RESUMO
The human Solute Carrier (SLC) family member, monocarboxylate transporter 1 (MCT1), transports lactic and pyruvic acid across biological membranes to regulate cellular pH and metabolism. Proper trafficking of MCT1 from the endoplasmic reticulum to the plasma membrane hinges on its interactions with the membrane-bound chaperone protein, CD147. Here, using AlphaFold2 modeling and copurification, we show how a conserved signature motif located in the flexible N-terminus of MCT1 is a crucial region of interaction between MCT1 and the C-terminus of CD147. Mutations to this motif-namely, the thymic cancer linked G19C and the highly conserved W20A-destabilize the MCT1-CD147 complex and lead to a loss of proper membrane localization and cellular substrate flux. Notably, the monomeric stability of MCT1 remains unaffected in mutants, thus supporting the role of CD147 in mediating the trafficking of the heterocomplex. Using the auxiliary chaperone, GP70, we demonstrated that W20A-MCT1 can be trafficked to the plasma membrane, while G19C-MCT1 remains internalized. Overall, our findings underscore the critical role of the MCT1 transmembrane one signature motif for engaging CD147 and identify altered chaperone binding mechanisms between the CD147 and GP70 glycoprotein chaperones.
Assuntos
Motivos de Aminoácidos , Basigina , Transportadores de Ácidos Monocarboxílicos , Transporte Proteico , Simportadores , Basigina/metabolismo , Basigina/genética , Basigina/química , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/química , Humanos , Simportadores/metabolismo , Simportadores/química , Simportadores/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Mutação de Sentido IncorretoRESUMO
A family of chelating aryl-functionalized germylene ligands has been developed and employed in the synthesis of their corresponding 16-electron Ni0 complexes (PhiPDippGeAr·Ni·IPr; PhiPDipp = {[Ph2PCH2Si(iPr)2](Dipp)N}-; IPr = [{(H)CN(Dipp)}2C:]; Dipp = 2,6-iPr2C6H3). These complexes demonstrate the ability to cooperatively and reversibly activate dihydrogen at the germylene-nickel interface under mild conditions (1.5 atm H2, 298 K). We show that the thermodynamics of the dihydrogen activation process can be modulated by tuning the electronic nature of the germylene ligands, with an increase in the electron-withdrawing character displaying more exergonic ΔG298 values, as ascertained through NMR spectroscopic Van't Hoff analyses for all systems. This is also shown to correlate with experimental 31P NMR and UV/vis absorption data as well as with computationally derived parameters such as Ge-Ni bond order and Ni/Ge NPA charge, giving a thorough understanding of the modulating effect of ligand design on this reversible, cooperative bond activation reaction. Finally, the utility of this modulation was demonstrated in the catalytic dehydrocoupling of phenylsilane, whereby systems that disfavor dihydrogen activation are more efficient catalysts, aligning with H2-elimination being the rate-limiting step. A density functional theory analysis supports cooperative activation of the Si-H moiety in PhSiH3.
RESUMO
Soluble fms-like tyrosine kinase-1 (sFlt-1) is a secreted protein that binds heparan sulfate expressed on the endothelial glycocalyx (eGC). In this paper we analyze how excess sFlt-1 causes conformational changes in the eGC, leading to monocyte adhesion, a key event triggering vascular dysfunction. In vitro exposure of primary human umbilical vein endothelial cells to excess sFlt-1 decreased eGC height and increased stiffness as determined by atomic force microscopy (AFM). Yet, structural loss of the eGC components was not observed, as indicated by Ulex europaeus agglutinin I and wheat germ agglutinin staining. Moreover, the conformation observed under excess sFlt-1, a collapsed eGC, is flat and stiff with unchanged coverage and sustained content. Functionally, this conformation increased the endothelial adhesiveness to THP-1 monocytes by about 35%. Heparin blocked all these effects, but the vascular endothelial growth factor did not. In vivo administration of sFlt-1 in mice also resulted in the collapse of the eGC in isolated aorta analyzed ex vivo by AFM. Our findings show that excess sFlt-1 causes the collapse of the eGC and favors leukocyte adhesion. This study provides an additional mechanism of action by which sFlt-1 may cause endothelial dysfunction and injury.
Assuntos
Pré-Eclâmpsia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Humanos , Animais , Camundongos , Feminino , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Glicocálix/metabolismo , Endotélio/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Pré-Eclâmpsia/metabolismoRESUMO
We report a facile synthetic method for accessing rare T-shaped Ni0 species, stabilised by low-coordinate cationic germylene and stannylene ligands which behave as Z-type ligands toward Ni0 . An in-depth computational analysis indicates significant Nid âEp donation (E=Ge, Sn), with essentially no EâNi donation. The tetrylene ligand's Lewis acidity can be modulated in situ through the addition of a donor ligand, which selectively binds at the Lewis acidic tetrylene site. This switches this binding centre from a Z-type to a classical L-type ligand, with a concomitant geometry switch at Ni0 from T-shaped to trigonal planar. Exploring the effects of this geometry switch in catalysis, isolated T-shaped complexes 3 a-c and 4 a-c are capable of the hydrogenation of alkenes under mild conditions, whilst the closely related trigonal planar and tetrahedral Ni0 complexes 5, D, and E, which feature L-type chloro- or cationic-tetrylene ligands, are inactive under these conditions. Further, addition of small amounts of N-bases to the catalytic systems involving T-shaped complexes significantly reduces turnover rates, giving evidence for the in situ modulation of ligand electronics for catalytic switching.
RESUMO
Soluble Fms-like tyrosine kinase (sFlt-1/sVEGFR1) is a naturally occurring antagonist of vascular endothelial growth factor (VEGF). Despite being a secreted, soluble protein lacking cytoplasmic and transmembrane domains, sFlt-1 can act locally and be protective against excessive microenvironmental VEGF concentration or exert autocrine functions independently of VEGF. Circulating sFlt-1 may indiscriminately affect endothelial function and the microvasculature of distant target organs. The clinical significance of excess sFlt-1 in kidney disease was first shown in preeclampsia, a major renal complication of pregnancy. However, circulating sFlt-1 levels appear to be increased in various diseases with varying degrees of renal impairment. Relevant clinical associations between circulating sFlt-1 and severe outcomes (e.g., endothelial dysfunction, renal impairment, cardiovascular disease, and all-cause mortality) have been observed in patients with CKD and after kidney transplantation. However, sFlt-1 appears to be protective against renal dysfunction-associated aggravation of atherosclerosis and diabetic nephropathy. Therefore, in this study, we provide an update on sFlt-1 in several kidney diseases other than preeclampsia, discuss clinical findings and experimental studies, and briefly consider its use in clinical practice.
Assuntos
Injúria Renal Aguda/sangue , Microvasos/patologia , Insuficiência Renal Crônica/sangue , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangue , Injúria Renal Aguda/cirurgia , Biomarcadores/sangue , Vasos Coronários/patologia , Endotélio/fisiopatologia , Feminino , Humanos , Rim/irrigação sanguínea , Rim/patologia , Transplante de Rim , Pré-Eclâmpsia/sangue , Gravidez , Diálise Renal , Insuficiência Renal Crônica/terapiaRESUMO
Endothelial cells are a critical target of the soluble Fms-like tyrosine kinase-1 (sFlt-1), a soluble factor increased in different diseases with varying degrees of renal impairment and endothelial dysfunction, including chronic kidney disease (CKD). Although the mechanisms underlying endothelial dysfunction are multifactorial and complex, herein, we investigated the damaging effects of sFlt-1 on structural and functional changes in endothelial cells. Our results evidenced that sera from patients with CKD stiffen the endothelial cell cortex in vitro, an effect correlated with sFlt-1 levels and prevented by sFlt-1 neutralization. Besides, we could show that recombinant sFlt-1 leads to endothelial stiffening in vitro and in vivo. This was accompanied by cytoskeleton reorganization and changes in the endothelial barrier function, as observed by increased actin polymerization and endothelial cell permeability, respectively. These results depended on the activation of the p38 MAPK and were blocked by the specific inhibitor SB203580. However, sFlt-1 only minimally affected the expression of stiffness-sensitive genes. These findings bring new insight into the mechanism of action of sFlt-1 and its biological effects that cannot be exclusively ascribed to the regulation of angiogenesis.
Assuntos
Pré-Eclâmpsia , Insuficiência Renal Crônica , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Feminino , Humanos , Células Endoteliais/metabolismo , Pré-Eclâmpsia/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Eight facultatively anaerobic rod-shaped bacteria were isolated from raw milk and two other dairy products. Results of phylogenetic analyses based on 16S rRNA gene sequences showed that the isolates are placed in a distinct lineage within the family Propionibacteriaceae with Propioniciclava sinopodophylli and Propioniciclava tarda as the closest relatives (94.6 and 93.5â% similarity, respectively). The cell-wall peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid and was of the A1γ type (meso-DAP-direct). The major cellular fatty acid was anteiso-C15â:â0 and the major polar lipids were diphosphatidylglycerol, phosphatidyglycerol and three unidentified glycolipids. The quinone system contained predominantly menaquinone MK-9(H4). The G+C content of the genomic DNA of strain VG341T was 67.7 mol%. The whole-cell sugar pattern contained ribose, rhamnose, arabinose and galactose. On the basis of phenotypic and genetic data, eight strains (VG341T, WS4684, WS4769, WS 4882, WS4883, WS4901, WS4902 and WS4904) are proposed to be classified as members of a novel species in a new genus of the family Propionibacteriaceae, for which the name Brevilactibacter flavus gen. nov., sp. nov. is proposed. The type strain is VG341T (=WS4900T=DSM 100885T=LMG 29089T) and seven additional strains are WS4684, WS4769, WS4882, WS4883, WS4901, WS4902 and WS4904. Furthermore, we propose the reclassification of P. sinopodophylli as Brevilactibacter sinopodophylli comb. nov.
Assuntos
Laticínios/microbiologia , Leite/microbiologia , Filogenia , Propionibacteriaceae/classificação , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Parede Celular/química , DNA Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Graxos/química , Microbiologia de Alimentos , Alemanha , Glicolipídeos/química , Peptidoglicano/química , Fosfolipídeos/química , Propionibacteriaceae/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
For the commercially established process of paclitaxel production with Taxus chinensis plant cell culture, the size of plant cell aggregates and phenotypic changes in coloration during cultivation have long been acknowledged as intangible parameters. So far, the variability of aggregates and coloration of cells are challenging parameters for any viability assay. The aim of this study was to investigate simple and non-toxic methods for viability determination of Taxus cultures in order to provide a practicable, rapid, robust and reproducible way to sample large amounts of material. A further goal was to examine whether Taxus aggregate cell coloration is related to general cell viability and might be exploited by microscopy and image analysis to gain easy access to general cell viability. The Alamar Blue assay was found to be exceptionally eligible for viability estimation. Moreover, aggregate coloration, as a morphologic attribute, was quantified by image analysis and found to be a good and traceable indicator of T. chinensis viability.
Assuntos
Colorimetria , Taxus/citologia , Reprodutibilidade dos Testes , Estresse MecânicoRESUMO
While particulate bone substitute materials are applied in a variety of augmentation procedures, standardized defects are being used for preclinical testing. This in vitro study evaluated the density and homogeneity of a particulate bone substitute in ridge preservation procedures. Premolars and molars were extracted in ten semimandibles of minipig cadavers. Light body impression material was used for determining the volume of the extraction sites followed by augmentation with particulate material, thereby weighing the graft material needed. Microradiographs and histologic sections were obtained for evaluating the homogeneity and density of the augmentation material. Statistical analyses were based on Shapiro-Wilk tests, Spearman's rho and one sample Wilcoxon test followed by Bonferroni-Holm correction for multiple testing (α = 0.05). Based on 103 single alveoli evaluated, the mean volume determined was 0.120 cm3 requiring a mean amount of graft material of 0.155 g. With only three exceptions, all parameters (volume, mass of augmentation material, density and homogeneity) correlated significantly (p < 0.020). The apical parts of the alveoli showed reduced density as compared to the middle parts (p < 0.001) and the homogeneity of the augmentation material was also lower as compared to the middle (p < 0.001) and cervical parts (p = 0.040). The packing of augmentation material is critical when non-standardized defects are treated.
RESUMO
SARS-CoV-2 Omicron subvariants with increased transmissibility and immune evasion are spreading globally with alarming persistence. Whether the mutations and evolution of spike (S) Omicron subvariants alter the viral hijacking of human TMPRSS2 for viral entry remains to be elucidated. This is particularly important to investigate because of the large number and diversity of mutations of S Omicron subvariants reported since the emergence of BA.1. Here we report that human TMPRSS2 is a molecular determinant of viral entry for all the Omicron clinical isolates tested in human lung cells, including ancestral Omicron subvariants (BA.1, BA.2, BA.5), contemporary Omicron subvariants (BQ.1.1, XBB.1.5, EG.5.1) and currently circulating Omicron BA.2.86. First, we used a co-transfection assay to demonstrate the endoproteolytic cleavage by TMPRSS2 of spike Omicron subvariants. Second, we found that N-0385, a highly potent TMPRSS2 inhibitor, is a robust entry inhibitor of virus-like particles harbouring the S protein of Omicron subvariants. Third, we show that N-0385 exhibits nanomolar broad-spectrum antiviral activity against live Omicron subvariants in human Calu-3 lung cells and primary patient-derived bronchial epithelial cells. Interestingly, we found that N-0385 is 10-20 times more potent than the repositioned TMPRSS2 inhibitor, camostat, against BA.5, EG.5.1, and BA.2.86. We further found that N-0385 shows broad synergistic activity with clinically approved direct-acting antivirals (DAAs), i.e., remdesivir and nirmatrelvir, against Omicron subvariants, demonstrating the potential therapeutic benefits of a multi-targeted treatment based on N-0385 and DAAs.
Assuntos
Benzotiazóis , COVID-19 , Sulfonamidas , Humanos , Anticorpos Neutralizantes , Anticorpos Antivirais , Antivirais , SARS-CoV-2 , Serina EndopeptidasesRESUMO
Recruiting the endogenous editing enzyme adenosine deaminase acting on RNA (ADAR) with tailored guide RNAs for adenosine-to-inosine (A-to-I) RNA base editing is promising for safely manipulating genetic information at the RNA level. However, the precision and efficiency of editing are often compromised by bystander off-target editing. Here, we find that in 5'-UAN triplets, which dominate bystander editing, Gâ¢U wobble base pairs effectively mitigate off-target events while maintaining high on-target efficiency. This strategy is universally applicable to existing A-to-I RNA base-editing systems and complements other suppression methods such as Gâ¢A mismatches and uridine (U) depletion. Combining wobble base pairing with a circularized format of the CLUSTER approach achieves highly precise and efficient editing (up to 87%) of a disease-relevant mutation in the Mecp2 transcript in cell culture. Virus-mediated delivery of the guide RNA alone realizes functional MeCP2 protein restoration in the central nervous system of a murine Rett syndrome model with editing yields of up to 19% and excellent bystander control in vivo.
RESUMO
PURPOSE: Participation and accessibility issues faced by gamers with multi-sensory disabilities are themes yet to be fully understood by accessible technology researchers. In this work, we examine the personal experiences and perceptions of individuals with deafblindness who play games despite their disability, as well as the reasons that lead some of them to stop playing games. MATERIALS AND METHODS: We conducted 60 semi-structured interviews with individuals living with deafblindness in five European countries: United Kingdom, Germany, Netherlands, Greece and Sweden. RESULTS: Participants stated that reasons for playing games included them being a fun and entertaining hobby, for socialization and meeting others, or for occupying the mind. Reasons for stop playing games included essentially accessibility issues, followed by high cognitive demand, changes in gaming experience due their disability, financial reasons, or because the accessible version of a specific game was not considered as fun as the original one. CONCLUSIONS: We identified that a considerable number of individuals with deafblindness enjoy playing casual mobile games such as Wordfeud and Sudoku as a pastime activity. Despite challenging accessibility issues, games provide meaningful social interactions to players with deafblindness. Finally, we introduce a set of user-driven recommendations for making digital games more accessible to players with a diverse combination of sensory abilities.IMPLICATIONS FOR REHABILITATIONDigital games were considered a fun and entertaining hobby by participants with deafblindness. Furthermore, participants play games for socialization and meeting others, or for occupying the mind.Digital games provide meaningful social interactions and past time to persons with deafblindness.On top of accessibility implications, our findings draw attention to the importance of the social element of gaming for persons with deafblindness.Based on interviews, we introduce a set of user-driven recommendations for making digital games more accessible to players with a diverse combination of sensory abilities.
Assuntos
Surdocegueira , Pessoas com Deficiência , Aplicativos Móveis , Jogos de Vídeo , Humanos , Surdocegueira/psicologia , GréciaRESUMO
The four compounds A3MO4H (A = Rb, Cs; M = Mo, W) are introduced as the first members of the new material class of the transition oxometalate hydrides. The compounds are accessible via a thermal synthesis route with carefully controlled conditions. Their crystal structures were solved by neutron diffraction of the deuterated analogues. Rb3MoO4D, Cs3MoO4D and Cs3WO4D crystallize in the antiperovskite-like K3SO4F-structure type, while Rb3WO4D adopts a different orthorhombic structure. 2H MAS NMR, Raman spectroscopy and elemental analysis prove the abundance of hydride ions next to oxometalate ions and experimental findings are supported by quantum chemical calculations. The tetragonal phases are direct and wide band gap semiconductors arising from hydride states, whereas Rb3WO4H shows a unique, peculiar valence band structure dominated by hydride states.
RESUMO
Optimising catalyst materials for visible light-driven fuel production requires understanding complex and intertwined processes including light absorption and catalyst stability, as well as mass, charge, and energy transport. These phenomena can be uniquely combined (and ideally controlled) in porous host-guest systems. Towards this goal we designed model systems consisting of molecular complexes as catalysts and porphyrin metal-organic frameworks (MOFs) as light-harvesting and hosting porous matrices. Two MOF-rhenium molecule hybrids with identical building units but differing topologies (PCN-222 and PCN-224) were prepared including photosensitiser-catalyst dyad-like systems integrated via self-assembled molecular recognition. This allowed us to investigate the impact of MOF topology on solar fuel production, with PCN-222 assemblies yielding a 9-fold turnover number enhancement for solar CO2-to-CO reduction over PCN-224 hybrids as well as a 10-fold increase compared to the homogeneous catalyst-porphyrin dyad. Catalytic, spectroscopic and computational investigations identified larger pores and efficient exciton hopping as performance boosters, and further unveiled a MOF-specific, wavelength-dependent catalytic behaviour. Accordingly, CO2 reduction product selectivity is governed by selective activation of two independent, circumscribed or delocalised, energy/electron transfer channels from the porphyrin excited state to either formate-producing MOF nodes or the CO-producing molecular catalysts.
RESUMO
RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing.
Assuntos
Edição de RNA , RNA Guia de Cinetoplastídeos , Animais , Sequência de Bases , Camundongos , RNA/metabolismo , Edição de RNA/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Pellicle is the initial proteinaceous layer that is formed almost instantaneously on all solid surfaces in the oral cavity. It is of essential relevance for any interactions and metabolism on the tooth surface. Up to now, there is no information on the metabolome of this structure. Accordingly, the present study aims to characterise the metabolomic profile of in-situ pellicle in children with different caries activity for the first time in comparison to saliva. Small molecules such as carbohydrates, amino acids, organic acids, and fatty acids, putatively involved in the formation of caries were quantified using mass spectrometry (MS)-based techniques, such as (stable isotope dilution analysis)-ultra-performance liquid chromatography-tandem MS and gas chromatography/electron ionisation-MS. Pellicle and corresponding saliva samples were collected from caries-active, caries-free and caries-rehabilitated 4- to 6-year-old children. The most abundant analytes in pellicle were acetic acid (1.2-10.5 nmol/cm2), propionic acid (0.1-8.5 nmol/cm2), glycine (0.7-3.5 nmol/cm2), serine (0.08-2.3 nmol/cm2), galactose (galactose + mannose; 0.035-0.078 nmol/cm2), lactose (0.002-0.086 nmol/cm2), glucose (0.018-0.953 nmol/cm2), palmitic acid (0.26-2.03 nmol/cm2), and stearic acid (0.34-1.81 nmol/cm2). Significant differences depending on caries activity were detected neither in saliva nor in the corresponding pellicle samples.
Assuntos
Cárie Dentária/metabolismo , Película Dentária/química , Metabolômica/métodos , Saliva/química , Ácido Acético/análise , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromatografia Líquida , Glicina/análise , Humanos , Masculino , Propionatos/análise , Espectrometria de Massas em TandemRESUMO
Nematodes are increasingly used as powerful bioindicators of soil food web composition and functioning in ecological studies. Todays' ecological research aims to investigate not only local relationships but global patterns, which requires consistent methodology across locations. Thus, a common and easy extraction protocol of soil nematodes is needed. In this study, we present a detailed protocol of the Baermann-funnel method and highlight how different soil pre-treatments and equipment (soil type, soil height, sieving, and filter type) can affect extraction efficiency and community composition by using natural nematode communities. We found that highest nematode extraction efficiency was achieved using lowest soil height as indicated by the thickness of the soil sample in the extractor (1, 2, or 4 cm soil height) in combination with soil sieving (instead of no sieving), and by using milk filters (instead of paper towels). PCA at the family level revealed that different pre-treatments significantly affected nematode community composition. Increasing the height of the soil sample by adding more soil increased the proportion of larger-sized nematodes likely because those are able to overcome long distances but selected against small nematodes. Sieving is suggested to break up soil aggregates and, therefore, facilitate moving in general. Interestingly, sieving did not negatively affect larger nematodes that are supposed to have a higher probability of getting bruised during sieving but, even if not significant, tended to yield more extracted nematodes than no sieving. We therefore recommend to use small heights of sieved soil with milk filter to extract free-living soil nematodes with the Baermann-funnel method. The present study shows that variations in the extraction protocol can alter the total density and community composition of extracted nematodes and provides recommendations for an efficient and standardized approach in future studies. Having a simple, cheap, and standardized extraction protocol can facilitate the assessment of soil biodiversity in global contexts.
RESUMO
OBJECTIVE: This study investigated, for the first time, pellicle enzymes with respect to their activity, distribution and fluorescence pattern in children with different caries experience. DESIGN: In-situ pellicles were collected from 41 children (aged 4-6 years) with different caries status; 17 of them were caries-free (dmfâ¯=â¯0), 12â¯had dental restorations but no current caries (dmf ≥ 2) and 12â¯had at least two carious lesions (dmf ≥ 2). Bovine enamel samples were fixed on individual upper jaw braces for pellicle formation. After 30â¯min of intraoral exposure, the pellicle and saliva samples were analysed for the activities of amylase, lysozyme, peroxidase and glucosyltransferase (GTF). The distribution of these enzymes, including GTF-isoforms B, C and D, and the pellicle ultrastructure were examined by gold-immunolabelling and transmission electron microscopy (TEM). Furthermore, interactions between pellicle enzymes and adherent bacteria were visualised using combined fluorescence and immunofluorescence labelling. RESULTS: There were no significant differences in the pellicle enzyme activities between the study groups. TEM analysis revealed the absence of GTF C and D in the pellicle of caries-active children. Amylase, peroxidase and GTF-isoforms showed a random distribution within the pellicle layer; lysozyme was found in the form of clusters. A similar ultrastructural pattern was observed for all subjects. Fluorescence labelling technique enabled visualisation of all enzymes, except for GTF B. CONCLUSION: Pellicle enzyme activities and ultrastructure are not associated with children's caries status. Further investigation is needed to assess the influence of individual GTF-isoforms on caries susceptibility in children.