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OBJECTIVES: Evaluate molecularly the role of P11-4 self-assembly peptide in dentin remineralization and its interaction with collagen I. METHODS: The calcium-responsive P11-4 peptide was analyzed by intrinsic fluorescence emission spectrum, circular dichroism spectrum (CD), and atomic force microscope (AFM). Differential light scattering was used to monitor the nucleation growth rate of calcium phosphate nanocrystals in the absence or in the presence of P11-4. AFM was used to analyze the radial size (nm) of calcium phosphate nanocrystals formed in the absence or in the presence of P11-4, as well as to verify the spatial structure of P11-4 in the absence or in the presence of Ca2+. RESULTS: The interaction of Ca2+ with the P11-4 (KD = 0.58 ± 0.06 mM) promotes the formation of ß-sheet antiparallel structure, leads to its precipitation in saturated solutions of Ca/P = 1.67 and induces the formation of parallel large fibrils (0.6 - 1.5 µm). P11-4 organized the HAP nucleation by reducing both the growth rate and size variability of nanocrystals, analyzed by the F test (p < 0.0001, N = 30). P11-4 interacts (KD = 0.75 ± 0.06 µM) with the KGHRGFSGL motif present at the C-terminal collagen telopeptide domain. P11-4 also increased the amount of HAP and collagen in the MDPC-23 cells. SIGNIFICANCE: The presented data propose a mechanism that will help future clinical and/or basic research to better understand a molecule able to inhibit structural collagen loss and help the impaired tissue to remineralize.
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Cálcio , Colágeno Tipo I , Peptídeos , Colágeno , Fosfatos de Cálcio/farmacologia , ÍonsRESUMO
Collagen, the most abundant structural protein found in mammals, plays a vital role as a constituent of the extracellular matrix (ECM) that surrounds cells. Collagen fibrils are strengthened through the formation of covalent cross-links, which involve complex enzymatic and non-enzymatic reactions. Lysyl oxidase (LOX) is responsible for catalyzing the oxidative deamination of lysine and hydroxylysine residues, resulting in the production of aldehydes, allysine, and hydroxyallysine. These intermediates undergo spontaneous condensation reactions, leading to the formation of immature cross-links, which are the initial step in the development of mature covalent cross-links. Additionally, non-enzymatic glycation contributes to the formation of abnormal cross-linking in collagen fibrils. During glycation, specific lysine and arginine residues in collagen are modified by reducing sugars, leading to the creation of Advanced Glycation End-products (AGEs). These AGEs have been associated with changes in the mechanical properties of collagen fibers. Interestingly, various studies have reported that plant polyphenols possess amine oxidase-like activity and can act as potent inhibitors of protein glycation. This review article focuses on compiling the literature describing polyphenols with amine oxidase-like activity and antiglycation properties. Specifically, we explore the molecular mechanisms by which specific flavonoids impact or protect the normal collagen cross-linking process. Furthermore, we discuss how these dual activities can be harnessed to generate properly cross-linked collagen molecules, thereby promoting the stabilization of highly organized collagen fibrils.
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Lisina , Proteína-Lisina 6-Oxidase , Animais , Proteína-Lisina 6-Oxidase/metabolismo , Lisina/metabolismo , Polifenóis/metabolismo , Matriz Extracelular/metabolismo , Colágeno/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Homeostase , Aminas/metabolismo , Mamíferos/metabolismoRESUMO
Infectious keratitis is a vision-threatening microbial infection. The increasing antimicrobial resistance and the fact that severe cases often evolve into corneal perforation necessitate the development of alternative therapeutics for effective medical management. Genipin, a natural crosslinker, was recently shown to exert antimicrobial effects in an ex vivo model of microbial keratitis, highlighting its potential to serve as a novel treatment for infectious keratitis. This study aimed to evaluate the antimicrobial and anti-inflammatory effects of genipin in an in vivo model of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) keratitis. Clinical scores, confocal microscopy, plate count, and histology were carried out to evaluate the severity of keratitis. To assess the effect of genipin on inflammation, the gene expression of pro- and anti-inflammatory factors, including matrix metalloproteinases (MMPs), were evaluated. Genipin treatment alleviated the severity of bacterial keratitis by reducing bacterial load and repressing neutrophil infiltration. The expression of interleukin 1B (IL1B), interleukin 6 (IL6), interleukin 8 (IL8), interleukin 15 (IL15), tumor necrosis factor-α (TNF-α), and interferon γ (IFNγ), as well as MMP2 and MMP9, were significantly reduced in genipin-treated corneas. Genipin promoted corneal proteolysis and host resistance to S. aureus and P. aeruginosa infection by suppressing inflammatory cell infiltration, regulating inflammatory mediators, and downregulating the expression of MMP2 and MMP9.
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Ceratite , Infecções por Pseudomonas , Humanos , Animais , Camundongos , Citocinas/metabolismo , Pseudomonas aeruginosa , Staphylococcus aureus/metabolismo , Projetos Piloto , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Ceratite/microbiologia , Córnea/metabolismo , Infecções por Pseudomonas/microbiologia , Camundongos Endogâmicos C57BLRESUMO
The latest advancements in dentin bonding have focused on strategies to impair degradation mechanisms in order to extend the longevity of bonded interfaces. Protease inhibitors can reduce collagen degradation within the hybrid layer (HL). Collagen cross-linkers allow better adhesive infiltration and also inhibit proteases activity. Particles added to adhesive can promote mineral precipitation within the HL, reducing nanoleakage and micropermeability, besides possible antimicrobial and enzymatic inhibition effects. Most of these approaches are still experimental, and aspects of the adhesive under the clinician's control are still determinant for the long-term stability of adhesive restorations.
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Colagem Dentária , Adesivos Dentinários , Colágeno/química , Dentina/metabolismo , Adesivos Dentinários/farmacologia , Humanos , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Cimentos de ResinaRESUMO
To evaluate the biomodification ability of lignin used as pre-treatment in human dentin before the application of an etch-and-rinse adhesive. Experimental hydroethanolic solutions with different cross-linking agents were used: 6.5% proanthocyanidins (PAC, from grape-seed extract); 2% cardanol (CARD, from cashew-nut shell liquid); lignin (LIG, from eucalyptus) at 1, 2 or 4% concentrations. The negative control (NC) was ethanol 50 v%. Extracted molars were prepared, and dentin microtensile bond strength (µTBS) was evaluated after 24 h water storage or 10,000 thermocycling aging. Further specimens were processed for SEM nanoleakage, micropermeability confocal microscopy evaluation and in situ degree of conversion (DC) through micro-Raman spectroscopy. Demineralized dentin sticks were submitted to a three-point bending test to evaluate the elastic modulus (E) before and after 1 min biomodification using the tested solutions. Moreover, it was also evaluated the mass changes and hydroxyproline (HYP) release after 4-weeks of water storage. Vibrational collagen crosslinking identification was evaluated through micro-Raman spectroscopy. The results were analyzed by analysis of variance (ANOVA) and Tukey's test (α = 0.05). A significant reduction in µTBS was observed in groups NC (p < 0.001) and CARD (p = 0.026). LIG-4% showed no significant reduction in µTBS after aging (p = 0.022). Nanoleakage micrographs showed hybrid layer protection with all agents, but reduced micropermeability was attained only with lignin. Polymerization was negatively affected in the presence of all tested cross-linking agents, except LIG-1%. Lignin and cardanol increased the dentin E values, but only lignin reduced the mass loss in dentin specimens. Effective collagen crosslinking (1117 cm−1 and 1235 cm−1) was detected for all agents. HYP release was significantly lower with LIG-1% than NC (p < 0.001). Lignin was able to perform collagen cross-linking and prevent the degradation of unprotected dentin collagen, thereby improving the bonding performance of the composite restorations performed in this study.
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Purpose: To compare management of postoperative pain after corneal collagen crosslinking (CXL) with oral gabapentin or ketorolac. Methods: Prospective interventional comparative case series in a single center. Patients undergoing epithelium-off (epi-off) or epithelium-on (epi-on) techniques performed by a single surgeon for progressive keratoconus were enrolled and randomly assigned to the ketorolac (10-mg tablets every 8 h) or the gabapentin (300-mg capsules every 8 h) group and instructed to take the medication for the first 3 postoperative days. Using a numeric scale of pain, scores were assessed for current pain (at the time of applying the questionnaire), and average pain over the preceding 24 h. Eye symptoms and systemic adverse events related to oral medication were also assessed. Results: Thirty-seven patients were included, with 22 (10 epi-on and 12 epi-off) assigned to the ketorolac group and 15 (7 epi-on and 8 epi-off) to the gabapentin group. No statistically significant differences were noted on the pain scale between groups at any point of the study, in the median pain scores of patients at the time of applying the questionnaire, nor in the severity of pain during the 24-h period before the assessment. Also, no differences were found among groups for the eye symptoms and the systemic adverse events. The median regression analysis showed no effect of the type of surgery or gender in the severity of pain. Conclusions: Both oral ketorolac and oral gabapentin can be used with similar results for pain and symptomatic control after epi-on or epi-off CXL procedures.
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Córnea/metabolismo , Gabapentina/administração & dosagem , Ceratocone/cirurgia , Cetorolaco/administração & dosagem , Dor Pós-Operatória/tratamento farmacológico , Administração Oral , Adolescente , Adulto , Analgésicos/administração & dosagem , Analgésicos/efeitos adversos , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/efeitos adversos , Colágeno/metabolismo , Reagentes de Ligações Cruzadas/administração & dosagem , Feminino , Gabapentina/efeitos adversos , Humanos , Cetorolaco/efeitos adversos , Masculino , Medição da Dor , Estudos Prospectivos , Inquéritos e Questionários , Resultado do Tratamento , Adulto JovemRESUMO
PURPOSE: To report the case of a 50-year-old woman with diabetes that presented with corneal melting and perforation 6 weeks after collagen cross-linking (CxL) for keratoconus (KC) and postoperative use of nepafenac eye drops, a nonsteroidal anti-inflammatory drug (NSAID). METHODS: This is a case report of a patient with diabetes, KC and a thin cornea that had undergone left eye corneal CxL at a different hospital followed by postoperative use of nepafenac eye drops for 6 weeks. RESULTS: The patient presented for the first time to our clinic with left corneal melting, perforation and iris prolapse 6 weeks after corneal CxL and topical nepafenac use. She was treated with a left eye tectonic penetrating keratoplasty, extracapsular cataract extraction, intraocular lens implantation and pupilloplasty. CONCLUSIONS: The corneal melting and perforation in this patient was associated with multiple risk factors: (1) nepafenac eye drop use, (2) CxL in a cornea thinner than 400 µm and (3) diabetes. The recommended corneal thickness limits should be respected. Topical NSAIDs should be used with caution if used as postoperative treatment after corneal CxL and in patients with diabetes, epithelial defect or delayed healing, because of the possible increased risk for corneal melting when multiple risk factors are observed.
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The molecular alterations involved in the capsule deformation presented in shoulder instability patients are poorly understood. Increased TGFß1 acts as a signal for production of matrix macromolecules by fibrogenic cells at joint injury sites. TGFß1, through its receptor TGFßR1, regulates genes involved in collagen cross-linking, such as LOX, PLOD1, and PLOD2. We evaluated TGFß1, TGFßR1, LOX, PLOD1, and PLOD2 gene expression in the antero-inferior (macroscopically injured region), antero-superior and posterior regions of the glenohumeral capsule of 29 shoulder instability patients and eight controls. We observed that PLOD2 expression was increased in the anterior-inferior capsule region of the patients compared to controls. LOX expression tended to be increased in the posterior portion of patients. Patients with recurrent shoulder dislocation presented upregulation of TGFßR1 in the antero-inferior capsule portion and of PLOD2 in the posterior region. Conversely, LOX was increased in the posterior portion of the capsule of patients with a single shoulder dislocation episode. In the antero-inferior, LOX expression was inversely correlated and TGFßR1 was directly correlated with the duration of symptoms. In the posterior region, PLOD2, TGFß1, and TGFßR1 were directly correlated with the duration of symptoms. In conclusion, PLOD2 expression was increased in the macroscopically injured region of the capsule of patients. Upregulation of TGFß1, TGFßR1, and PLOD2 seems to be related with the maintenance of disease symptoms, especially in the posterior region. LOX upregulation seems to occur only in the initial phase of the affection. Therefore, TGFß1, TGFßR1, LOX, and PLOD2 may play a role in shoulder instability.
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Colágeno/metabolismo , Luxação do Ombro/metabolismo , Articulação do Ombro/metabolismo , Adulto , Estudos de Casos e Controles , Feminino , Expressão Gênica , Humanos , Masculino , Adulto JovemRESUMO
Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C-type lectin-like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin-based contractility [myosin-light chain-2 (MLC2) phosphorylation], loss of cell polarity, loss of cell-cell junctions, luminal infiltration and basal invasion induced by AGE-modified basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180(Δ) (Ex2-6/) (Δ) (Ex2-6) mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour in non-transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Membrana Basal/metabolismo , Movimento Celular , Células Epiteliais/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Mitogênicos/metabolismo , Animais , Membrana Basal/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Elasticidade , Humanos , Estimativa de Kaplan-Meier , Lectinas Tipo C/metabolismo , Masculino , Mecanotransdução Celular , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos Knockout , Invasividade Neoplásica , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Estrutura Terciária de Proteína , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Fatores de TempoRESUMO
Studies suggest that standard corneal collagen crosslinking (CXL) is a safe and effective treatment to stiffen the cornea for keratoconus and other ectatic corneal disorders. The purpose of the present study was to compare the biomechanical effects of transepithelial benzalkonium chloride-EDTA (BAC-EDTA) riboflavin-UVA crosslinking to standard epithelium-off riboflavin-UVA crosslinking in a rabbit model. Corneal stiffness was quantified using optical coherence elastography at two months after treatment. The mean lateral-to-axial displacement ratio for the BAC-EDTA transepithelial CXL group was lower (greater stiffness) [0.062 ± 0.042, mean ± SD] than epithelium-off CXL (mean ± SD: 0.065 ± 0.045) or untreated control eyes (0.069 ± 0.044). Using ANOVA with Tukey correction, a statistically significant difference was found between the BAC-EDTA transepithelial CXL group and standard epithelium-off CXL (p = 0.0019) or the untreated control (p < 0.0001) groups. A graph of the probability density functions for biomechanical stiffness also showed a greater shift in stiffening in the BAC-EDTA transepithelial CXL group than the standard epithelium-off CXL or untreated control group. These results demonstrated that the biomechanical stiffening effect produced by BAC-EDTA transepithelial CXL was greater than that produced by standard epithelium-off CXL in a rabbit model.
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Córnea/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Elasticidade/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Riboflavina/farmacologia , Análise de Variância , Animais , Compostos de Benzalcônio/farmacologia , Fenômenos Biomecânicos/fisiologia , Quelantes/farmacologia , Córnea/fisiopatologia , Elasticidade/fisiologia , Técnicas de Imagem por Elasticidade , Modelos Animais , Coelhos , Raios UltravioletaRESUMO
This work evaluated the relationship of charqui meat (CHM) chemical composition with the tenderness throughout its production. CHM was prepared from beef Vastus lateralis of 4-5 years old. Shear force of fresh CHM showed an approx. 3-fold increase in toughness compared to the raw material while, in the case of cooked CHM it was 6-fold increased in relation to the raw charqui. The moisture content decreased by 39.0 and 58.0 percent (p<0.05) for uncooked and cooked CHM, respectively, in relation to the raw material. Mathematical modeling of the influence of these meat components showed that shear force increased exponentially with the loss of moisture. The texture of CHM was the result of a multitude of factors involving myofibril proteins which promoted dynamic biochemical events such as the binding of water molecules. It was the amount of the latter which ultimately determine the final charqui meat texture.
Este trabalho avaliou a relação entre a composição química aproximada do charque (CHM) com a maciez durante todas as etapas de sua produção. CHM foi produzida do m. Vastus lateralis bovino (patinho) de aproximadamente 4-5 anos de idade. A força de cisalhamento do charque cru mostrou o valor aproximado de 3 vezes maior em dureza comparada à matéria prima enquanto que no caso do CHM cozido houve 6 vezes maior em aumento sob as mesmas condições.O índice de umidade diminui de 39 a 58 por cento (p<0,05) para CHM cru e cozido, respectivamente, em relação à matéria prima. O modelo matemático da influência destes componentes mostrou que a força de cisalhamento aumentou exponencialmente com a perda de umidade. A textura do charque é o resultado da associação de multifatores envolvendo proteínas miofibrilares que provocam eventos bioquímicos dinâmicos como a sua ligação com as moléculas da água. É a quantidade destas que determina a textura final do charque.