whether lncRNA-CIR regulated the expression of MMP13 as a sponge of miR-27 in OA. Methods: Primary cultured chondrocytes were challenged by IL-1β and TNF-α to simulate OA conditions. qRT-PCR was performed to detect the miR-27, lncRNA-CIR, MMP13 mRNA expression levels. Western blot was applied to detect MMP13 protein expression. Soluble sGAG secretion/ formation was analysed by the dimethylmethylene blue (DMMB) assay. lncRNA-CIR overexpression or inhibition was performed using overexpression plasmid and small interfering RNAs (siRNAs), respectively. Results: lncRNA-CIR significantly up-regulated in OA patients, concomitantly down-regulated miR-27 and up-regulated MMP13. Bioinformatics analysis predicted miR-27 was the target of both lncRNA-CIR and MMP13. Overexpression of lncRNA-CIR significantly increased the expression of MMP13, while miR-27 remarkably suppressed the expression of MMP13, Accompanying with the increases of mRNA level, protein level and relative luciferase activity. Conclusion: The present findings indicated that lncRNA-CIR/miR-27/MMP13 axis involved in the degradation of the ECM of chondrocyte in OA.
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Ahuja, CS, M. Khazaei, P. Chan, J. Bhavsar, Y. Yao, Z. Lou, J. Wang, and M. Fehlings. "GP.04 Smart human neural stem cells to degrade scar and optimize regeneration after traumatic cervical spinal cord injury." Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques 45, s2 (June 2018): S9. http://dx.doi.org/10.1017/cjn.2018.81.
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Background: Human induced pluripotent stem cell-derived neural stem cells (hiPS-NSCs) represent an exciting therapeutic approach for traumatically spinal cord injury (SCI). Unfortunately, most patients are the in chronic injury phase where a dense perilesional chondroitin sulfate proteoglycan (CSPG) scar significantly hinders regeneration. CSPG-degrading enzymes can enhance NSC-mediated recovery, however, nonspecific intrathecal administration causes off-target effects. We aimed to genetically engineer hiPS-NSCs to express a scar-degrading ENZYME into their local environment to enhance functional recovery. Methods: A bicistronic scar-degrading ENZYME and RFP reporter vector was non-virally integrated into hiPS-NSCs and monoclonalized. ENZYME activity was assessed by WST-1 and DMMB biochemical assays and an in vitro CSPG spot assay with hiPS-NSC-derived neurons. To assess in vivo efficacy, T-cell deficient rats (N=60) with chronic (8wk) C6-7 SCIs were randomized to receive (1)SMaRT cells, (2)hiPS-NSCs, (3)vehicle, or (4)sham surgery. Results: SMaRT cells retained key hiPS-NSC characteristics while stably expressing ENZYME. The expressed ENZYME could appropriately degrade in vitro and ex vivo CSPGs. While blinded neurobehavioural and immunohistochemical assessments are ongoing at 40wks post-injury, an interim analysis demonstrated human cells extending remarkably long (≥20,000µm) axons along host white matter tracts. Conclusions: This work provides exciting proof-of-concept data that genetically-engineered SMaRT cells can degrade CSPGs and human NSCs can extend long-distance processes in chronic SCI.
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Toffoletto, Odaly, Agostinho Tavares, Dulce Elena Casarini, Beata Marie Redublo, and Artur Beltrame Ribeiro. "Farmacocinética da associação de glucosamina e sulfato de condroitina em humanos sadios do sexo masculino." Acta Ortopédica Brasileira 13, no. 5 (2005): 235–37. http://dx.doi.org/10.1590/s1413-78522005000500005.
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A osteoartrose é uma doença crônica das articulações que, uma vez instalada, leva seus portadores a uma incapacidade funcional progressiva. Como os proteocondroitins sulfato são os maiores constituintes das cartilagens, espera-se que com a ingestão de glucosamina e condroitina haja uma melhora das condições biológicas desse tecido. Uma vez que não temos conhecimento de estudo da farmacocinética da administração oral dessa associação em seres humanos, o objetivo deste trabalho foi avaliá-la utilizando a associação entre o sulfato de glucosamina (SG) e o sulfato de condroitina (SC) administrada a dois grupos de doze voluntários sadios do sexo masculino (grupo I uma cápsula de (500 mg SG; 400 mg SC) e grupo II quatro cápsulas). Amostras de sangue foram retiradas a intervalos de tempo pré-definidos até 48 horas pós-dose. O SG e o SC foram dosados no plasma pelo método de DMMB (azul de 1,9,dimetildimetileno). A concentração máxima foi atingida em 2 horas (média ±SE; 0,893±0,093 µg/mL, grupo I e 2,222±0,313 µg/mL, grupo II). As áreas sob a curva até 48 horas foram de 10,803±0,965 µg-hr/mL e 38,776±2,981 µg-hr/mL, respectivamente para os grupos I e II. Os dois grupos apresentaram um segundo pico após 18 horas, indicando circulação êntero-hepática. Os nossos resultados indicam que essa associação é absorvida por via oral por mecanismo saturável, o que pode facilitar o seu uso em tratamentos clínicos.
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Clutterbuck, Abigail L., David Allaway, Pat Harris, and Ali Mobasheri. "Curcumin reduces prostaglandin E2, matrix metalloproteinase-3 and proteoglycan release in the secretome of interleukin 1β-treated articular cartilage." F1000Research 2 (July 4, 2013): 147. http://dx.doi.org/10.12688/f1000research.2-147.v1.
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Objective: Curcumin (diferuloylmethane) is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA). The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β)-stimulated inflammation and catabolism in an explant model of cartilage inflammation.Methods: Articular cartilage explants and primary chondrocytes were obtained from equine metacarpophalangeal joints. Curcumin was added to monolayer cultured primary chondrocytes and cartilage explants in concentrations ranging from 3μM-100μM. Prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)-3 release into the secretome of IL-1β-stimulated explants was measured using a competitive ELISA and western blotting respectively. Proteoglycan (PG) release in the secretome was measured using the 1,9-dimethylmethylene blue (DMMB) assay. Cytotoxicity was assessed with a live/dead assay in monolayer cultures after 24 hours, 48 hours and five days, and in explants after five days.Results: Curcumin induced chondrocyte death in primary cultures (50μM p<0.001 and 100μM p<0.001) after 24 hours. After 48 hours and five days, curcumin (≥25μM) significantly increased cell death (p<0.001 both time points). In explants, curcumin toxicity was not observed at concentrations up to and including 25μM after five days. Curcumin (≥3μM) significantly reduced IL-1β-stimulated PG (p<0.05) and PGE2 release (p<0.001) from explants, whilst curcumin (≥12μM) significantly reduced MMP-3 release (p<0.01).Conclusion: Non-cytotoxic concentrations of curcumin exert anti-catabolic and anti-inflammatory effects in cartilage explants.
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Clutterbuck, Abigail L., David Allaway, Pat Harris, and Ali Mobasheri. "Curcumin reduces prostaglandin E2, matrix metalloproteinase-3 and proteoglycan release in the secretome of interleukin 1β-treated articular cartilage." F1000Research 2 (August 20, 2013): 147. http://dx.doi.org/10.12688/f1000research.2-147.v2.
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Objective: Curcumin (diferuloylmethane) is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA). The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β)-stimulated inflammation and catabolism in an explant model of cartilage inflammation.Methods: Articular cartilage explants and primary chondrocytes were obtained from equine metacarpophalangeal joints. Curcumin was added to monolayer cultured primary chondrocytes and cartilage explants in concentrations ranging from 3μM-100μM. Prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)-3 release into the secretome of IL-1β-stimulated explants was measured using a competitive ELISA and western blotting respectively. Proteoglycan (PG) release in the secretome was measured using the 1,9-dimethylmethylene blue (DMMB) assay. Cytotoxicity was assessed with a live/dead assay in monolayer cultures after 24 hours, 48 hours and five days, and in explants after five days.Results: Curcumin induced chondrocyte death in primary cultures (50μM p<0.001 and 100μM p<0.001) after 24 hours. After 48 hours and five days, curcumin (≥25μM) significantly increased cell death (p<0.001 both time points). In explants, curcumin toxicity was not observed at concentrations up to and including 25μM after five days. Curcumin (≥3μM) significantly reduced IL-1β-stimulated PG (p<0.05) and PGE2 release (p<0.001) from explants, whilst curcumin (≥12μM) significantly reduced MMP-3 release (p<0.01).Conclusion: Non-cytotoxic concentrations of curcumin exert anti-catabolic and anti-inflammatory effects in cartilage explants.
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Fitzpatrick, Carrie A., Nikolai V. Sharkov, Gary Ramsay, and Alisa L. Katzen. "Drosophila myb exerts opposing effects on S phase, promoting proliferation and suppressing endoreduplication." Development 129, no. 19 (October 1, 2002): 4497–507. http://dx.doi.org/10.1242/dev.129.19.4497.
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Drosophila melanogaster possesses a single gene, Dm myb, that is closely related to the vertebrate family of Myb genes, which encode transcription factors that are involved in regulatory decisions affecting cell proliferation, differentiation and apoptosis. The vertebrate Myb genes have been specifically implicated in regulating the G1/S transition of the cell cycle. Dm myb is expressed in all proliferating tissues, but not at detectable levels in endoreduplicating cells. Analysis of loss-of-function mutations in Dm myb revealed a block at the G2/M transition and mitotic defects, but did not directly implicate Dm myb function in the G1/S transition. We have used the Gal4-UAS binary system of ectopic expression to further investigate the function of Dm myb. Our results demonstrate that depending upon the type of cell cycle, ectopic Dm myb activity can exert opposing effects on S phase: driving DNA replication and promoting proliferation in diploid cells, even when developmental signals normally dictate cell cycle arrest; but suppressing endoreduplication in endocycling cells, an effect that can be overcome by induction of E2F. We also show that a C-terminally truncated DMyb protein, which is similar to an oncogenic form of vertebrate Myb, has more potent effects than the full-length protein, especially in endoreduplicating tissues. This finding indicates that the C terminus acts as a negative regulatory domain, which can be differentially regulated in a tissue-specific manner. Our studies help to resolve previous discrepancies regarding myb gene function in Drosophila and vertebrates. We conclude that in proliferating cells, Dm myb has the dual function of promoting S phase and M phase, while preserving diploidy by suppressing endoreduplication.
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Wu, Shun-Cheng, Hsu-Feng Hsiao, Mei-Ling Ho, Yung-Li Hung, Je-Ken Chang, Gwo-Jaw Wang, and Chau-Zen Wang. "Suppression of discoidin domain receptor 1 expression enhances the chondrogenesis of adipose-derived stem cells." American Journal of Physiology-Cell Physiology 308, no. 9 (May 1, 2015): C685—C696. http://dx.doi.org/10.1152/ajpcell.00398.2014.
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Effectively directing the chondrogenesis of adipose-derived stem cells (ADSCs) to engineer articular cartilage represents an important challenge in ADSC-based articular cartilage tissue engineering. The discoidin domain receptor 1 (DDR1) has been shown to affect cartilage homeostasis; however, little is known about the roles of DDR1 in ADSC chondrogenesis. In this study, we used the three-dimensional culture pellet culture model system with chondrogenic induction to investigate the roles of DDR1 in the chondrogenic differentiation of human ADSCs (hADSCs). Real-time polymerase chain reaction and Western blot were used to detect the expression of DDRs and chondrogenic genes. Sulfated glycosaminoglycan (sGAG) was detected by Alcian blue and dimethylmethylene blue (DMMB) assays. Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to assess cell death. During the chondrogenesis of hADSCs, the expression of DDR1 but not DDR2 was significantly elevated. The depletion of DDR1 expression in hADSCs using short hairpin RNA increased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and cartilaginous matrix deposition (collagen type II and sGAG) and only slightly increased cell death (2–8%). DDR1 overexpression in hADSCs decreased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and sGAG and enhanced hADSC survival. Moreover, DDR1-depleted hADSCs showed decreased expression of the terminal differentiation genes runt-related transcription factor 2 (Runx2) and matrix metalloproteinase 13 (MMP-13). These results suggest that DDR1 suppression may enhance ADSC chondrogenesis by enhancing the expression of chondrogenic genes and cartilaginous matrix deposition. We proposed that the suppression of DDR1 in ADSCs may be a candidate strategy of genetic modification to optimize ADSC-based articular cartilage tissue engineering.
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Kuhlmann, Constanze, Thilo L. Schenck, Attila Aszodi, Riccardo E. Giunta, and Paul Severin Wiggenhauser. "Zone-Dependent Architecture and Biochemical Composition of Decellularized Porcine Nasal Cartilage Modulate the Activity of Adipose Tissue-Derived Stem Cells in Cartilage Regeneration." International Journal of Molecular Sciences 22, no. 18 (September 14, 2021): 9917. http://dx.doi.org/10.3390/ijms22189917.
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Previous anatomical studies have shown different functional zones in human nasal septal cartilage (NC). These zones differ in respect to histological architecture and biochemical composition. The aim of this study was to investigate the influence of these zones on the fate of stem cells from a regenerative perspective. Therefore, decellularized porcine septal cartilage was prepared and subjected to histological assessment to demonstrate its equivalence to human cartilage. Decellularized porcine NC (DPNC) exposed distinct surfaces depending on two different histological zones: the outer surface (OS), which is equivalent to the superficial zone, and the inner surface (IS), which is equivalent to the central zone. Human adipose tissue-derived stem cells (ASCs) were isolated from the abdominal fat tissue of five female patients and were seeded on the IS and OS of DPNC, respectively. Cell seeding efficiency (CSE), vitality, proliferation, migration, the production of sulfated glycosaminoglycans (sGAG) and chondrogenic differentiation capacity were evaluated by histological staining (DAPI, Phalloidin, Live-Dead), biochemical assays (alamarBlue®, PicoGreen®, DMMB) and the quantification of gene expression (qPCR). Results show that cell vitality and CSE were not influenced by DPNC zones. ASCs, however, showed a significantly higher proliferation and elevated expression of early chondrogenic differentiation, as well as fibrocartilage markers, on the OS. On the contrary, there was a significantly higher upregulation of hypertrophy marker MMP13 (p < 0.0001) and GAG production (p = 0.0105) on the IS, whereas cell invasion into the three-dimensional DPNC was higher in comparison to the OS. We conclude that the zonal-dependent distinct architecture and composition of NC modulates activities of ASCs seeded on DPNC. These findings might be used for engineering of cartilage substitutes needed in facial reconstructive surgery that yield an equivalent histological and functional structure, such as native NC.
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Jiang, Wei, Pei Zhao, and Xuemei Zhang. "Apelin Promotes ECM Synthesis by Enhancing Autophagy Flux via TFEB in Human Degenerative NP Cells under Oxidative Stress." BioMed Research International 2020 (February 14, 2020): 1–8. http://dx.doi.org/10.1155/2020/4897170.
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Background. Apelin alleviates oxidative stress which contributes to the development of aging. IVDD is a disease closely correlated to aging and oxidative stress which is known to be harmful to NP cells’ matrix synthesis. The purpose of the present study was to investigate the role and underlying mechanism of Apelin in NP cells’ matrix degradation under oxidative stress. Methods. First, the mRNA and protein expressions of Apelin were checked by RT-PCR and Western blot in NP from normal and degenerative IVD to explore the relationship between Apelin and IVDD preliminarily. Then, H2O2 was used to mimic oxidative stress of NP cells. After treated with Apelin 13 and CQ, the GAG content was assessed by DMMB and the mRNA/protein expressions of NP matrix macromolecules (Collagen II and Aggrecan) and autophagy-related markers (LC3 and p62) were assessed by RT-PCR/Western blot. Finally, TFEB was knocked down by esiRNA-TFEB transfection and the nucleoprotein expression of TFEB and autophagy-related markers (LC3 and p62) were assessed by Western blot to discuss whether TFEB is involved in Apelin regulating autophagy flux in NP cells under oxidative stress. Results. Our data first confirmed that the mRNA and protein expressions of Apelin were decreased with IVDD. Furthermore, Apelin increased GAG content of NP cells and mRNA/protein expressions of NP matrix macromolecules (Collagen II and Aggrecan) and promoted autophagic flux (LC3II/I increased and p62 decreased) under oxidative stress. Finally, after transfected with esiRNA-TFEB, Apelin cannot promote autophagic flux any more in human degenerative NP cells. Conclusion. Our data indicated that Apelin promotes ECM synthesis by enhancing autophagy flux via TFEB in human degenerative NP cells under oxidative stress. This viewpoint may provide a new therapeutic idea for IVDD.
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Rambacher, K., J. Gennrich, R. Schewior, S. Lang, G. Pattappa, C. Zihlmann, N. Stiefel, J. Zellner, D. Docheva, and P. Angele. "INTERLEUKIN-1B (IL-1B)-INHIBITED MENISCUS DEPOSITION IS COUNTERED BY CULTURE UNDER PHYSIOXIA TO A GREATER EXTENT COMPARED WITH CELECOXIB-TREATED MENISCUS CELLS." Orthopaedic Proceedings 105-B, SUPP_9 (April 17, 2023): 81. http://dx.doi.org/10.1302/1358-992x.2023.9.081.
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Meniscus tears have been treated using partial meniscectomy to relieve pain in patients, although this leads to the onset of early osteoarthritis (OA). Cell-based therapies can help preserve the meniscus, although the presence of inflammatory cytokines compromises clinical outcomes. Anti-inflammatory drugs (e.g. celecoxib), can help to reduce pain in patients and in vitro studies suggest a beneficial effect on cytokine inhibited matrix content. Previously, we have demonstrated that the inhibitory effects of IL-1β can be countered by culture under low oxygen tension or physioxia. The present study sought to understand whether physioxia, celecoxib or combined application can counter the inhibitory effects IL-1β inhibited meniscus cells.Human avascular and vascular meniscus cells (n =3) were isolated and expanded under 20% (hyperoxia) or 2% (physioxia) oxygen. Cells were seeded into collagen scaffolds (Geistlich, Wolhusen) and cultured for 28 days either in the presence of 0.1ng/mL IL-1β, 5µg/mL celecoxib or both under their expansion oxygen conditions. Histological (DMMB, collagen I and collagen II immunostaining), GAG content and gene expression analysis was evaluated for the scaffolds.Under hyperoxia, meniscus cells showed a significant reduction in GAG content in the presence of IL-1β (*p < 0.05). Celecoxib alone did not significantly increase GAG content in IL-1β treated cultures. In contrast, physioxic culture showed a donor dependent increase in GAG content in control, IL-1β and celecoxib treated cultures with corresponding histological staining correlating with these results. Additionally, gene expression showed an upregulation in COL1A1, COL2A1 and ACAN and a downregulation in MMP13 and ADAMTS5 under physioxia for all experimental groups.Physioxia alone had a stronger effect in countering the inhibitory effects of IL-1β treated meniscus cells than celecoxib under hyperoxia. Preconditioning meniscus cells under physioxia prior to implantation has the potential to improve clinical outcomes for cell-based therapies of the meniscus.
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Kim, Keun Su, S. Tim Yoon, Jin Soo Park, Jun Li, Moon Soo Park, and William C. Hutton. "Inhibition of proteoglycan and type II collagen synthesis of disc nucleus cells by nicotine." Journal of Neurosurgery: Spine 99, no. 3 (October 2003): 291–97. http://dx.doi.org/10.3171/spi.2003.99.3.0291.
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Object. Systemic nicotine has been hypothesized to cause degeneration of the intervertebral disc which in turn decreases vascular supply to the disc through a cholinergic receptor—mediated process. Another possible mechanism may be through direct regulatory effects on disc cells. In this study, the authors tested the hypothesis that nicotine adversely affects nucleus pulposus cells by directly inhibiting proteoglycan synthesis and gene expression of type II collagen (Phase I study). They also assessed the hypothesis that nicotine inhibits the bone morphogenetic protein (BMP)—2-induced upregulation of extracellular matrix (Phase II study). Methods. Cells were isolated from nucleus pulposus obtained in rat lumbar discs and cultured on a monolayer. Media were treated with nicotine and/or recombinant human (rh)BMP-2 for 7 days. Sulfated glycosaminoglycan (SO4-GAG) in media was quantified using 1,9-dimethylmethylene blue (DMMB) assay. Gene assay of types I and II collagen, Sox9, and glyceraldehyde-3-phosphate dehydrogenase were quantified using reverse transcriptase—polymerase chain reaction (RT-PCR) and real time PCR. In the Phase I study, nicotine-treated (100 µg/ml) and nontreated cells were compared. The s-GAG production and messenger RNA (mRNA) of type II collagen and Sox9 decreased significantly in the nicotine-treated group. In the Phase II study, five groups were compared: 1) nontreatment; 2) rhBMP-2 only (100 ng/ml); and 3–5) with rhBMP-2 (100 ng/ml) and increasing doses of nicotine (1 [third group], 10, [fourth group], 100 [fifth group] µg/ml). The SO4-GAG production and mRNA of type II collagen and Sox9 decreased significantly in the groups treated with rhBMP-2 combined with 10 and 100 µg/ml of nicotine compared with the group treated with rhBMP-2. Conclusions. The results of this study raise the possibility that nicotine may contribute to the process of disc degeneration by a direct effect on the nucleus pulposus cells, possibly by antagonizing the effect of BMP-2.
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Clements, Kristen M., Jo K. Flannelly, Jonathan Tart, Sarah M. V. Brockbank, John Wardale, Jim Freeth, Andrew E. Parker, and Peter Newham. "Matrix metalloproteinase 17 is necessary for cartilage aggrecan degradation in an inflammatory environment." Annals of the Rheumatic Diseases 70, no. 4 (January 7, 2011): 683–89. http://dx.doi.org/10.1136/ard.2010.130757.
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ObjectiveAggrecan is a critical component of cartilage extracellular matrix. Several members of the ‘a disintegrin and metalloproteinase with thrombospondin motifs’ (ADAMTS) family have been characterised as aggrecanases by their ability to generate fragments containing the NITEGE neoepitope from aggrecan. Increased NITEGE fragments in synovial fluid and articular cartilage are a hallmark of osteoarthritis (OA) and it is hypothesised that the enhanced rate of aggrecan degradation is critical for cartilage destruction in OA. Recently, matrix metalloproteinase 17 (MMP17, also known as MT4-MMP) has been implicated in the activation of one of the key aggrecanases: ADAMTS4. In the present work, the hypothesis that MMP17 mediates the interleukin 1β (IL-1β) induced release of NITEGE neoepitope from human and murine articular cartilage is investigated.MethodsMMP17 was quantified at the protein and RNA level and NITEGE neoepitope generation by immunohistochemistry. Human postmortem articular cartilage explants were treated with recombinant MMP17, or IL-1β in the presence or absence of an MMP17 inhibitor. Glycosaminoglycan (GAG) loss into the media was quantified using the 1,9-dimethylmethylene blue (DMMB) assay. Intra-articular injection (IAI) of IL-1β or meniscotibial ligament transaction was carried out in MMP17 null mice.ResultsThe data reveal an association between increased MMP17 protein and NITEGE staining in areas of OA cartilage damage. Ex vivo treatment of normal human cartilage with recombinant MMP17 protein increased NITEGE generation in the cartilage and GAG loss into the media. In addition, IL-1β mediated cartilage GAG loss, and increased NITEGE neoepitope expression, were attenuated with an MMP17 inhibitor.IAI of IL-1β into C57BL6/Jax mice resulted in increased MMP17 expression in articular cartilage and increased GAG content in the synovial fluid. MMP17 null mice were protected against this increase. However, aggrecan loss driven by mechanical stress following medial meniscotibial ligament transection was not dependent on MMP17.ConclusionThese data further implicate MMP17 in the control of articular cartilage extracellular matrix aggrecan integrity in an inflammatory environment.
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Vadalà, G., G. Di Giacomo, L. Ambrosio, C. Cicione, V. Tilotta, F. Russo, R. Papalia, and V. Denaro. "IRISIN STIMULATES ANABOLISM AND MATRIX SYNTHESIS IN HUMAN NUCLEUS PULPOSUS CELLS IN VITRO: NEW INSIGHTS INTO A CROSS-TALK BETWEEN THE MUSCLE AND THE INTERVERTEBRAL DISC." Orthopaedic Proceedings 105-B, SUPP_8 (April 11, 2023): 53. http://dx.doi.org/10.1302/1358-992x.2023.8.053.
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This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation.hNPCs were isolated from intervertebral discs and cultured in alginate beads. hNPCs were exposed to phosphate-buffered saline (PBS) or recombinant irisin (r-irisin) at 5, 10 and 25 ng/mL (n=4). Each experiment was performed in triplicate. Cell proliferation was assessed with trypan blue staining-automated cell counting and PicoGreen assay. Glycosaminoglycan (GAG) content was measured using the DMMB assay. Metabolic activity was assessed with the MTT assay and the Griess Reagent System. Gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and −3, aggrecan, interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 was measured by RT-PCR. MTT assay and ADAMTS-5, COL2, TIMP-1 and IL-1β gene expression were evaluated following incubation with 5, 10 and 25 ng/mL r-irisin for 24 hours and subsequent culture with 10 ng/ml IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin).Irisin increased hNPC proliferation (p<0.001), metabolic activity (p<0.05), GAG content (p<0.01), as well as COL2 (p<0.01), aggrecan (p<0.05), TIMP-1 and −3 (p<0.01) gene expression, while decreasing MMP-13 (p<0.05) and IL-1β (p<0.001) mRNA levels. r-irisin pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p<0.001) and a decrease of IL-1β (p<0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-irisin increased hNPC metabolic activity (p<0.001), COL2 gene expression (p<0.05) and decreased IL-1β (p<0.05) and ADAMTS-5 levels (p<0.01).Irisin stimulates hNPC proliferation, metabolic activity, and anabolism by reducing IL-1β and catabolic enzyme expression while promoting matrix synthesis.
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Lin, Y.-C., A. C. Hall, and A. H. R. W. Simpson. "A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage." Bone & Joint Research 7, no. 3 (March 2018): 205–12. http://dx.doi.org/10.1302/2046-3758.73.bjr-2017-0320.
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Objectives The purpose of this study was to create a novel ex vivo organ culture model for evaluating the effects of static and dynamic load on cartilage. Methods The metatarsophalangeal joints of 12 fresh cadaveric bovine feet were skinned and dissected aseptically, and cultured for up to four weeks. Dynamic movement was applied using a custom-made machine on six joints, with the others cultured under static conditions. Chondrocyte viability and matrix glycosaminoglycan (GAG) content were evaluated by the cell viability probes, 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), and dimethylmethylene blue (DMMB) assay, respectively. Results Chondrocyte viability in the static model decreased significantly from 89.9% (sd 2.5%) (Day 0) to 66.5% (sd 13.1%) (Day 28), 94.7% (sd 1.1%) to 80. 9% (sd 5.8%) and 80.1% (sd 3.0%) to 46.9% (sd 8.5%) in the superficial quarter, central half and deep quarter of cartilage, respectively (p < 0.001 in each zone; one-way analysis of variance). The GAG content decreased significantly from 6.01 μg/mg (sd 0.06) (Day 0) to 4.71 μg/mg (sd 0.06) (Day 28) (p < 0.001; one-way analysis of variance). However, with dynamic movement, chondrocyte viability and GAG content were maintained at the Day 0 level over the four-week period without a significant change (chondrocyte viability: 92.0% (sd 4.0%) (Day 0) to 89.9% (sd 0.2%) (Day 28), 93.1% (sd 1.5%) to 93.8% (sd 0.9%) and 85.6% (sd 0.8%) to 84.0% (sd 2.9%) in the three corresponding zones; GAG content: 6.18 μg/mg (sd 0.15) (Day 0) to 6.06 μg/mg (sd 0.09) (Day 28)). Conclusion Dynamic joint movement maintained chondrocyte viability and cartilage GAG content. This long-term whole joint culture model could be of value in providing a more natural and controlled platform for investigating the influence of joint movement on articular cartilage, and for evaluating novel therapies for cartilage repair. Cite this article: Y-C. Lin, A. C. Hall, A. H. R. W. Simpson. A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage. Bone Joint Res 2018;7:205–212. DOI: 10.1302/2046-3758.73.BJR-2017-0320.
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Coulson-Thomas, Vivien, and Tarsis Gesteira. "Dimethylmethylene Blue Assay (DMMB)." BIO-PROTOCOL 4, no. 18 (2014). http://dx.doi.org/10.21769/bioprotoc.1236.
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de Ávila Narciso Gomes, Raphael, Alejandro Marmolejo-Garza, Floris-Jan Haan, Teresa Mitchell García, Tingting Chen, Mario Mauthe, Yollanda E. Moreira Franco Parisotto, et al. "Mitochondrial dysfunction mediates neuronal cell response to DMMB photodynamic therapy." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, January 2023, 119429. http://dx.doi.org/10.1016/j.bbamcr.2022.119429.
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"Electrochemical Detection of Polyanions on Polypyrrole/DMMB Nanowire Composite Film Modified Electrode." ECS Meeting Abstracts, 2014. http://dx.doi.org/10.1149/ma2014-02/24/1408.
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Hattori, Kyosuke, Nobunori Takahashi, Kenya Terabe, Yoshifumi Ohashi, Kenji Kishimoto, Yutaka Yokota, Mochihito Suzuki, Toshihisa Kojima, and Shiro Imagama. "Activation of transient receptor potential vanilloid 4 protects articular cartilage against inflammatory responses via CaMKK/AMPK/NF-κB signaling pathway." Scientific Reports 11, no. 1 (July 30, 2021). http://dx.doi.org/10.1038/s41598-021-94938-3.
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AbstractTransient 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.
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Sun, Yi, Yuen-kee Tsui, Mengqi Yu, Minmin Lyu, Kenneth Cheung, Richard Kao, and Victor Leung. "Integration of a miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism." Scientific Reports 12, no. 1 (January 20, 2022). http://dx.doi.org/10.1038/s41598-022-04805-y.
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AbstractDefective 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.
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Alemu, Tadele Ageru, Delele Worku Ayele, Shahitha Parveen J, Ababay Ketema Worku, Minbale Admas Teshager, Praveen Ramamurthy, and Dhakshnamoorthy M. "Highly sensitive detection of bacteria (E. Coli) Endotoxin using novel PANI-benzimidazole-Ag nanocomposite by DMMB dye displacement assay." Materials Research Express, June 27, 2023. http://dx.doi.org/10.1088/2053-1591/ace239.
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Abstract In the present study, a new biochemical sensor material of conductive Silver (Ag) reinforced polyaniline (PANI)-Benzimidazole copolymer nanocomposite was fabricated via in-situ chemical oxidative polymerization method for the detection of endotoxin. The fabricated PANI-Benz-Ag nanocomposite was characterized by FTIR, XRD, UV-Visible spectrometer, DSC, TGA, Zeta-potential, SEM, TEM, and Confocal fluorescence imaging microscopy. The measured particle size, zeta-potential, and conductivity of the PANI-Benz-Ag nanocomposite were 4.942nm, -10.4mV, and 73.7μS/cm respectively. The crystallite size of Ag nanoparticles was around 67 nm calculated by XRD analysis and TGA analysis was used to determine weight losses and thermal stabilities of PANI-Benz and PANI-Benz-Ag nanocomposite. The endotoxin E. Coli bacteria detection ability of the synthesized PANI-Benz-Ag nanocomposite-based biochemical sensor using DMMB dye displacement assay through the hitchhiking method by confocal fluorescence microscopy was found to be effective. The synthesized electrochemical biosensor can avoid the diagnostic methods for the next generation by applying too early detection of bacteria from adulterated and putrefied food.
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Soares, Luiz G. P., Cristiane Galdino de Almeida, Pedro J. L. Crugeira, Iago P. F. Nunes, Anna Paula L. T. da Silva, Jeovana A. Almeida, Maria C. T. Cangussú, Paulo F. de Almeida, Fernando A. L. Habib, and Antônio L. B. Pinheiro. "Photodynamic antimicrobial therapy (AmPDT) using 1,9-Dimethyl-Methylene Blue zinc chloride double salt - DMMB and λ640 ± 5ηm LED light in patients undertaking orthodontic treatment." Photodiagnosis and Photodynamic Therapy, March 2023, 103503. http://dx.doi.org/10.1016/j.pdpdt.2023.103503.
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Pereira, Luiz Miguel, Gisele Bulhões Portapilla, Guilherme Thomaz Pereira Brancini, Bruna Possato, Cássia Mariana Bronzon da Costa, Péricles Gama Abreu-Filho, Mark Wainwright, Ana Patrícia Yatsuda, and Gilberto Úbida Leite Braga. "The potential of phenothiazinium dyes as cytotoxicity markers in cisplatin-treated cells." Scientific Reports 13, no. 1 (June 23, 2023). http://dx.doi.org/10.1038/s41598-023-36721-0.
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AbstractAssessing 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.
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Lama, Polly, Harry Claireaux, Luke Flower, Ian J. Harding, Trish Dolan, Christine L. Le Maitre, and Michael A. Adams. "Physical disruption of intervertebral disc promotes cell clustering and a degenerative phenotype." Cell Death Discovery 5, no. 1 (December 2019). http://dx.doi.org/10.1038/s41420-019-0233-z.
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AbstractTo test the hypothesis that physical disruption of an intervertebral disc disturbs cell-matrix binding, leading to cell clustering and increased expression of matrix degrading enzymes that contribute towards degenerative disc cell phenotype. Lumbar disc tissue was removed at surgery from 21 patients with disc herniation, 11 with disc degeneration, and 8 with adolescent scoliosis. 5 μm sections were examined with histology, and 30-µm sections by confocal microscopy. Antibodies were used against integrin α5beta1, matrix metalloproteinases (MMP) 1, MMP-3, caspase 3, and denatured collagen types I and II. Spatial associations were sought between cell clustering and various degenerative features. An additional, 11 non-herniated human discs were used to examine causality: half of each specimen was cultured in a manner that allowed free ‘unconstrained’ swelling (similar to a herniated disc in vivo), while the other half was cultured within a perspex ring that allowed ‘constrained’ swelling. Changes were monitored over 36 h using live-cell imaging. 1,9-Di-methyl methylene blue (DMMB) assay for glycosaminoglycan loss was carried out from tissue medium. Partially constrained specimens showed little swelling or cell movement in vitro. In contrast, unconstrained swelling significantly increased matrix distortion, glycosaminoglycan loss, exposure of integrin binding sites, expression of MMPs 1 and 3, and collagen denaturation. In the association studies, herniated disc specimens showed changes that resembled unconstrained swelling in vitro. In addition, they exhibited increased cell clustering, apoptosis, MMP expression, and collagen denaturation compared to ‘control’ discs. Results support our hypothesis. Further confirmation will require longitudinal animal experiments.
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Devaraj, IC, Dhiraj J. Trivedi, Vidya S. Patil, V. Indumati, and NB Sanjeevini. "Study of Urinary Glycosaminoglycans among Essential Hypertensive Patients." JOURNAL OF CLINICAL AND DIAGNOSTIC RESEARCH, 2021. http://dx.doi.org/10.7860/jcdr/2021/51777.15695.
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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 <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.
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Shamdani, Sara, Sandrine Chantepie, Camille Flageollet, Nadia Henni-Chebra, Yohann Jouan, Florent Eymard, Eric Hay, et al. "Heparan sulfate functions are altered in the osteoarthritic cartilage." Arthritis Research & Therapy 22, no. 1 (December 2020). http://dx.doi.org/10.1186/s13075-020-02352-3.
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Abstract Background Heparan sulfate (HS) proteoglycans (PG) may be found at the chondrocyte surface and in the pericellular cartilage matrix, and are involved in cell-cell and cell-matrix interactions. An important function of HS chains is to regulate cell fate through specific interactions with heparin-binding proteins (HBP) modulated by their complex sulfation pattern. Osteoarthritis (OA) is a joint disorder characterized by the degradation of articular cartilaginous extracellular matrix. The aim of this study was to investigate HS structure and functions in osteoarthritic cartilages compared to normal cartilages (controls). Methods Glycosaminoglycans (GAG) were extracted from human macroscopically normal cartilages (controls, n = 7) and (OA cartilages n = 11). HS were isolated and quantified using the DMMB quantification method. Their structure and functions were then compared using respectively a HPLC analysis and HBP binding tests and their phenotypic effects on murine chondrocytes were studied by RQ-PCR. Statistical analyzes were performed using a one-way ANOVA followed by a Dunnett’s test or a t test for pairwise comparisons. Results In OA, HS were characterized by increased sulfation levels compared to controls. Moreover, the capacity of these HS to bind HBP involved in the OA pathophysiological process such as FGF2 and VEGF was reduced. Chondroitin sulfates and keratan sulfates regulated these binding properties. Finally, HS from OA cartilages induced the mRNA levels of catabolic markers such as MMP3, MMP13, and TS4 and inhibited the mRNA levels of anabolic markers such as COL2, ACAN, SOX9, and VEGF in murine articular chondrocytes. Conclusion The sulfation of HS chains was increased in OA cartilages with changes in HBP binding properties and biological effects on chondrocyte phenotypes. Thus, modified HS present in altered cartilages could be a novel therapeutic target in OA.
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Zuncheddu, Daniele, Elena Della Bella, Dalila Petta, Cecilia Bärtschi, Sonja Häckel, Moritz C. Deml, Martin J. Stoddart, Sibylle Grad, and Valentina Basoli. "Effect of glucose depletion and fructose administration during chondrogenic commitment in human bone marrow-derived stem cells." Stem Cell Research & Therapy 13, no. 1 (December 27, 2022). http://dx.doi.org/10.1186/s13287-022-03214-2.
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Abstract Background Bone marrow mesenchymal stromal cells (BMSCs) are promising for therapeutic use in cartilage repair, because of their capacity to differentiate into chondrocytes. Often, in vitro differentiation protocols employ the use of high amount of glucose, which does not reflect cartilage physiology. For this reason, we investigated how different concentrations of glucose can affect the chondrogenic differentiation of BMSCs in cell culture pellets. Additionally, we investigated how fructose could influence the chondrogenic differentiation in vitro. Methods BMSC were isolated from six donors and cultured in DMEM containing glucose at either 25 mM (HG), 5.5 mM (LG) or 1 mM (LLG), and 1% non-essential amino acids, 1% ITS+, in the presence of 100 nM dexamethasone, 50 µg/ml ascorbic acid-2 phosphate and 10 ng/ml TGF-β1. To investigate the effect of different metabolic substrates, other groups were exposed to additional 25 mM fructose. The media were replaced every second day until day 21 when all the pellets were harvested for further analyses. Biochemical analysis for glycosaminoglycans into pellets and released in medium was performed using the DMMB method. Expression of GLUT3 and GLUT5 was assayed by qPCR and validated using FACS analysis and immunofluorescence in monolayer cultures. Chondrogenic differentiation was further confirmed by qPCR analysis of COL2A1, COL1A1, COL10A1, ACAN, RUNX2, SOX9, SP7, MMP13, and PPARG, normalized on RPLP0. Type 2 collagen expression was subsequently validated by immunofluorescence analysis. Results We show for the first time the presence of fructose transporter GLUT5 in BMSC and its regulation during chondrogenic commitment. Additionally, decreasing glucose concentration during chondrogenesis dramatically decreased the yield of differentiation. However, the use of fructose alone or together with low glucose concentrations does not limit cell differentiation, but on the contrary it might help in maintaining a stable chondrogenic phenotype comparable with the standard culture conditions (high glucose). Conclusion This study provides evidence that BMSC express GLUT5 and differentially regulate GLUT3 in the presence of glucose variation. This study gives a better comprehension of BMSCs sugar use during chondrogenesis.