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The effective protection and delivery of antisense oligomers to its site of action is a challenge without an optimal strategy. Some of the most promising approaches encompass the complexation of nucleic acids, which are anionic, with liposomes of fixed or ionizable cationic charge. Thus, the main purpose of this work was to study the complexation of cationic liposomes with anti-EFG1 2'OMe oligomers and evaluate the complex efficacy to control Candida albicans filamentation in vitro and in vivo using a Galleria mellonella model. To accomplish this, cationic dioleoyl-trimethylammoniumpropane (DOTAP) was mixed with three different neutral lipids dioleoyl-phosphocholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) and monoolein (MO) and used as delivery vectors. Fluorescence Cross Correlation Spectroscopy measurements revealed a high association between antisense oligomers (ASO) and cationic liposomes confirming the formation of lipoplexes. In vitro, all cationic liposome-ASO complexes were able to release the anti-EFG1 2'OMe oligomers and consequently inhibit C. albicans filamentation up to 60% after 72 h. In vivo, from all formulations the DOTAP/DOPC 80/20 ρchg = 3 formulation proved to be the most effective, enhancing the G. mellonella survival by 40% within 48 h and by 25% after 72 h of infection. In this sense, our findings show that DOTAP-based lipoplexes are very good candidates for nano-carriers of anti-EFG1 2'OMe oligomers.
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Candida albicans , Lipossomos , Animais , Candida albicans/genética , Lipossomos/químicaRESUMO
Allergic diseases are a major health concern worldwide. Pollens are important triggers for allergic rhinitis, conjunctivitis and asthma. Proteases released upon pollen grain hydration appear to play a major role in the typical immunological and inflammatory responses that occur in patients with allergic disorders. In this study, we aimed to identify specific proteolytic activity in a set of pollens with diverse allergenic potential. Diffusates from Chenopodium album, Plantago lanceolata and Eucalyptus globulus were added to a confluent monolayer of Calu-3 cells grown in an air-liquid interface system. We identified serine proteases and metalloproteinases in all pollen diffusates investigated. Proteases found in these pollen diffusates were shown to compromise the integrity of the lung epithelial barrier by disrupting transmembrane adhesion proteins E-cadherin, claudin-1 and Occludin, as well as, the cytosolic complex zonula occludens-1 (ZO-1) resulting in a time-dependent increase in transepithelial permeability. Tight junction disruption and increased transepithelial permeability facilitates allergen exposure to epithelial sub-layers contributing to the sensitization to a wide range of allergens. These pollen extracts also induced an increase in the release of interleukin 6 (IL-6) and interleukin 8 (IL-8) cytokines measured by flow cytometry possibly as a result of the activation of protease-activated receptors 2 (PAR-2).
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Hipersensibilidade/enzimologia , Peptídeo Hidrolases/metabolismo , Pólen/enzimologia , Linhagem Celular , Chenopodium/enzimologia , Eucalyptus/enzimologia , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Plantago/enzimologia , Receptor PAR-2/metabolismo , ÁguaRESUMO
Cancer is an extremely complex disease, typically caused by mutations in cancer-critical genes. By delivering therapeutic nucleic acids (NAs) to patients, gene therapy offers the possibility to supplement, repair or silence such faulty genes or to stimulate their immune system to fight the disease. While the challenges of gene therapy for cancer are significant, the latter approach (a type of immunotherapy) starts showing promising results in early-stage clinical trials. One important advantage of NA-based cancer therapies over synthetic drugs and protein treatments is the prospect of a more universal approach to designing therapies. Designing NAs with different sequences, for different targets, can be achieved by using the same technologies. This versatility and scalability of NA drug design and production on demand open the way for more efficient, affordable and personalized cancer treatments in the future. However, the delivery of exogenous therapeutic NAs into the patients' targeted cells is also challenging. Membrane-type lipids exhibiting permanent or transient cationic character have been shown to associate with NAs (anionic), forming nanosized lipid-NA complexes. These complexes form a wide variety of nanostructures, depending on the global formulation composition and properties of the lipids and NAs. Importantly, these different lipid-NA nanostructures interact with cells via different mechanisms and their therapeutic potential can be optimized to promising levels in vitro. The complexes are also highly customizable in terms of surface charge and functionalization to allow a wide range of targeting and smart-release properties. Most importantly, these synthetic particles offer possibilities for scaling-up and affordability for the population at large. Hence, the versatility and scalability of these particles seem ideal to accommodate the versatility that NA therapies offer. While in vivo efficiency of lipid-NA complexes is still poor in most cases, the advances achieved in the last three decades are significant and very recently a lipid-based gene therapy medicine was approved for the first time (for treatment of hereditary transthyretin amyloidosis). Although the path to achieve efficient NA-delivery in cancer therapy is still long and tenuous, these advances set a new hope for more treatments in the future. In this review, we attempt to cover the most important biophysical and physicochemical aspects of non-viral lipid-based gene therapy formulations, with a perspective on future cancer treatments in mind.
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Fenômenos Químicos , Lipídeos/química , Neoplasias/tratamento farmacológico , Ácidos Nucleicos/química , Animais , Humanos , Ácidos Nucleicos/uso terapêuticoRESUMO
Parkinson's disease (PD) is characterized by proteinaceous aggregates named Lewy Bodies and Lewy Neurites containing α-synuclein fibrils. The underlying aggregation mechanism of this protein is dominated by a secondary process at mildly acidic pH, as in endosomes and other organelles. This effect manifests as a strong acceleration of the aggregation in the presence of seeds and a weak dependence of the aggregation rate on monomer concentration. The molecular mechanism underlying this process could be nucleation of monomers on fibril surfaces or fibril fragmentation. Here, we aim to distinguish between these mechanisms. The nature of the secondary processes was investigated using differential sedimentation analysis, trap and seed experiments, quartz crystal microbalance experiments and super-resolution microscopy. The results identify secondary nucleation of monomers on the fibril surface as the dominant secondary process leading to rapid generation of new aggregates, while no significant contribution from fragmentation was found. The newly generated oligomeric species quickly elongate to further serve as templates for secondary nucleation and this may have important implications in the spreading of PD.
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Amiloide/química , Biocatálise , Multimerização Proteica , alfa-Sinucleína/química , Cinética , Imagem Molecular , Estrutura Secundária de Proteína , Propriedades de SuperfícieRESUMO
The deposition of α-synuclein fibrils is one hallmark of Parkinson's disease. Here, we investigate how ganglioside lipids, present in high amounts in neurons and exosomes, influence the aggregation kinetics of α-synuclein. Gangliosides, as well as, other anionic lipid species with small or large headgroups were found to induce conformational changes of α-synuclein monomers and catalyse their aggregation at mildly acidic conditions. Although the extent of this catalytic effect was slightly higher for gangliosides, the results imply that charge interactions are more important than headgroup chemistry in triggering aggregation. In support of this idea, uncharged lipids with large headgroups were not found to induce any conformational change and only weakly catalyse aggregation. Intriguingly, aggregation was also triggered by free ganglioside headgroups, while these caused no conformational change of α-synuclein monomers. Our data reveal that partially folded α-synuclein helical intermediates are not required species in triggering of α-synuclein aggregation.
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The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that α-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of α-synuclein aggregation and aggregate spreading in the context of Parkinson disease.
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Amiloide/biossíntese , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Microscopia de Força Atômica , Eletricidade EstáticaRESUMO
Exosomes are small vesicles released from cells into extracellular space. We have isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein associated with Parkinson disease pathology. Using cryo-transmission electron microscopy of exosomes, we found spherical unilamellar vesicles with a significant protein content, and Western blot analysis revealed that they contain, as expected, the proteins Flotillin-1 and Alix. Using thioflavin T fluorescence to monitor aggregation kinetics, we found that exosomes catalyze the process in a similar manner as a low concentration of preformed α-synuclein fibrils. The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation. The catalytic effects of exosomes derived from naive cells and cells that overexpress α-synuclein do not differ. Vesicles prepared from extracted exosome lipids accelerate aggregation, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise. Using mass spectrometry, we found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and the gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. We then prepared vesicles from corresponding pure lipids or defined mixtures, most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles containing ganglioside lipids GM1 or GM3 accelerate the process. Understanding how α-synuclein interacts with biological membranes to promote neurological disease might lead to the identification of novel therapeutic targets.
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Exossomos/metabolismo , alfa-Sinucleína/metabolismo , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M2) , Gangliosídeo G(M3)/metabolismo , Humanos , Doença de Parkinson/metabolismo , Fosfolipídeos/metabolismo , Lipossomas Unilamelares/metabolismoRESUMO
UNLABELLED: This cross-sectional study was performed to examine the prevalence of hypovitaminosis D in infants with acute bronchiolitis compared with control subjects and to evaluate the relationship between serum 25-hydroxyvitamin D (25(OH) D) and the severity of bronchiolitis. Serum 25(OH) D levels were measured by radioimmunoassay in 48 infants with acute bronchiolitis (2.5 ± 2.0 months) and in 30 healthy infants (3.2 ± 2.3 months). 25(OH) D levels (ng/ml) in children with acute bronchiolitis were significantly lower than in the control group (median 29.9 ng/ml (interquartile range (IQR) 21.4-37.5) versus median 38.2 ng/ml ((IQR 26.1-48.1), p = 0.022), mainly in infants with moderate-severe bronchiolitis (median 29.8 ng/ml, IQR 19.2-35.9). The prevalence of hypovitaminosis D was remarkably greater among infants with bronchiolitis than in control subjects (52.1 versus 26.6%). A significant inverse correlation was found between serum 25-hydroxyvitamin D levels and disease severity (rho = -0.457, p < 0.001). CONCLUSION: The prevalence of hypovitaminosis D is high in Spanish infants with bronchiolitis. The severity of acute bronchiolitis increases with a decline in serum 25 (OH) D level.
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Bronquiolite/epidemiologia , Bronquiolite/fisiopatologia , Deficiência de Vitamina D/epidemiologia , Vitamina D/análogos & derivados , Doença Aguda , Bronquiolite/sangue , Estudos Transversais , Feminino , Humanos , Lactente , Masculino , Prevalência , Índice de Gravidade de Doença , Espanha/epidemiologia , Vitamina D/sangue , Deficiência de Vitamina D/sangueRESUMO
Hybrid core-shell lipid-polycation-nucleic acid nanoparticles (LPNPs) provide unique delivery strategies for nonviral gene therapeutics. Since LPNPs consist of multiple components, involving different pairwise interactions between them, they are challenging to characterize and understand. Here, we propose a method based on fluorescence cross-correlation spectroscopy to elucidate the association between the three LPNP components. Through this lens, we demonstrate that cationic lipid shells (liposomes) do not displace polycations or DNA from the polycation-DNA cores (polyplexes). Hence, polyplexes and liposomes must be oppositely charged to associate into LPNPs. Furthermore, we identify the liposome:polyplex number ratio (ρN), which was hitherto an intangible quantity, as the primary parameter predicting stable LPNPs. We establish that ρN ≥ 1 ensures that every polyplex is enveloped by a liposome, thus avoiding coexisting oppositely charged species prone to aggregation.
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Nanopartículas , Ácidos Nucleicos , Polímeros/química , Lipossomos , DNA/química , Nanopartículas/química , Lipídeos/químicaRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome−DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.
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The impact of nanotechnology on the exponential growth of several research areas, particularly nanomedicine, is undeniable. The ability to deliver active molecules to the desired site could significantly improve the efficiency of medical treatments. One of the nanocarriers developed which has drawn researchers' attention are cubosomes, which are nanosized dispersions of lipid bicontinuous cubic phases in water, consisting of a lipidic interior and aqueous domains folded in a cubic lattice. They stand out due to their ability to incorporate hydrophobic, hydrophilic, and amphiphilic compounds, their tortuous internal configuration that provides a sustained release, and the capacity to protect and safely deliver molecules. Several approaches can be taken to prepare this structure, as well as different lipids like monoolein or phytantriol. This review paper describes the different methods to prepare nanocarriers. As it is known, the physicochemical properties of nanocarriers are very important, as they influence their pharmacokinetics and their ability to incorporate and deliver active molecules. Therefore, an extensive characterization is essential to obtain the desired effect. As a result, we have extensively described the most common techniques to characterize cubosomes, particularly nanocarriers. The exceptional properties of the cubosomes make them suitable to be used in several applications in the biomedical field, from cancer therapeutics to imaging, which will be described. Taking in consideration the outstanding properties of cubosomes, their application in several research fields is envisaged.
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α-Synuclein is a membrane-interacting protein involved in Parkinson's disease. Here we have investigated the co-association of α-synuclein and lipids from ganglioside-containing model membranes. Our study relies on the reported importance of ganglioside lipids, which are found in high amounts in neurons and exosomes, on cell-to-cell prion-like transmission of misfolded α-synuclein. Samples taken along various stages of the aggregation process were imaged using cryogenic transmission electron microscopy, and the composition of samples corresponding to the final state analyzed using NMR spectroscopy. The combined data shows that α-synuclein co-assembles with lipids from the ganglioside (GM1)-containing model membranes. The lipid-protein samples observed during the aggregation process contain non-vesicular objects not present at the final stage, thus capturing the co-existence of species under non-equilibrium conditions. A range of different lipid-protein co-assemblies are observed during the time course of the reaction and some of these appear to be transient assemblies that evolve into other co-aggregates over time. At the end of the aggregation reaction, the samples become more homogeneous, showing thin fibrillar structures heavily decorated with small vesicles. From the NMR analysis, we conclude that the ratio of GM1 to phosphatidyl choline (PC) in the supernatant of the co-aggregated samples is significantly reduced compared to the GM1/PC ratio of the lipid dispersion from which these samples were derived. Taken together, this indicates a selective uptake of GM1 into the fibrillar aggregates and removal of GM1-rich objects from the solution.
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The development of nonviral gene delivery vehicles for therapeutic applications requires methods capable of quantifying the association between the genes and their carrier counterparts. Here we investigate the potential of fluorescence cross-correlation spectroscopy (FCCS) to characterize and optimize the assembly of nonviral cationic liposome (CL)-DNA complexes based on a CL formulation consisting of the cationic lipid DOTAP and zwitterionic lipid DOPC. We use a DNA plasmid for lipoplex loading encoding the Oct4 gene, critically involved in reprogramming somatic cells into induced pluripotent stem cells. We demonstrate that FCCS is able to quantitatively determine the extent of the association between DNA and the liposomes and assess its loading capacity. We also establish that the cationic lipid fraction, being proportional to the liposome membrane charge density, as well as charge ratio between the CLs and anionic DNA play an important role in the degree of interaction between the liposomes and DNA.
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Lipossomos , Nanopartículas , DNA/genética , Espectrometria de Fluorescência , TransfecçãoRESUMO
Parkinson´s disease is characterized by the accumulation of proteinaceous aggregates in Lewy bodies and Lewy Neurites. The main component found in such aggregates is α-synuclein. Here, we investigate how bovine eye lens crystallin proteins influence the aggregation kinetics of α-synuclein at mildly acidic pH (5.5) where the underlying aggregation mechanism of this protein is dominated by secondary nucleation of monomers on fibril surface providing an autocatalytic amyloid amplification process. Bovine α-, ßH- and γB-crystallins were found to display chaperone-like activity inhibiting α-synuclein aggregation. This effect was shown to be time-dependent, with early additions of α-crystallin capable of retarding and even inhibiting aggregation during the time frame of the experiment. The inhibitory nature of crystallins was further investigated using trap and seed kinetic experiments. We propose crystallins interact with mature α-synuclein fibrils, possibly binding along the surfaces and at fibril free ends, inhibiting both elongation and monomer-dependent secondary nucleation processes in a mechanism that may be generic to some chaperones that prevent the onset of protein misfolding related pathologies.
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Doença de Parkinson/metabolismo , Agregação Patológica de Proteínas , alfa-Cristalinas/metabolismo , alfa-Sinucleína/metabolismo , beta-Cristalinas/metabolismo , gama-Cristalinas/metabolismo , Amiloide/metabolismo , Animais , Bovinos , Clonagem Molecular , Escherichia coli/genética , Humanos , Cinética , Cristalino/metabolismo , Ligação ProteicaRESUMO
α-Synuclein (α-syn) is an intrinsically disordered protein with a highly asymmetric charge distribution, whose aggregation is linked to Parkinson's disease. The effect of ionic strength was investigated at mildly acidic pH (5.5) in the presence of catalytic surfaces in the form of α-syn seeds or anionic lipid vesicles using thioflavin T fluorescence measurements. Similar trends were observed with both surfaces: increasing ionic strength reduced the rate of α-syn aggregation although the surfaces as well as α-syn have a net negative charge at pH 5.5. This anomalous salt dependence implies that short-range attractive electrostatic interactions are critical for secondary nucleation as well as heterogeneous primary nucleation. Such interactions were confirmed in Monte Carlo simulations of α-syn monomers interacting with surface-grafted C-terminal tails, and found to be weakened in the presence of salt. Thus, nucleation of α-syn aggregation depends critically on an attractive electrostatic component that is screened by salt to the extent that it outweighs the screening of the long-range repulsion between negatively charged monomers and negative surfaces. Interactions between the positively charged N-termini of α-syn monomers on the one hand, and the negatively C-termini of α-syn on fibrils or vesicles surfaces on the other hand, are thus critical for nucleation.
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BACKGROUND: Long-term studies of community and population dynamics indicate that abrupt disturbances often catalyse changes in vegetation and carbon stocks. These disturbances include the opening of clearings, rainfall seasonality, and drought, as well as fire and direct human disturbance. Such events may be super-imposed on longer-term trends in disturbance, such as those associated with climate change (heating, drying), as well as resources. Intact neotropical forests have recently experienced increased drought frequency and fire occurrence, on top of pervasive increases in atmospheric CO2 concentrations, but we lack long-term records of responses to such changes especially in the critical transitional areas at the interface of forest and savanna biomes. Here, we present results from 20 years monitoring a valley forest (moist tropical forest outlier) in central Brazil. The forest has experienced multiple drought events and includes plots which have and which have not experienced fire. We focus on how forest structure (stem density and aboveground biomass carbon) and dynamics (stem and biomass mortality and recruitment) have responded to these disturbance regimes. RESULTS: Overall, the biomass carbon stock increased due to the growth of the trees already present in the forest, without any increase in the overall number of tree stems. Over time, both recruitment and especially mortality of trees tended to increase, and periods of prolonged drought in particular resulted in increased mortality rates of larger trees. This increased mortality was in turn responsible for a decline in aboveground carbon toward the end of the monitoring period. CONCLUSION: Prolonged droughts influence the mortality of large trees, leading to a decline in aboveground carbon stocks. Here, and in other neotropical forests, recent droughts are capable of shutting down and reversing biomass carbon sinks. These new results add to evidence that anthropogenic climate changes are already adversely impacting tropical forests.
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An amendment to this paper has been published and can be accessed via the original article.
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The deposition of α-synuclein fibrils is one hallmark of Parkinson's disease. Here, we investigate how ganglioside lipids, present in high amounts in neurons and exosomes, influence the aggregation kinetics of α-synuclein. Gangliosides, as well as, other anionic lipid species with small or large headgroups were found to induce conformational changes of α-synuclein monomers and catalyse their aggregation at mildly acidic conditions. Although the extent of this catalytic effect was slightly higher for gangliosides, the results imply that charge interactions are more important than headgroup chemistry in triggering aggregation. In support of this idea, uncharged lipids with large headgroups were not found to induce any conformational change and only weakly catalyse aggregation. Intriguingly, aggregation was also triggered by free ganglioside headgroups, while these caused no conformational change of α-synuclein monomers. Our data reveal that partially folded α-synuclein helical intermediates are not required species in triggering of α-synuclein aggregation.
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PURPOSE: Studies have suggested that corneal biomechanical properties influence intraocular pressure (IOP) measurements, namely central corneal thickness (CCT) and corneal hysteresis (CH). The present study aimed to investigate the associations of CH and CCT with glaucoma development. METHODS: We performed a review of the literature and meta-analysis of observational studies (2006-2016) that included both adult glaucoma patients and controls and reported CCT and CH as outcomes. Nineteen studies were conside red eligible, and the mean difference (MD) between groups (patient and control) for both variables was used for statistical analyses. RESULTS: A total of 1,213 glaucoma and 1,055 healthy eyes were studied. Quan titative analysis suggested that CH was significantly lower in the glaucoma group than in the control group (MD=-1.54 mmHg, 95% CI [-1.68, -1.41], P<0.0001). Additionally, CCT was significantly lower in the glaucoma group than in the control group (MD=-8.49 µm, 95% CI [-11.36, -5.62], P<0.001). CONCLUSION: Corneal properties appear to differ between glaucoma patients and healthy controls. Our results emphasize the importance of corneal biomechanical properties in IOP interpretation and should support further studies on the influence of CH and CCT in glaucoma screening and diagnosis.
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Córnea/fisiopatologia , Paquimetria Corneana , Glaucoma/fisiopatologia , Pressão Intraocular/fisiologia , Fenômenos Biomecânicos/fisiologia , Estudos de Casos e Controles , Córnea/patologia , Glaucoma/patologia , Humanos , Estudos Observacionais como Assunto , Valores de Referência , Fatores de RiscoRESUMO
The cork stopper manufacturing process includes an operation, known as stabilisation, by which humid cork slabs are extensively colonised by fungi. The effects of fungal growth on cork are not completely understood although they are considered to be involved in the so-called "cork taint" of wine. It is essential to (a) identify environmental constraints which define the appearance of the colonising fungal species and (b) trace their origin to the forest and/or the manufacturing space. The present article correlates two sets of data, from consecutive years and the same season, of systematic sampling of two manufacturing units, located in the North and South of Portugal. Chrysonilia sitophila dominance was confirmed, followed by a high diversity of Penicillium species. Penicillium glabrum, which was found in all samples, was the most frequently isolated species. P. glabrum intra-species variability was investigated using DNA fingerprinting techniques revealing highly discriminative polymorphic markers in the genome. Cluster analysis of P. glabrum data was discussed in relation to the geographical location of strains, and results suggest that P. glabrum arise from predominantly the manufacturing space, although cork specific fungi can contribute.