RESUMO
Photosynthesis, as the core of solar energy biotransformation, is driven by photosynthetic membrane protein complexes in plants and algae. Current methods for intracellular photosynthetic membrane protein complex analysis mostly require the separation of specific chloroplasts or the change of the intracellular environment, which causes the missing of real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membrane protein complexes in the chloroplasts of living Chlamydomonas reinhardtii (C. reinhardtii) cells under cultural conditions. Poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles were fabricated to deliver bis(succinimidyl)propargyl with a nitro compound (BSPNO) into the chloroplasts to crosslink photosynthetic membrane protein complexes. After the in vivo crosslinked protein complexes were extracted and digested, mass spectrometry was employed to detect lysine-specific crosslinked peptides for further elucidating the protein conformations and interactions. With this method, the weak interactions between extrinsic proteins in the luminal side (PsbL and PsbH) and the core subunits (CP47 and CP43) in photosynthetic protein complexes were directly captured in living cells. Additionally, the previously uncharacterized protein (Cre07.g335700) was bound to the light-harvesting proteins, which was related to the biosynthesis of light-harvesting antennae. These results indicated that in vivo analysis of photosynthetic protein complexes based on crosslinker nanocarriers was expected to not only figure out the difficulty in the study of photosynthetic protein complexes in living cells but also provide an approach to explore transient and weak interactions and the function of uncharacterized proteins.
Assuntos
Chlamydomonas reinhardtii , Complexo de Proteínas do Centro de Reação Fotossintética , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Membrana/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Complexos de Proteínas Captadores de Luz/metabolismo , CloroplastosRESUMO
The purpose of this study was to develop a chewing gum containing a novel antimicrobial peptide GH12 and evaluate its biocompatibility, antimicrobial activity, and caries-preventive effects in vivo and in vitro. GH12 chewing gum was developed using a conventional method and its extracts were prepared in artificial saliva. GH12 concentration in the extracts was determined by high-performance liquid chromatography; extracts were used for growth curve assay, time-kill assay, crystal violet staining assay, scanning electron microscopy, and Cell Counting Kit-8 assay. A rat caries model was established, and molars were treated topically with extracts for 5 weeks. Weight gain monitoring, hematoxylin-eosin staining, micro-computed tomography, and Keyes scoring were conducted. Significant inhibition of Streptococcus mutans growth and biofilm formation was observed. Extracts displayed low cytotoxicity against human gingival epithelial cells. No significant differences in weight gain or signs of harm to the mucosal tissues in any of the rats were observed. Keyes scores of caries lesions in the GH12 chewing gum group were lower than those of the negative control group. It was concluded that GH12 chewing gum showed good biocompatibility, antimicrobial activity, and caries-preventive effects, exhibiting great potential to prevent dental caries as an adjuvant to regular oral hygiene.
Assuntos
Anti-Infecciosos , Cárie Dentária , Animais , Anti-Infecciosos/farmacologia , Peptídeos Antimicrobianos , Goma de Mascar/análise , Cárie Dentária/prevenção & controle , Suscetibilidade à Cárie Dentária , Amarelo de Eosina-(YS)/farmacologia , Violeta Genciana/farmacologia , Hematoxilina/farmacologia , Humanos , Ratos , Saliva Artificial/farmacologia , Streptococcus mutans , Aumento de Peso , Microtomografia por Raio-XRESUMO
Due to the complex microecology and microenvironment of dental plaque, novel caries prevention strategies require modulating the microbial communities ecologically and reducing the cariogenic properties effectively. Antimicrobial peptide GH12 reduced the lactic acid production and exopolysaccharide (EPS) synthesis of a Streptococcus mutans biofilm and a three-species biofilm in vitro in previous studies. However, the anticaries effects and microecological effects of GH12 remained to be investigated in a complex biofilm model in vitro and an animal caries model in vivo In the present study, GH12 at 64 mg/liter showed the most effective inhibition of lactic acid production, EPS synthesis, pH decline, and biofilm integrity of human dental plaque-derived multispecies biofilms in vitro, and GH12 at 64 mg/liter was therefore chosen for use in subsequent in vitro and in vivo assays. When treated with 64-mg/liter GH12, the dental plaque-derived multispecies biofilms sampled from healthy volunteers maintained its microbial diversity and showed a microbial community structure similar to that of the control group. In the rat caries model with a caries-promoting diet, 64-mg/liter GH12 regulated the microbiota of dental plaque, in which the abundance of caries-associated bacteria was decreased and the abundance of commensal bacteria was increased. In addition, 64-mg/liter GH12 significantly reduced the caries scores of sulcal and smooth surface caries in all locations. In conclusion, GH12 inhibited the cariogenic properties of dental plaque without perturbing the dental plaque microbiota of healthy individuals and GH12 regulated the dysbiotic microbial ecology and arrested caries development under cariogenic conditions.IMPORTANCE The anticaries effects and microecological regulation effects of the antimicrobial peptide GH12 were evaluated systematically in vitro and in vivo GH12 inhibited the cariogenic virulence of dental plaque without overintervening in the microbial ecology of healthy individuals in vitro GH12 regulated the microbial ecology of dental plaque to a certain extent in vivo under cariogenic conditions, increased the proportion of commensal bacteria, and decreased the abundance of caries-associated bacteria. GH12 significantly suppressed the incidence and severity of dental caries in vivo This study thus describes an alternative antimicrobial therapy for dental caries.
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Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Cárie Dentária/prevenção & controle , Placa Dentária/microbiologia , Microbiota/efeitos dos fármacos , Adulto , Animais , Biofilmes/crescimento & desenvolvimento , Cárie Dentária/genética , Feminino , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Organismos Livres de Patógenos Específicos , Adulto JovemRESUMO
Malignant gliomas are the most common primary brain tumors and are associated with aggressive growth, high morbidity, and mortality. Aberrant mesenchymal-epithelial transition factor (MET) activation occurs in approximately 30% of glioma patients and correlates with poor prognosis, elevated invasion, and increased drug resistance. Therefore, MET has emerged as an attractive target for glioma therapy. In this study, we developed a novel nanoinhibitor by conjugating MET-targeting cMBP peptides on the G4 dendrimer. Compared to the binding affinity of the free peptide ( KD = 3.96 × 10-7 M), the binding affinity of the nanoinhibitor to MET increased 3 orders of magnitude to 1.32 × 10-10 M. This nanoinhibitor efficiently reduced the proliferation and invasion of human glioblastoma U87MG cells in vitro by blocking MET signaling with remarkably attenuated levels of phosphorylated MET ( pMET) and its downstream signaling proteins, such as pAKT and pERK1/2. Although no obvious therapeutic effect was observed after treatment with free cBMP peptide, in vivo T2-weighted magnetic resonance imaging (MRI) showed a significant delay in tumor growth after intravenous injection of the nanoinhibitor. The medium survival in mouse models was extended by 59%, which is similar to the effects of PF-04217903, a small molecule MET inhibitor currently in clinical trials. Immunoblotting studies of tumor homogenate verified that the nanoinhibitor restrained glioma growth by blocking MET downstream signaling. pMET and its downstream proteins pAKT and pERK1/2, which are involved in the survival and invasion of cancer cells, decreased in the nanoinhibitor-treated group by 44.2%, 62.2%, and 32.3%, respectively, compared with those in the control group. In summary, we developed a peptide-functionalized MET nanoinhibitor that showed extremely high binding affinity to MET and effectively inhibited glioma growth by blocking MET downstream signaling. To the best of our knowledge, this is the first report of therapeutic inhibition of glioma growth by blocking MET signaling with a novel nanoinhibitor. Compared to antibodies and chemical inhibitors in clinical trials, the nanoinhibitor blocks MET signaling and provides a new approach for the treatment of glioma with the advantages of high efficiency, affordability, and, most importantly, potentially reduced drug resistance.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Dendrímeros/uso terapêutico , Glioma/tratamento farmacológico , Nanoconjugados/uso terapêutico , Peptídeos/uso terapêutico , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dendrímeros/química , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Camundongos , Nanoconjugados/química , Nylons/química , Peptídeos/químicaRESUMO
Dental caries is a biofilm-mediated disease that occurs when acidogenic/aciduric bacteria obtain an ecological advantage over commensal species. In previous studies, the effects of the antimicrobial peptide GH12 on planktonic bacteria and monospecies biofilms were confirmed. The objectives of this study were to investigate the effects of GH12 on a cariogenic multispecies biofilm and to preliminarily explain the mechanism. In this biofilm model, Streptococcus mutans ATCC 70061 was the representative of cariogenic bacteria, while Streptococcus gordonii ATCC 35105 and Streptococcus sanguinis JCM 5708 were selected as healthy microbiota. The results showed that GH12 was more effective in suppressing S. mutans than the other two species, with lower MIC and minimal bactericidal concentration (MBC) values among diverse type strains and clinical isolated strains. Therefore, GH12, at no more than 8 mg/liter, was used to selectively suppress S. mutans in the multispecies biofilm. GH12 at 4 mg/liter and 8 mg/liter reduced the cariogenic properties of the multispecies biofilm in biofilm formation, glucan synthesis, and lactic acid production. In addition, GH12 suppressed S. mutans within the multispecies biofilm and changed the bacterial composition. Furthermore, 8 mg/liter GH12 showed a selective bactericidal impact on S. mutans, and GH12 promoted hydrogen peroxide production in S. sanguinis and S. gordonii, which improved their ecological advantages. In conclusion, GH12 inhibited the cariogenic properties and changed the composition of the multispecies biofilm through a two-part mechanism by which GH12 directly suppressed the growth of S. mutans as well as enhanced the ecological competitiveness of S. sanguinis and S. gordoniiIMPORTANCE Dental caries is one of the most prevalent chronic infectious diseases worldwide, with substantial economic and quality-of-life impacts. Streptococcus mutans has been considered the principal pathogen of dental caries. To combat dental caries, an antimicrobial peptide, GH12, was designed, and its antibacterial effects on planktonic S. mutans and the monospecies biofilm were confirmed. As etiological concepts of dental caries evolved to include microecosystems, the homeostasis between pathogenic and commensal bacteria and a selective action on cariogenic virulence have increasingly become the focus. The novelty of this research was to study the effects of the antimicrobial peptides on a controlled cariogenic multispecies biofilm model. Notably, the role of an antimicrobial agent in regulating interspecific competition and composition shifts within this multispecies biofilm was investigated. With promising antibacterial and antibiofilm properties, the use of GH12 might be of importance in preventing and controlling caries and other dental infections.
Assuntos
Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Cariogênicos/farmacologia , Peptídeos/farmacologia , Biofilmes/crescimento & desenvolvimento , Cárie Dentária/microbiologia , Placa Dentária/microbiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Ácido Láctico/metabolismo , Testes de Sensibilidade Microbiana , Microbiota/efeitos dos fármacos , Streptococcus gordonii/efeitos dos fármacos , Streptococcus gordonii/crescimento & desenvolvimento , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/crescimento & desenvolvimento , Streptococcus sanguis/efeitos dos fármacos , Streptococcus sanguis/crescimento & desenvolvimentoRESUMO
Pulpitis is a common dental emergency that presents with intense pain; there is still no specific medicine to treat pulpitis-induced pain to date. Herein, differentially expressed genes in mouse anterior cingulate cortex (ACC) were investigated 7 days after pulp exposure via a combination of high-throughput transcriptomic and proteomic analyses. We screened 34 key genes associated with 8 critical pathways. Among these, genes (Elovl5, Ikbke, and Nbeal2) involved in immune or inflammatory responses exhibited exclusive regulation at the transcriptomic level, as confirmed by qRT-PCR. We also investigated the comprehensive expression profiles of genes (Erg1, Shank2, Bche, Serinf1, and Pax6) related to synaptic plasticity. Furthermore, the underlying mechanisms for pulpitis-induced pain through immune or inflammatory responses and synaptic plasticity were discussed. Taken together, our findings shed light on the mechanisms underlying pulpitis-induced pain, deepening our understanding of the molecular pathways and providing potential therapeutic and diagnostic targets.
RESUMO
Mass spectrometry (MS)-based proteomics can identify and quantify the differential abundance of expressed proteins in parallel, and bottom-up proteomic approaches are even approaching comprehensive coverage of the complex eukaryotic proteome. Protein-nanoparticle (NP) interactions have been extensively studied owing to their importance in biological applications and nanotoxicology. However, the proteome-level effects of NPs on cells have received little attention, although changes in protein abundance can reflect the direct effects of nanocarriers on protein expression. Herein, we investigated the effect of PLGA-based NPs on protein expression in HepG2 cells using a label-free quantitative proteomics approach with data independent acquisition (DIA). The percentage of two-fold change in the protein expression of cells treated with PLGA-based NPs was less than 10.15% during a 6 hour observation period. Among the changed proteins, we found that dynamic proteins involved in cell division, localization, and transport are more likely to be more susceptible to PLGA-based NPs.
Assuntos
Nanopartículas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteômica , Humanos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Nanopartículas/química , Proteômica/métodos , Células Hep G2 , Tamanho da PartículaRESUMO
Uniform hollow spherical rhombohedral LaMO3 and solid spherical cubic MOx (M = Mn and Co) NPs were fabricated using the PMMA-templating strategy. Hollow spherical LaMO3 and solid spherical MOx NPs possessed surface areas of 21-33 and 21-24 m(2)/g, respectively. There were larger amounts of surface-adsorbed oxygen species and better low-temperature reducibility on/of the hollow spherical LaMO3 samples than on/of the solid spherical MOx samples. Hollow spherical LaMO3 and solid spherical MOx samples outperformed their nanosized counterparts for oxidation of CO and toluene, with the best catalytic activity being achieved over the solid spherical Co3O4 sample for CO oxidation (T50% = 81 °C and T90% = 109 °C) at space velocity = 10,000 mL/(g h) and the hollow spherical LaCoO3 sample for toluene oxidation (T50% = 220 °C and T90% = 237 °C) at space velocity = 20,000 mL/(g h). It is concluded that the higher surface areas and oxygen adspecies concentrations and better low-temperature reducibility are responsible for the excellent catalytic performance of the hollow spherical LaCoO3 and solid spherical Co3O4 NPs. We believe that the PMMA-templating strategy provides an effective route to prepare uniform perovskite-type oxide and transition-metal oxide NPs.
Assuntos
Monóxido de Carbono/química , Metais/química , Nanopartículas/química , Tolueno/química , Catálise , Cobalto/química , Cristalografia por Raios X , Lantânio/química , Manganês/química , Oxirredução , Oxigênio/química , Polietilenoglicóis/química , Polimetil Metacrilato/química , Propriedades de Superfície , Tensoativos/químicaRESUMO
Dental caries is associated with caries-related streptococci and antimicrobial agents have been widely used for caries control, but troubled by antibiotic resistance. This study aimed to investigate the intrinsic and acquired resistance of caries-related streptococci to antimicrobial peptide GH12, which was proven promising for caries control, and preliminarily explore the phenotypic changes and whole genome of stable acquired resistant strains. In this study, susceptibility assays and resistance assays were performed, followed by stability assays of resistance, to evaluate the intrinsic resistance and the potential resistance of caries-related streptococci. Then, the phenotypic changes of the stable acquired resistant strain were explored. The whole genome of the resistant strain was sequenced and analyzed by second-generation and third-generation high-throughput sequencing technologies. Streptococcus gordonii and Streptococcus sanguinis were intrinsically resistant to GH12 compared to cariogenic Streptococcus mutans. Acquired GH12 resistance in one S. sanguinis and four S. mutans clinical strains was transient but stable in one S. mutans strain (COCC33-14). However, acquired resistance to daptomycin (DAP) and chlorhexidine in all strains was stable. Furthermore, the COCC33-14 showed cross-resistance to DAP and delayed growth rates and a lower population. However, no drug-resistant gene mutation was detected in this strain, but 6 new and 5 missing genes were found. Among them, annotation of one new gene (gene 1782|COCC33-14R) is related to the integral component of the membrane, and one missing gene rpsN is associated with the metabolism and growth of bacteria. The results indicate that stable resistant mutants of caries-related streptococci could hardly be selected by exposure to consecutive sublethal GH12, but the risk still existed. Resistance in COCC33-14R is mainly related to changes in the cell envelope.
Assuntos
Anti-Infecciosos , Daptomicina , Cárie Dentária , Anti-Infecciosos/farmacologia , Peptídeos Antimicrobianos , Biofilmes , Clorexidina , Suscetibilidade à Cárie Dentária , Resistência a Medicamentos , Humanos , Streptococcus/genética , Streptococcus mutansRESUMO
Dental pulpitis often induces severe pain, and the molecular immune response is remarkable in both peripheral and central nervous system. Accumulating evidence indicates that activated microglia in the medullary dorsal horn (MDH) contribute to dental pulpitis induced pain. The P2X7 receptor plays an important role in driving pain and inflammatory processes, and its downstream target hypoxia-induced factor-1α (HIF-1α) has a crucial role in maintaining inflammation. However, the relationship between P2X7 and HIF-1α in dental inflammatory pain remains unclear. This study demonstrated that the degree of inflammation in the dental pulp tissue became more severe in a time-dependent manner by establishing a rat dental pulpitis model via pulp exposure. Meanwhile, the expression of P2X7, HIF-1α, IL-1ß, and IL-18 in the MDH increased most on the seventh day when the pain threshold was the lowest in the dental pulpitis model. Furthermore, lipopolysaccharides (LPS) increased P2X7-mediated HIF-1α expression in microglia. Notably, the suppression of P2X7 caused less IL-1ß and IL-18 release and lower HIF-1α expression, and P2X7 antagonist Brilliant Blue G (BBG) could alleviate pain behaviors of the dental pulpitis rats. In conclusion, our results provide further evidence that P2X7 is a key molecule, which regulates HIF-1α expression and inflammation in dental pulpitis-induced pain.
RESUMO
OBJECTIVES: To evaluate the effects of antimicrobial peptide GH12 designed de novo on the structure, morphology, and composition of a cariogenic three-species biofilm. METHODS: The cariogenic three-species biofilm consis-ted of the cariogenic Streptococcus mutans (S. mutans) and commensal bacteria Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii). The biofilm was treated using GH12 (2, 4, and 8 mg·L-1), and untreated biofilm was used as the control. Changes in the morphology and structure of the three-species biofilm were evaluated through crystal violet staining, scanning electron microscopy (SEM), and fluorescent in situ hybridization (FISH). Moreover, S. mutans in the biofilm was selectively cultured, and its colony-forming units were counted. RESULTS: The biomass and density of the cariogenic three-species biofilm treated with GH12 decreased compared with those of the control. The number of S. mutans decreased gradually and eventually became undetectable, whereas the number of S. gordonii and S. sanguinis increased and became predominant in the biofilm. CONCLUSIONS: GH12 can reduce the number of S. mutans within the cariogenic three-species biofilm, destroys its integrity, and consequently makes the biofilm easy to remove.
Assuntos
Cárie Dentária , Biofilmes , Humanos , Hibridização in Situ Fluorescente , Proteínas Citotóxicas Formadoras de Poros , Streptococcus mutansRESUMO
OBJECTIVES: The aim of this study was to analyse the protein profiles of acquired enamel pellicle (AEP) at two time points (5â¯min and 2â¯h) between caries-susceptible and caries-free subjects using label-free and parallel reaction monitoring (PRM) proteomic approaches, and to discover potential biomarkers for dental caries. METHODS: Sixty participants (30 caries-susceptible (DMFT>5) and 30 caries-free subjects (DMFTâ¯=â¯0)) were included. Their AEP at 5â¯min and 2â¯h was separately sampled, and the AEP materials were quantitatively analysed using label-free proteomics. Bioinformatics analysis of differentially expressed proteins was subsequently conducted and target proteins were verified using PRM analysis. RESULTS: A total of 82 and 39 differentially expressed proteins were identified at the 5-minute and 2â¯-h points, respectively, between the caries-susceptible and caries-free groups. Some proteins related to immune response and antibacterial activity were identified, and the characteristic enriched biological processes of up- and down-regulated proteins were presented using bioinformatics analysis. Subsequently, five intriguing target proteins of each group were chosen for PRM analysis and their presence was successfully verified in the 5-minute and 2â¯-h AEPs, respectively. CONCLUSIONS: The proteins in the AEPs of caries-susceptible and caries-free subjects at both time points presented unique protein profiles related to immune response and antibacterial activity. Mucin-7 can be regarded as an important potential AEP biomarker for dental caries. CLINICAL SIGNIFICANCE: The results of the study identified potential biomarkers, which can facilitate the design of new bio-functional agents for the diagnosis and treatment of dental caries in future.
Assuntos
Cárie Dentária , Proteômica , Biomarcadores , Película Dentária , Humanos , ProteínasRESUMO
Nonionic surfactant Triton X-100 was shown to aggregate and form micellar aggregation in ionic liquids (ILs), 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF(4)) and 1-butyl-3-methylimidazolium hexafluorophosphate (bmimPF(6)). The surface tension measurements revealed that the dissolution of Triton X-100 in ILs depressed the surface tension in a manner analogous to aqueous solutions, and a relatively higher critical micellar concentration (CMC) was obtained compared to that of water. Freeze-fracture transmission electron microscopy (FFTEM) shows that the micelles have an irregular droplet shape, which is larger than that formed in water. The micellar droplets preferred to assemble into larger clusters. (1)H NMR and two-dimensional rotating frame nuclear Overhauser effect (NOE) experiments (2D ROESY) show that the addition of Triton X-100 destroyed the ion pairs of pure ILs due to the electrostatic interaction between the positively charged imidazolium cation of ILs and the electronegative oxygen atoms of oxyethylene (OE) units of Triton X-100. The electrostatic interaction behaves similar to hydrogen bond that occurred between the OE units of nonionic surfactants and water molecules in aqueous micelles and cooperates with solvatophobicity, leading to the formation of IL micelles. The 2D ROESY analysis reveals that the microstructures of Triton X-100-based micelles in ILs are not regular spherical, which accords with the FFTEM image. Similar to the aqueous micellar systems, the hydrophobic interaction or solvatophobicity was found to drive the formation of micelles.