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Gelatin and chitosan nanoparticles have been widely used in pharmaceutical, biomedical, and nanofood applications due to their high biocompatibility and biodegradability. This study proposed a highly efficient synthesis method for type B gelatin and low-molecular-weight (LMW) chitosan nanoparticles. Gelatin nanoparticles (GNPs) were synthesized by the double desolvation method and the chitosan nanoparticles (CNPs) by the ionic gelation method. The sizes of the obtained CNPs and GNPs (373 ± 71 nm and 244 ± 67 nm, respectively) and zeta potential (+36.60 ± 3.25 mV and -13.42 ± 1.16 mV, respectively) were determined via dynamic light scattering. Morphology and size were verified utilizing SEM and TEM images. Finally, their biocompatibility was tested to assure their potential applicability as bioactive molecule carriers and cell-penetrating agents.
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In this work, previously synthesized and characterized core-shell silica nanoparticles (FCSNP) functionalized with immobilized molecular bait, Cibacron blue, and a porous polymeric bis-acrylamide shell were incubated with pooled urine samples from adult women or men with normal weight, overweight or obesity for the isolation of potential biomarkers. A total of 30 individuals (15 woman and 15 men) were included. FCSNP allowed the capture of a variety of low molecular weight (LMW) proteins as evidenced by mass spectrometry (MS) and the exclusion of high molecular weight (HMW) proteins (>34 kDa) as demonstrated by SDS-PAGE and 2D SDS-PAGE. A total of 36 proteins were successfully identified by MS and homology database searching against the Homo sapiens subset of the Swiss-Prot database. Identified proteins were grouped into different clusters according to their abundance patterns. Four proteins were found only in women and five only in men, whereas 27 proteins were in urine from both genders with different abundance patterns. Based on these results, this new approach represents an alternative tool for isolation and identification of urinary biomarkers.
Assuntos
Obesidade/urina , Proteinúria/urina , Proteômica , Adulto , Biomarcadores/urina , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: During periodontitis, chronic inflammation triggers soft tissue breakdown, and hyaluronan is degraded into fragments of low molecular weight (LMW-HA). This investigation aimed to elucidate whether LMW-HA fragments with immunogenic potential on T lymphocytes remain in periodontal tissues after periodontal treatment. MATERIALS AND METHODS: GCF samples were obtained from 15 periodontitis-affected patients and the LMW-HA, RANKL, and OPG levels were analyzed before and after 6 months of periodontal treatment by ELISA. Eight healthy individuals were analyzed as controls. Besides, human T lymphocytes were purified, exposed to infected dendritic cells, and pulsed with LMW-HA. Non-treated T lymphocytes were used as control. The expression levels of the transcription factors and cytokines that determine the Th1, Th17, and Th22 lymphocyte differentiation and function were analyzed by RT-qPCR. Similarly, the expression levels of RANKL and CD44 were analyzed. RESULTS: In the GCF samples of periodontitis-affected patients, higher levels of LMW-HA were detected when compared with those of healthy individuals (52.1 ± 15.4 vs. 21.4 ± 12.2, p < 0.001), and these increased levels did not decrease after periodontal therapy (52.1 ± 15.4 vs. 45.7 ± 15.9, p = 0.158). Similarly, the RANKL levels and RANKL/OPG ratios did not change after periodontal therapy. Furthermore, in human T lymphocytes, LMW-HA induced higher expression levels of the Th1, Th17, and Th22-related transcription factors and cytokines, as well as CD44 and RANKL, as compared with non-treated cells. CONCLUSIONS: In some patients, increased levels of LMW-HA persist in periodontal tissues after conventional periodontal therapy, and these remaining LMW-HA fragments with immunostimulatory potential could induce the polarization of a pathologic Th1/Th17/Th22-pattern of immune response on T lymphocytes. CLINICAL RELEVANCE: The persistence of increased levels of LMW-HA in periodontal tissues after periodontal therapy could favor the recurrence of the disease and further breakdown of periodontal supporting tissues.
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Ácido Hialurônico , Periodontite , Citocinas , Humanos , Peso Molecular , Periodontite/tratamento farmacológico , Ligante RANK , Células Th17RESUMO
Cinnamic acid derivatives, particularly α-cyano-4-hydroxycinnamic acid (E-α-cyano-4-hydroxycinnamic acid or (E)-2-cyano-3-(4-hydroxyphenyl)prop-2-enoate; CHCA), have been extensively used especially for protein and peptide analysis. Together with the introduction of ionic liquid MALDI matrix (ILM) started the study of applications of IL prepared with CHCA and a counter organic base (ie, aliphatic amines) in which CHCA moiety is the chromophore responsible of UV-laser absorption. Despite the extensive studies of norharmane (9H-pyrido[3,4-b]indole; nHo) applications as matrix and its peculiar basic properties in the ground and electronic excited state, nHo containing ILM was never tested in MALDI-MS experiments. This pyrido-indole compound was introduced as MALDI matrix 22 years ago for different applications including low molecular weight (LMW) carbohydrates (neutral, acidic, and basic carbohydrates). These facts encouraged us to use it as a base, for the first time, for ILM preparation. As a rational design of new IL MALDI matrices, E-α-cyanocinnamic acid.nHo and E-cinnamic acid.nHo were prepared and their properties as matrices studied. Their performance was compared with that of (a) the corresponding IL prepared with butylamine as basic component, (b) the corresponding crystalline E-α-cyanocinnamic and E-cinnamic acid, and (c) the classical crystalline matrices (2,5-dihydroxybenzoic acid, DHB; nHo) used in the analysis of neutral/sulfated carbohydrates. The IL DHB.nHo was tested, too. Herein, we demonstrate the outstanding performance for the IL CHCA.nHo for LMW carbohydrate in positive and negative ion mode (linear and reflectron modes). Sulfated oligosaccharides were detected in negative ion mode, and although the dissociation of sulfate groups was not completely suppressed the relative intensity (RI) of [M - Na]- peak was quite high. Additionally, to better understand the quite different performance of each IL tested as matrix, the physical and morphological properties in solid state were studied (optical image; MS image).
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Carbolinas/química , Ácidos Cumáricos/química , Líquidos Iônicos/química , Oligossacarídeos/análise , Gentisatos/química , Íons , Limite de Detecção , Peso Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , EstereoisomerismoRESUMO
OBJECTIVES: Periodontitis is a chronic inflammatory disease characterized by tooth-supporting tissue destruction, which is elicited by the host's immune response triggered against periodonto-pathogen bacteria. During periodontal tissue destruction, extracellular matrix components are metabolized and fragmented. Some extracellular matrix component-derived fragments, such as low-molecular-weight hyaluronan (LMW-HA), have potent immunogenic potential, playing a role as damage-associated molecular patterns (DAMPs) during activation of immune cells. Dendritic cells (DCs) play a central role in the host's immune response displayed during periodontitis; thus, this study aimed to analyze whether LMW-HA has an immunostimulatory activity on DCs when stimulated with periodonto-pathogen bacteria. MATERIALS AND METHODS: LMW-HA-treated and non-treated DCs were stimulated with Aggregatibacter actinomycetemcomitans or Porphyromonas gingivalis and the mRNA expression for cytokines tumor necrosis factor-α (TNF-alpha), interleukin-1ß (IL-1B), interleukin-6 (IL-6), and interleukin-23 (IL-23A) was quantified by RT-qPCR. In addition, transcription factors interferon regulatory factor 4 (IRF4), interferon regulatory factor 8 (IRF8), neurogenic locus notch homolog protein 2 (NOTCH2), and basic leucine zipper ATF-like transcription factor 3 (BATF3), involved in DC activation, were analyzed. RESULTS: Higher expression levels of TNF-alpha, IL-1B, IL-6, and IL-23A were detected in LMW-HA-treated DCs after bacterial infection, as compared with non-treated DCs. When LMW-HA-treated DCs were infected with A. actinomycetemcomitans, higher levels of IRF4, NOTCH2, and BATF3 were detected compared with non-treated cells; whereas against P. gingivalis infection, increased levels of IRF4 and NOTCH2 were detected. CONCLUSION: LMW-HA plays an immunostimulatory role on the immune response triggered by DCs during infection with A. actinomycetemcomitans or P. gingivalis. CLINICAL RELEVANCE: Detection of extracellular matrix component-derived fragments produced during periodontal tissue destruction, such as LMW-HA, could explain at least partly unsuccessful periodontal treatment and the chronicity of the disease.
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Adjuvantes Imunológicos/farmacologia , Aggregatibacter actinomycetemcomitans , Células Dendríticas/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Porphyromonas gingivalis , Células Cultivadas , Citocinas/imunologia , Células Dendríticas/microbiologia , Matriz Extracelular , Humanos , Peso Molecular , PeriodontiteRESUMO
Exposition of neutrophils (polymorphonuclear neutrophils, PMNs) to bacterial products triggers exacerbated activation of these cells, increasing their harmful effects on host tissues. We evaluated the possibility of interfering with the classic immune innate responses of human PMNs exposed to bacterial endotoxin (lipopolysaccharide, LPS), and further stimulated with bacterial formyl peptide (N-formyl-methionine-leucine-phenylalanine, fMLP). We showed that the low- molecular-weight fucoidan (LMW-Fuc), a polysaccharide extracted from brown algae, attenuated the exacerbated activation induced by fMLP on LPS-primed PMNs, in vitro, impairing chemotaxis, NET formation, and the pro-survival and pro-oxidative effects. LMW-Fuc also inhibited the activation of canonical signaling pathways, AKT, bad, p47phox and MLC, activated by the exposition of PMN to bacterial products. The activation of PMN by sequential exposure to LPS and fMLP induced the release of L-selectin+ microparticles, which were able to trigger extracellular reactive oxygen species production by fresh PMNs and macrophages. Furthermore, we observed that LMW-Fuc inhibited microparticle release from activated PMN. In vivo experiments showed that circulating PMN-derived microparticles could be detected in mice exposed to bacterial products (LPS/fMLP), being downregulated in animals treated with LMW-Fuc. The data highlight the autocrine and paracrine role of pro-inflammatory microparticles derived from activated PMN and demonstrate the anti-inflammatory effects of LMW-Fuc on these cells.
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Micropartículas Derivadas de Células/metabolismo , Armadilhas Extracelulares/metabolismo , Selectina L/metabolismo , Neutrófilos/imunologia , Polissacarídeos/farmacologia , Animais , Sobrevivência Celular , Células Cultivadas , Quimiotaxia , Humanos , Imunidade Inata , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , N-Formilmetionina Leucil-Fenilalanina/imunologia , Ativação de Neutrófilo , Estresse Oxidativo , Phaeophyceae/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Banana is one of the most popular fruits in the world but has been substantially impaired by Panama disease in the last years. Fusarium oxysporum f. sp. cubense (Foc) is the causal agent and colonizes banana cultivars from many subgroups with different aggressiveness levels, often leading to plant death while compromising new crops in infested areas. This study has evaluated the ability of MALDI-MS protein and lipid fingerprinting to provide intraspecies classification of Foc isolates and to screen biomolecules related to host-pathogen relationship. The MS data, when inspected via partial least square discriminant analysis (PLS-DA), distinguished the isolates by aggressiveness as well as by specific location and host. Although both lipids and proteins show discriminating tendencies, these differences were more clearly perceived via the protein profiles. Considering that Cavendish cultivar is the more resistant option to endure Foc presence in the field, the lipids and proteins related to this subgroup might have an important role in pathogen adaptation. This study reports a new application of MALDI-MS for the analysis of a banana pathogen with intraspecies classification ability. Graphical abstract MALDI-MS classified Foc isolates by aggressiveness level on banana revealing the additional influence of location and host cultivar on the expression of lipids and proteins.
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Proteínas Fúngicas/química , Fusarium/química , Fusarium/classificação , Lipídeos/química , Mapeamento de Peptídeos , Doenças das Plantas/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Low molecular weight protein tyrosine phosphatases (LMW-PTP, EC 3.1.3.48) are a family of single-domain enzymes with molecular weight up to 18 kDa, expressed in different tissues and considered attractive pharmacological targets for cancer chemotherapy. Despite this, few LMW-PTP inhibitors have been described to date, and the structural information on LMW-PTP druggable binding sites is scarce. In this study, a small series of phosphonic acids were designed based on a new crystallographic structure of LMW-PTP complexed with benzylsulfonic acid, determined at 2.1Å. In silico docking was used as a tool to interpret the structural and enzyme kinetics data, as well as to design new analogs. From the synthesized series, two compounds were found to act as competitive inhibitors, with inhibition constants of 0.124 and 0.047 mM. We also report the 2.4Å structure of another complex in which LMW-PTP is bound to benzylphosphonic acid, and a structure of apo LMW-PTP determined at 2.3Å resolution. Although no appreciable conformation changes were observed, in the latter structures, amino acid residues from an expression tag were found bound to a hydrophobic region at the protein surface. This regions is neighbored by positively charged residues, adjacent to the active site pocket, suggesting that this region might be not a mere artefact of crystal contacts but an indication of a possible anchoring region for the natural substrate-which is a phosphorylated protein.
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Ácidos Fosforosos/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Cinética , Simulação de Acoplamento Molecular , Ácidos Fosforosos/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Ácidos Sulfônicos/química , Ácidos Sulfônicos/metabolismoRESUMO
Reactive oxygen species (ROS) modulate a variety of intracellular events, but their role in osteoblast adhesion and spreading remains unclear. ROS is a very-known physiological modulators of Protein Tyrosine Phosphatases activities, mainly to low molecular weight protein tyrosine phosphatase (LMW-PTP) activity. As this biological mechanism is not clear in osteoblast adhesion, we decided to investigate ROS levels and phosphorylations of FAK and Src, identifying these proteins as potential substrates to LMW-PTP activity. Our results showed that during osteoblast adhesion/spreading (30 min and 2 h of seeding) the intracellular ROS content (hydrogen peroxide) is finely regulated by an effective anti-oxidant system [catalase and Superoxide Dismutase (SOD) activities were evaluated]. During the first 30 min of adhesion, there was an increase in ROS production and a concomitant increase in focal adhesion kinase (FAK) activity after its phosphorylation at Tyrosine 397 (Y397 ). Moreover, after 2 h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW-PTP expression at 30 min or 2 h. In order to validate our hypothesis that LMW-PTP is able to control FAK activity by modulating its phosphorylation status, we decided to overexpress and silence LMW-PTP in this context. Our results showed that FAK phosphorylation at Y397 was increased and decreased in osteoblasts with silenced or overexpressed LMW-PTP, respectively. Together, these data show that ROS modulate FAK phosphorylation by an indirect way, suggesting that a LMW-PTP/FAK supra-molecular complex is involved in transient responses during osteoblast adhesion and spreading.
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Osteoblastos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Catalase/metabolismo , Adesão Celular , Linhagem Celular , Citometria de Fluxo , Quinase 1 de Adesão Focal/metabolismo , Peróxido de Hidrogênio/metabolismo , Immunoblotting , Cinética , Camundongos , Microscopia Confocal , Osteoblastos/citologia , Fosforilação , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Interferência de RNA , Superóxido Dismutase/metabolismo , Fatores de Tempo , Tirosina/metabolismoRESUMO
OBJECTIVE: To examine associations of urinary phthalate levels with blood pressure (BP) and serum triglyceride and lipoprotein levels in children. STUDY DESIGN: We performed a cross-sectional analysis of a subsample of US children aged 6-19 years who participated in the National Health and Nutrition Examination Survey between 2003 and 2008. We quantified exposure to 3 families of phthalates--low molecular weight, high molecular weight and di-2-ethylhexylphthalate (DEHP)--based on molar concentration of urinary metabolites. We assessed descriptive, bivariate, and multivariate associations with BP and lipid levels. RESULTS: Controlling for an array of sociodemographic and behavioral factors, as well as diet and body mass index, levels of metabolites of DEHP, a phthalate commonly found in processed foods, were associated with higher age-, sex-, and height-standardized BP. For each log unit (roughly 3-fold) increase in DEHP metabolites, a 0.041 SD unit increase in systolic BP z-score was identified (P = .047). Metabolites of low molecular weight phthalates commonly found in cosmetics and personal care products were not associated with BP. Phthalate metabolites were not associated with triglyceride levels, high-density lipoprotein level, or prehypertension. CONCLUSIONS: Dietary phthalate exposure is associated with higher systolic BP in children and adolescents. Further work is needed to confirm these associations, as well as to evaluate opportunities for intervention.