RESUMO
Background: Conventional microtiter plates lack the surface strength needed for effective binding of pneumococcal polysaccharide antigens. This study tackles the limitation by altering the surface of polystyrene plates through carbodiimide activation under acidic pH conditions.Method: The microtiter plates were activated with carbodiimide coupling agents, N,N'-Dicyclohexylcarbodiimide (DCC) and N-Hydroxysuccinimide (NHS). They were subsequently coated with 13 pneumococcal antigens at a concentration of 5 µg/ml with a pH of 3.5. The IgG antibody titer was assessed utilizing the World Health Organization (WHO) ELISA protocol for 30 human serum samples. In addition, validation experiments were conducted to evaluate specificity and precision.Results: The modified plates exhibited two-times higher antibody titers compared to conventional plates across all 13 serotypes. Observations revealed elevated antibody levels, with geometric concentrations ranging between 0.96 µg/ml and 4.24 µg/ml.Conclusion: Carbodiimide activation and acidic pH modification of microtiter plates enhance sensitivity and specificity in detecting pneumococcal antibodies, critical for vaccination planning and immunity assessment.
[Box: see text].
Assuntos
Carbodi-Imidas , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G , Streptococcus pneumoniae , Streptococcus pneumoniae/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Humanos , Carbodi-Imidas/química , Polissacarídeos Bacterianos/imunologia , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Concentração de Íons de HidrogênioRESUMO
To enhance the stability and adsorption performance of chitosan in Cr(VI)-contaminated acidic wastewater, a novel EDAC-modified-EDTA-crosslinked chitosan derivative (CSEC) was synthesized via a one-pot method with chitosan, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), and Na2EDTA as raw materials. To further improve the mechanical strength and separation performance of CSEC, a novel composite bead (CSEP) of CSEC and imidazolium-functionalized polysulfone (IMPSF) was prepared through a phase inversion method. The chemical composition and microstructure of CSEC and CSEP were characterized by FESEM, FTIR, NMR and XPS techniques. The maximum adsorption capacities of CSEC and CSEP for Cr(VI) were 145.96 and 135.82 mg g-1 at pH 3, respectively, and the equilibrium time for Cr(VI) adsorption by CSEC and CSEP was 5 min and 8 h, respectively. The adsorption process of Cr(VI) by both CSEC and CSEP was exothermic and spontaneous. Compared to CSEC, CSEP has significantly enhanced resistance to interference from coexisting anions. The removal mechanism of Cr(VI) by CSEP might involve redox reaction as well as electrostatic attraction between Cr(VI) oxyanions and various nitrogen cations, including protonated amino groups, guanidinium groups, protonated tertiary amine groups, and imidazolium cations. The CSEP beads have potential application value in the treatment of acidic wastewater containing Cr(VI).
Assuntos
Quitosana , Cromo , Imidazóis , Polímeros , Sulfonas , Poluentes Químicos da Água , Purificação da Água , Quitosana/química , Cromo/isolamento & purificação , Cromo/química , Sulfonas/química , Adsorção , Poluentes Químicos da Água/química , Poluentes Químicos da Água/isolamento & purificação , Imidazóis/química , Polímeros/química , Purificação da Água/métodos , Concentração de Íons de Hidrogênio , Cinética , Águas Residuárias/química , Carbodi-Imidas/química , Água/química , Soluções , MicroesferasRESUMO
Carbodiimides are important crosslinkers in organic synthesis and are used in the isocyanate industry as modifier additives. Therefore, the understanding of their formation is of high importance. In this work, we present a theoretical B3LYP/6-31G(d) and SMD solvent model and experimental investigation of the formation of diphenylcarbodiimide (CDI) from phenyl isocyanate using a phosphorus-based catalyst (MPPO) in ortho-dichlorobenzene (ODCB) solvent. Kinetic experiments were based on the volumetric quantitation of CO2 evolved, at different temperatures between 40 and 80 °C. Based on DFT calculations, we managed to construct a more detailed reaction mechanism compared to previous studies which is supported by experimental results. DFT calculations revealed that the mechanism is composed of two main parts, and the rate determining step of the first part, controlling the CO2 formation, is the first transition state with a 52.9 kJ mol-1 enthalpy barrier. The experimental activation energy was obtained from the Arrhenius plot (ln k vs. 1/T) using the observed second-order kinetics, and the obtained 55.8 ± 2.1 kJ mol-1 was in excellent agreement with the computational one, validating the complete mechanism, giving a better understanding of carbodiimide production from isocyanates.
Assuntos
Carbodi-Imidas , Isocianatos , Carbodi-Imidas/química , Isocianatos/química , Cinética , Termodinâmica , Catálise , Dióxido de Carbono/química , Solventes/química , TemperaturaRESUMO
BACKGROUND: This study investigated the effect of carbodiimide (EDC) combined with Clearfil SE self-etch adhesive on the shear bond strength (SBS), crosslinking degree, denaturation temperature, and enzyme activity of dentin in vitro. MATERIALS AND METHODS: Collected human sound third molars were randomly divided into different groups with or without EDC treatment (0.01-1 M). The specimens (n = 16)were stored for 24 h (immediate) or 12 months (aging) before testing the SBS. Fine dentin powder was obtained and treated with the same solutions. Then the crosslinking degree, denaturation temperature (Td), and enzyme activity were tested. Statistical analysis was performed using a one-way analysis of variance (ANOVA) to compare the differences of data between groups (α = 0.05). RESULTS: There was a significant drop in immediate SBS and more adhesive fracture of 1.0 M EDC group, while there were no significant differences among the other groups. SEM showed a homogeneous interface under all treatments. After 12 months of aging, the SBS significantly decreased. Less decreases of SBS in the 0.3 and 0.5 M groups were found. Due to thermal and enzymatical properties consideration, the 0.3 and 0.5 M treatments also showed higher cross-link degree and Td with lower enzyme activity. CONCLUSION: 0.3 and 0.5 M EDC may be favorable for delaying the aging of self-etch bond strength for 12 months. But it is still needed thoroughly study.
Assuntos
Carbodi-Imidas , Cimentos de Resina , Resistência ao Cisalhamento , Humanos , Carbodi-Imidas/química , Cimentos de Resina/química , Teste de Materiais , Dentina , Microscopia Eletrônica de Varredura , Adesivos Dentinários/química , Análise do Estresse Dentário , Reagentes de Ligações Cruzadas/química , Colagem Dentária/métodos , Técnicas In Vitro , Condicionamento Ácido do Dente/métodos , Dente Serotino , Temperatura , Fatores de Tempo , Propriedades de SuperfícieRESUMO
The problem of antimicrobial resistance is becoming a daunting challenge for human society and healthcare systems around the world. Hence, there is a constant need to develop new antibiotics to fight resistant bacteria, among other important social and economic measures. In this regard, murepavadin is a cyclic antibacterial peptide in development. The synthesis of murepavadin was undertaken in order to optimize the preparative protocol and scale-up, in particular, the use of new activation reagents. In our hands, classical approaches using carbodiimide/hydroxybenzotriazole rendered low yields. The use of novel carbodiimide and reagents based on OxymaPure® and Oxy-B is discussed together with the proper use of chromatographic conditions for the adequate characterization of peptide crudes. Higher yields and purities were obtained. Finally, the antimicrobial activity of different synthetic batches was tested in three Pseudomonas aeruginosa strains, including highly resistant ones. All murepavadin batches yielded the same highly active MIC values and proved that the chiral integrity of the molecule was preserved throughout the whole synthetic procedure.
Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/efeitos dos fármacos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/síntese química , Peptídeos Antimicrobianos/farmacologia , Carbodi-Imidas/química , HumanosRESUMO
The process of crosslinking improves the physicochemical properties of biopolymer-based composites, making them valuable for biomedical applications. EDC/NHS-crosslinked collagen materials have a significant potential for tissue engineering applications, due to their enhanced properties and biocompatibility. Chemical crosslinking of samples can be carried out in several ways, which is crucial and has a direct effect on the final properties of the obtained material. In this study, the effect of crosslinking conditions on the properties of collagen films using EDC and NHS was investigated. Studies included FTIR spectroscopy, AFM, swelling and degradation tests, mechanical testing and contact angle measurements. Evaluation of prepared collagen films indicated that both crosslinking agents and crosslinking conditions influenced film properties. Notable alternations were observed in the infrared spectrum of the sample, to which EDC was added directly to the fish collagen solution. The same sample indicated the lowest Young modulus, tensile strength and breaking force parameters and the highest elongation at break. All samples reached the maximum swelling degree two hours after immersion in PBS solution; however, the immersion-crosslinked samples exhibited a significantly lower degree of swelling and were highly durable. The highest roughness was observed for the collagen film crosslinked with EDC, whereas the lowest was observed for the specimen crosslinked with EDC with NHS addition. The crosslinking agents increased the surface roughness of the collagen film, except for the sample modified with the addition of EDC and NHS mixture. All films were characterized by hydrophilic character. The films' modification resulted in a decrease in their hydrophilicity and wettability. Our research allows for a comparison of proposed EDC/NHS crosslinking conditions and their influence on the physicochemical properties of fish collagen thin films. EDC and NHS are promising crosslinking agents for the modification of fish collagen used in biomedical applications.
Assuntos
Materiais Biocompatíveis , Colágeno , Reagentes de Ligações Cruzadas , Peixes , Animais , Reagentes de Ligações Cruzadas/química , Colágeno/química , Materiais Biocompatíveis/química , Resistência à Tração , Engenharia Tecidual/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Teste de Materiais , Carbodi-Imidas/químicaRESUMO
Crosslinking is usually required to improve the mechanical properties and stability of collagen-based scaffolds. Introducing exogenous crosslinks into collagen may however affect the collagen structure. Since the architecture of collagen is tied to its functionality, it is important to study the effect of crosslinking and to select a crosslinking method that preserves both the collagen structure and mechanical properties. The objective of this study is to compare the effect of various crosslinking methods on the structure and mechanical properties of bioartificial tendon-like materials (collagen multifilament bundles) fabricated by contact drawing. We examine both physical (ultraviolet light, UVC) and chemical (genipin, carbodiimide (EDC), and glutaraldehyde) crosslinking methods. The presence of collagen and the formation of well-ordered collagen structures are confirmed by attenuated total reflectance Fourier-transform infrared spectromicroscopy and wide-angle X-ray scattering for all crosslinking methods. The morphology of the collagen multifilament bundles is similar across crosslinking methods. Swelling of the multifilament bundles is dramatically reduced following crosslinking and varies by crosslinking method, with genipin- and carbodiimide-crosslinked specimens swelling the least. Ultimate tensile strength (UTS) and Young's modulus significantly improve for all crosslinked specimens compared to non-crosslinked specimens. Glutaraldehyde crosslinked collagen multifilament bundles display the highest UTS values ranging from 33.82±0.0â MPa to 45.59±0.76â MPa.
Assuntos
Colágeno , Reagentes de Ligações Cruzadas , Reagentes de Ligações Cruzadas/química , Colágeno/química , Glutaral/química , Raios Ultravioleta , Carbodi-Imidas/química , Iridoides/química , Animais , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
A key to understanding the roles of RNA in regulating gene expression is knowing their structures in vivo. One way to obtain this information is through probing the structures of RNA with chemicals. To probe RNA structure directly in cells, membrane-permeable reagents that modify the Watson-Crick (WC) face of unpaired nucleotides can be used. Although dimethyl sulfate (DMS) has led to substantial insight into RNA structure, it has limited nucleotide specificity in vivo, with WC face reactivity only at adenine (A) and cytosine (C) at neutral pH. The reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was recently shown to modify the WC face of guanine (G) and uracil (U). Although useful at lower concentrations in experiments that measure chemical modifications by reverse transcription (RT) stops, at higher concentrations necessary for detection by mutational profiling (MaP), EDC treatment leads to degradation of RNA. Here, we demonstrate EDC-stimulated degradation of RNA in Gram-negative and Gram-positive bacteria. In an attempt to overcome these limitations, we developed a new carbodiimide reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide (ETC), which we show specifically modifies unpaired Gs and Us in vivo without substantial degradation of RNA. We establish ETC as a probe for MaP and optimize the RT conditions and computational analysis in Escherichia coli Importantly, we demonstrate the utility of ETC as a probe for improving RNA structure prediction both alone and with DMS.
Assuntos
Guanina , Conformação de Ácido Nucleico , Ésteres do Ácido Sulfúrico , Uracila , Ésteres do Ácido Sulfúrico/química , Uracila/química , Uracila/análogos & derivados , Uracila/metabolismo , Guanina/química , Guanina/metabolismo , RNA/química , RNA/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Carbodi-Imidas/química , RNA Bacteriano/química , RNA Bacteriano/genética , Estabilidade de RNA , Indicadores e Reagentes/químicaRESUMO
Particle-based systems have become a state-of-the-art method for in vitro expanding cytotoxic T cells by tailoring their surface with activating molecules. However, commonly used methods utilize facile carbodiimide chemistry leading to uncontrolled orientation of the immobilized antibodies on the particle surface that can lead to poor binding to target cells. To address this, selective coupling strategies utilizing regioselective chemical groups such as disulfide bridges offer a simple approach. In this work we present a set of methods to investigate the effect of polymeric nanoparticles, conjugated with either regioselective- or randomly-immobilized antiCD3 and antiCD28 antibodies, on the activation potential, expansion and expression of activation markers in T cells. We show that nanoparticles with well-oriented monovalent antibodies conjugated via maleimide require fewer ligands on the surface to efficiently expand T cells compared to bivalent antibodies randomly-immobilized via carbodiimide conjugation. Analysis of the T cell expression markers reveal that the T cell phenotype can be fine-tuned by adjusting the surface density of well-oriented antibodies, while randomly immobilized antibodies showed no differences despite their ligand density. Both conjugation techniques induced cytotoxic T cells, evidenced by analyzing their Granzyme B secretion. Furthermore, antibody orientation affects the immunological synapse and T cell activation by changing the calcium influx profile upon activation. Nanoparticles with well-oriented antibodies showed lower calcium influx compared to their bivalent randomly-immobilized counterparts. These results highlight the importance of controlling the antibody density and orientation on the nanoparticle surface via controlled coupling chemistries, helping to develop improved particle-based expansion protocols to enhance T cell therapies.
Assuntos
Anticorpos Imobilizados , Nanopartículas , Humanos , Cálcio , Anticorpos , Linfócitos T CD8-Positivos , Complexo CD3 , Nanopartículas/química , Carbodi-ImidasRESUMO
The utilization of contrast agents in magnetic resonance imaging (MRI) has become increasingly important in clinical diagnosis. However, the low diagnostic specificity of this technique is a limiting factor for the early detection of tumors. To develop a new contrast agent with a specific target for early stage tumors, we present the synthesis and characterization of a nanocontrast composed of gold nanoparticles (AuNPs), gadopentetic acid (Gd-DTPA), and epidermal growth factor (EGF). Carbodiimide-based chemistry was utilized to modify Gd-DTPA for functionalization with AuNPs. This resulted in the formation of the Au@Gd-EGF nanocontrast. The relaxation rate (1/T1) of the nanocontrast was analyzed using MRI, and cytotoxicity was determined based on cell viability and mitochondrial activity in a human breast adenocarcinoma cell line. Fourier-transform infrared spectroscopy analysis confirmed the effectiveness of carbodiimide in the formation of the Gd-DTPA-cysteamine complex in the presence of bands at 930, 1042, 1232, 1588, and 1716 cm-1. The complexes exhibited good interactions with the AuNPs. However, the signal intensity of the Au@Gd-EGF nanocontrast was lower than that of the commercial contrast agent because the r1/r2 relaxivities of the Gd-DTPA-based contrast agents were lower than those of the gadoversetamide-based molecules. The Au@Gd-EGF nanocontrast agent exhibited good biocompatibility, low cytotoxicity, and high signal intensity in MRI with active targeted delivery, suggesting significant potential for future applications in the early diagnosis of tumors.
Assuntos
Nanopartículas Metálicas , Neoplasias , Humanos , Meios de Contraste , Gadolínio DTPA/química , Ouro/química , Fator de Crescimento Epidérmico , Gadolínio/química , Nanopartículas Metálicas/química , Imageamento por Ressonância Magnética/métodos , Carbodi-ImidasRESUMO
A pH-responsive amphiphilic chitosan derivative, N-lauric-O-carboxymethyl chitosan (LA-CMCh), is synthesized. Its molecular structures are characterized by FTIR, 1H NMR, and XRD methods. The influencing factors are investigated, including the amount of lauric acid (LA), carboxymethyl chitosan (CMCh), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS), and their molar ratio, reaction time, and reaction temperature on the substitution. The degrees of substitution (DS) of the lauric groups on the -NH2 groups are calculated based on the integrated data of 1H NMR spectra. The optimum reaction condition is obtained as a reaction time of 6 h, a reaction temperature of 80 °C, and a molar ratio of lauric acid to O-carboxymethyl chitosan to N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide of 1:3:4.5:4.5, respectively. The crystallinity and initial decomposition temperature of LA-CMCh decrease, but the maximum decomposition temperature increases. The crystallinity is reduced due to the introduction of LA and the degree of hydrogen bonding among LA-CMCh molecules. LA-CMCh could self-aggregate into particles, which size and critical aggregation concentration depend on the degree of substitution and medium pH. LA-CMCh aggregates could load curcumin up to 21.70 %, and continuously release curcumin for >200 min. LA-CMCh shows nontoxicity to fibroblast HFF-1 cells and good antibacterial activity against S. aureus and E. coli, indicating that it could be used as an oil-soluble-drug carrier.
Assuntos
Carbodi-Imidas , Quitosana , Curcumina , Metilaminas , Succinimidas , Quitosana/química , Curcumina/farmacologia , Escherichia coli , Staphylococcus aureus , Concentração de Íons de HidrogênioRESUMO
ß-Lactum antibiotics are broad class of antibiotics which kills bacteria by inhibiting the formation of peptidoglycan that constitutes the bacterial cell wall. The resistance that develops in bacteria for antibiotics led the scientific world to think about the future aspects for modifying the way through which antibiotics are acted on the bacteria and become lethal for them. In this consequence, the potential of latest marketed antibiotics e.g. Amoxiciline (I), ceftazidim (II) have been evaluated after being conjugated with quantum dots. The surface of quantum dots has been conjugated with antibiotics by carbodiimide coupling with the help of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as conjugating agent between antibiotic and functionalized quantum dots. The antibacterial properties of QD-conjugated antibiotics have been determined by disc diffusion assay. The potency of QD-conjugated antibiotics has been estimated by determining their MIC50 for the selected strain of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. Minimum inhibitory concentration study, minimum bactericidal concentration and growth pattern analysis revealed that QD-antibiotic conjugates showed slightly more prospective than pure native antibiotics against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria.
Assuntos
Compostos de Cádmio , Pontos Quânticos , Antibacterianos/farmacologia , Compostos de Cádmio/farmacologia , Estudos Prospectivos , Telúrio , Bactérias , Escherichia coli , Carbodi-Imidas , Testes de Sensibilidade MicrobianaRESUMO
In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between 17α-Methyltestosterone-3-Carboxymethyloxime and Horseradish peroxidase label through N-hydroxysuccinimide mediated carbodiimide reaction method. The immunogen 17α-Methyltestosterone-3-Carboxymethyloxime-Bovine serum albumin used to generate the antibody was also prepared by the N-hydroxysuccinimide mediated carbodiimide reaction without using any spacer. We have studied the impact of bridge/aromatic spacers on functional parameters i.e. sensitivity, affinity and ED50 of the bridge heterologous assay and compared it with homologous assay. The five combinations of bridge heterologous assay using 17α-Methyl testosterone-3-CMO-BSA antiserum and 17α-MT-3-CMO-4,4'-Diaminodiphenyl sulphide-HRP, 17α MT-3-CMO-4,4'-Oxydianiline-HRP, 17α-MT-3-CMO-Benzidine-HRP, 17α- MT-3-CMO-p-Phenylenediamine-HRP and 17α-MT-3-CMO-Dapson-HRP enzyme conjugates were evaluated. Out of these five combinations, the combination 17α-MT-3-CMO-BSA with 17α-MT-3-CMO-Benzidine-HRP showed the best results. Sensitivity, affinity and ED50 were improved and found to be 0.02 ng/mL, 0.086 × 10-8 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED50 were 0.11 ng/mL, 0.02 × 10-8 L/mol and 5.78 ng/mL respectively. The cross-reactivity for this bridge heterologous assay combination was seen with only 4 steroids (6-hydrotestosterone- 6%, Testosterone-5.14%, Danazol-0.9% and Nandrolone-0.85%) instead of eight steroids (6-hydrotestosterone-43.75%, Testosterone-38.3%, Danazol-25.14%, Androstenediol-19.16%, Nandrolone-19%, Metandienone-5%, Androstenedione-3.52%, and 17α dimethyltestosterone-2%) as in homologous assay out of 59 structurally related steroids. Thus, the results of this study conclude that the incorporation of aromatic spacer (bridge) in enzyme conjugate has a crucial role in improving sensitivity, specificity, ED50 and affinity of the developed assay. The assay was then studied for parameters such as recovery (97.4%-108.6%), precision (Inter and Intra-assay coefficient of variation <10%), correlation coefficient (R2 = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.
Assuntos
Metiltestosterona , Nandrolona , Animais , Ratos , Danazol , Ensaio de Imunoadsorção Enzimática/métodos , Antígenos , Esteroides , Testosterona , Benzidinas , Carbodi-ImidasRESUMO
Chitosan (Ch) and different Ch derivatives have been applied in tissue engineering (TE) because of their biocompatibility, favored mechanical properties, and cost-effectiveness. Most of them, however, lack cell adhesive properties that are crucial for TE. In this study, we aimed to design an S-protected thiolated Ch derivative exhibiting high cell adhesive properties serving as a scaffold for TE. 3-((2-Acetamido-3-methoxy-3-oxopropyl)dithio) propanoic acid was covalently attached to Ch via a carbodiimide-mediated reaction. Low-, medium-, and high-modified Chs (Ch-SS-1, Ch-SS-2, and Ch-SS-3) with 54, 107 and 140 µmol of ligand per gram of polymer, respectively, were tested. In parallel, three thiolated Chs, namely Ch-SH-1, Ch-SH-2, and Ch-SH-3, were prepared by conjugating N-acetyl cysteine to Ch at the same degree of modification to compare the effectiveness of disulfide versus thiol modification on cell adhesion. Ch-SS-1 showed better cell adhesion capability than Ch-SS-2 and Ch-SS-3. This can be explained by the more lipophilic surfaces of Ch-SS as a higher modification was made. Although Ch-SH-1, Ch-SH-2, and Ch-SH-3 were shown to be good substrates for cell adhesion, growth, and proliferation, Ch-SS polymers were superior to Ch-SH polymers in the formation of 3D cell cultures. Cryogels structured by Ch-SS-1 (SSg) resulted in homogeneous scaffolds with tunable pore size and mechanical properties by changing the mass ratio between Ch-SS-1 and heparin used as a cross-linker. SSg scaffolds possessing interconnected microporous structures showed good cell migration, adhesion, and proliferation. Therefore, Ch-SS can be used to construct tunable cryogel scaffolds that are suitable for 3D cell culture and TE.
Assuntos
Quitosana , Materiais Biocompatíveis/farmacologia , Engenharia Tecidual , Acetilcisteína , Carbodi-Imidas , CriogéisRESUMO
Tendon tears are common and healing often occurs incompletely and by fibrosis. Tissue engineering seeks to improve repair, and one approach under investigation uses cell-seeded scaffolds containing biomimetic factors. Retention of biomimetic factors on the scaffolds is likely critical to maximize their benefit, while minimizing the risk of adverse effects, and without losing the beneficial effects of the biomimetic factors. The aim of the current study was to evaluate cross-linking methods to enhance the retention of tendon-derived matrix (TDM) on electrospun poly(ε-caprolactone) (PCL) scaffolds. We tested the effects of ultraviolet (UV) or carbodiimide (EDC:NHS:COOH) crosslinking methods to better retain TDM to the scaffolds and stimulate tendon-like matrix synthesis. Initially, we tested various crosslinking configurations of carbodiimide (2.5:1:1, 5:2:1, and 10:4:1 EDC:NHS:COOH ratios) and UV (30 s 1 J/cm2 , 60 s 1 J/cm2 , and 60 s 4 J/cm2 ) on PCL films compared to un-crosslinked TDM. We found that no crosslinking tested retained more TDM than coating alone (Kruskal-Wallis: p > .05), but that human adipose stem cells (hASCs) spread most on the 60 s 1 J/cm2 UV- and 2.5:1:1 EDC-crosslinked films (Kruskal-Wallis: p < .05). Next, we compared the effects of 60 s 1 J/cm2 UV- and 2.5:1:1 EDC-crosslinked to TDM-coated and untreated PCL scaffolds on hASC-induced tendon-like differentiation. UV-crosslinked scaffolds had greater modulus and stiffness than PCL or TDM scaffolds, and hASCs spread more on UV-crosslinked scaffolds (ANOVA: p < .05). Fourier transform infrared spectra revealed that UV- or EDC-crosslinking TDM did not affect the peaks at wavenumbers characteristic of tendon. Crosslinking TDM to electrospun scaffolds improves tendon-like matrix synthesis, providing a viable strategy for improving retention of TDM on electrospun PCL scaffolds.
Assuntos
Colágeno , Engenharia Tecidual , Humanos , Engenharia Tecidual/métodos , Adipócitos , Tendões , Carbodi-Imidas , Alicerces Teciduais , PoliésteresRESUMO
RNA structure can be essential for its cellular function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structure probing is an indirect method to asess the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. Dimethyl sulfate (DMS) is a well-established compound that reports on base pairing context of adenine (A) and cytidine (C) in-vitro and in-vivo, but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in plant, bacteria, and human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyze the effectiveness of guanine modification by the glyoxal family in Saccharomyces cerevisiae and Candida albicans. We show that within glyoxal family of compounds, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. We also explore the effectiveness of uracil modification by Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluenesulfonate (CMCT) in vivo and demonstrate that uracils can be modified by CMCT in S. cerevisiae in vivo. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil nucleotides in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.
Assuntos
RNA , Saccharomyces cerevisiae , Humanos , RNA/genética , Saccharomyces cerevisiae/genética , Guanina/química , Nucleotídeos de Uracila , Conformação de Ácido Nucleico , Glioxal , Carbodi-Imidas , UracilaRESUMO
The objective of this study is to evaluate the effect of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and chitosan (CHI) on the adhesive interface of resin cements to root dentine. Forty-five upper canines were sectioned, endodontically treated, prepared and divided into three groups according to dentine treatment (distilled water-DW, CHI 0.2% and EDC 0.5) and in three subgroups according to resin cement: RelyX ARC, Panavia F 2.0 or RelyX U200. Slices were obtained, with five slices of each third submitted to the analysis of the adaptation of the adhesive interface through scores and the perimeter with gaps in confocal laser scanning microscopy and one slice of each third later evaluated qualitatively in scanning electron microscopy. The results were analyzed using with Kruskal-Wallis and Spearman correlation tests. There was no difference in adaptation for the different resin cements (p = .438). EDC presented better adaptation when compared to the groups treated with DW and CHI (p < .001), while the CHI and DW presented similar adaptation values (p = .365). No difference was observed in the perimeter referring to the gap areas for the different resin cements (p = .510). EDC showed a lower percentage of perimeters with gaps when compared to CHI (p < .001), with the percentage of perimeter with gaps of teeth treated with CHI being lower than DW (p < .001). A positive correlation coefficient equal to 0.763 was obtained between the perimeter with gaps and the adaptation data of the adhesive interface (p < .001). EDC resulted in better adaptation of the adhesive interface and a lower percentage of perimeters with gaps compared to chitosan.
Assuntos
Quitosana , Colagem Dentária , Técnica para Retentor Intrarradicular , Cimentos de Resina , Cimentação/métodos , Carbodi-Imidas , Dentina , Teste de MateriaisRESUMO
A cascade protocol for the synthesis of aminotetrazoles have been developed by treating isonitriles, N,N-dibromoarylsulfonamides, and sodium azides in the presence of K2CO3. This metal-free process proceeds via an isolable carbodiimide intermediate at room temperature, which could further react with sodium azide and subsequently cyclizes intermolecularly to provide 5-aminotetrazoles within a short reaction time. The present protocol's remarkable achievements are the wide substrate scope, good to excellent yields, and good functional group tolerance.
Assuntos
Azidas , Carbodi-Imidas , TetrazóisRESUMO
BACKGROUND: To investigate the effect of 0.3 M 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) aqueous solution pretreatment on push-out bond strength (PBS) and matrix-metalloproteinases (MMPs) activity within radicular dentin when different post cementation strategies were employed. METHODS: One hundred and twenty monoradicular human teeth were endodontically treated and randomly divided into six groups, depending on the cementation strategy and root dentin pretreatment (n = 20): EAR: cementation with an etch-and-rinse adhesive (LuxaBond Total Etch, DMG) and resin cement (LuxaCore Z Dual, DMG); EAR/EDC: 1 min EDC pretreatment after etching + EAR; SE: cementation with a self-etch primer (Multilink Primer, Ivoclar Vivadent) and corresponding cement (Multilink Automix, Ivoclar Vivadent); SE/EDC: self-etch primer + EDC pretreatment + SE; SA: cementation with a universal self-adhesive cement (RelyX Universal, 3 M); SA/EDC: EDC pretreatment + SA. Slices were submitted to PBS test and interfacial nanoleakage evaluation 24 h after cementation or after thermocycling (40.000 cycles, 5-55 °C). To investigate the effect of EDC on MMPs activity, 4 additional first maxillary premolars per group were processed for in situ zymography analysis. Multivariate ANOVA and post hoc Tukey tests were used to analyze PBS values. The data from in situ zymography were analyzed with Kruskal-Wallis test and Dunn's pairwise multiple comparison procedures (α = 0.05). RESULTS: The variables "EDC pretreatment", "root region" and "thermocycling" significantly influenced PBS (p < 0.05), while the variable "cementation strategy" had no influence (p > 0.05). Thermocycling significantly reduced PBS in SE and SA groups (p < 0.05). EDC was effective in preserving PBS after artificial aging. EDC pretreatment significantly reduced enzymatic activity at baseline in EAR and SE groups, and in SA group after thermocycling (p < 0.05). CONCLUSIONS: The use of EDC prevents the reduction of bond-strength values after artificial aging and silences endogenous enzymatic activity within radicular dentin when different cementation strategies were employed.
Assuntos
Colagem Dentária , Humanos , Carbodi-Imidas/química , Dentina , Cimentos de Resina/uso terapêutico , Cimentos de Resina/química , Metaloproteinases da Matriz , Teste de MateriaisRESUMO
PURPOSE: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) can increase dentin bonding longevity. This study aimed to evaluate the effect of final irrigation of the root canal with EDC on the bond strength (BS) longevity of an epoxy resin-based root-canal sealer. MATERIALS AND METHODS: Twenty maxillary canines were sectioned and standardized for root length at 17 mm. Roots were instrumented and distributed into 2 groups according to the final irrigation protocol: EDTA 17%+NaOCl 2.5% (C) and EDTA 17%+NaOCl 2.5%+EDC 0.5M (EDC). The canals were dried and filled with AH Plus (Dentsply Sirona). Three slices were obtained per third, and the first slice from each third was used for the immediate push-out test (i) followed by analysis of the failure pattern (n = 10); the second slice from each third was used for the push-out test after 6-month aging (A) followed by analysis of the failure pattern (n = 10); the third slice from each third was used to examine the adhesive interface under confocal laser scanning microscopy (CLSM) (n = 10). Data were analyzed with ANOVA, Fisher's exact and Kruskal-Wallis tests. RESULTS: Higher BSs were found for EDC-A (5.6 ± 1.9) than for EDC-I (3.3 ± 0.7), C-i (2.5 ± 1.0) and C-i (2.6 ± 1.0) (p = 0.0001), while C-A values were in some cases similar to C-i and in others similar to EDC-i. No statistically significant difference was observed between the thirds (p > 0.05), except for EDC-i, which showed lower BS for the cervical (2.79 ± 0.46) compared to the apical third (3.8 ± 0.5), while the middle third in some cases had values similar to those of the apical and in others to the cervical third (3.2 ± 0.7) (p = 0.032). More mixed adhesive failures were found in the cervical third, and more adhesive failures to the sealer occurred in the middle and apical thirds (p = 0.014). A significant difference was observed between treatments in terms of adaptation of the adhesive interface, with a higher percentage of good adaptation using EDC (66.7%) than using C (40%), and a lower percentage of poor adaptation with EDC (10%) compared to C (20%) (p < 0.05). CONCLUSION: Root canal irrigation with EDC increased the longevity of the adhesive interface of an epoxy resin-based root-canal sealer.