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Lipid nanovectors (LNVs) represent potent and versatile tools in the field of drug delivery for a wide range of medical applications including cancer therapy and vaccines. With this Technical Note, we introduce a novel "portable", easy-to-use, and low-cost strategy for double use: (1) it allows one to both quantify the amount of cargo in LNV formulation and (2) classify the nature of formulation with the aim of chemometrics. In particular, an electrochemical strip, based on a screen-printed electrode, was exploited to detect methylene blue (MB) as the model cargo encapsulated in various liposomes (used as model LNV). The experimental setup, including release of the MB content and its electrochemical quantification were optimized through a multivariate design of experiment (DoE), obtaining a satisfactory 88-95% accuracy in comparison to standard methods. In addition, the use of principal component analysis-linear discriminant analysis (PCA-LDA) highlighted the satisfactory differentiation of liposomes. The combination of portable electroanalysis and multivariate analysis is a potent tool for enhancing quality control in the field of pharmaceutical technologies, and also in the field of diagnostics, this approach might be useful for application toward naturally occurring lipid nanoparticles, i.e., exosomes.
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Técnicas Eletroquímicas , Lipossomos , Lipossomos/química , Azul de Metileno/química , Nanopartículas/química , Lipídeos/química , Análise de Componente Principal , Análise DiscriminanteRESUMO
Prolonged B cells stimulation due to the Hepatitis C virus (HCV) can result in autoimmunity, stigmatized by rising levels of cryoglobulins (CGs), the rheumatoid factor (RF), and free light chains (FLC) of immunoglobulins (Ig) associated with a range of symptoms, from their absence to severe cryoglobulinemic vasculitis and lymphoma. Here, we aimed to identify an immunological signature for the earliest stages of vasculitis when cryoprecipitate is still not detectable. We firstly analyzed the IgG subclasses, FLC, and RF in 120 HCV-RNA-positive patients divided into four groups according to the type of cryoprecipitate and symptoms: 30 asymptomatic without cryoprecipitate (No Cryo), 30 with vasculitis symptoms but without CGs that we supposed were circulating but still not detectable (Circulating), 30 type II and 30 type III mixed cryoglobulinemia (Cryo II and Cryo III, respectively). Our results revealed that patients with supposed circulating CGs displayed a pattern of serological parameters that closely resembled Cryo II and Cryo III, with a stronger similarity to Cryo II. Accordingly, we analyzed the groups of Circulating and Cryo II for their immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements, finding a similar mixed distribution of monoclonal, oligoclonal, and polyclonal responses compared to a control group of ten HCV-RNA-negative patients recovered from infection, who displayed a 100% polyclonal response. Our results strengthened the hypothesis that circulating CGs are the origin of symptoms in HCV-RNA-positive patients without cryoprecipitate and demonstrated that an analysis of clonal IGH and TCR rearrangements is the best option for the early diagnosis of extrahepatic complications.
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Crioglobulinemia , Crioglobulinas , Hepatite C Crônica , Vasculite , Vasculite/diagnóstico , Vasculite/imunologia , Vasculite/virologia , Humanos , Masculino , Feminino , Crioglobulinemia/diagnóstico , Crioglobulinemia/virologia , Crioglobulinas/análise , Fator Reumatoide/sangue , Imunoglobulinas/sangue , Hepatite C Crônica/sangue , Hepatite C Crônica/complicaçõesRESUMO
BACKGROUND: Extracellular Vesicles (EVs) are sub-micrometer lipid-bound particles released by most cell types. They are considered a promising source of cancer biomarkers for liquid biopsy and personalized medicine due to their specific molecular cargo, which provides biochemical information on the state of parent cells. Despite this potential, EVs translation process in the diagnostic practice is still at its birth, and the development of novel medical devices for their detection and characterization is highly required. RESULTS: In this study, we demonstrate mid-infrared plasmonic nanoantenna arrays designed to detect, in the liquid and dry phase, the specific vibrational absorption signal of EVs simultaneously with the unspecific refractive index sensing signal. For this purpose, EVs are immobilized on the gold nanoantenna surface by immunocapture, allowing us to select specific EV sub-populations and get rid of contaminants. A wet sample-handling technique relying on hydrophobicity contrast enables effortless reflectance measurements with a Fourier-transform infrared (FTIR) spectro-microscope in the wavelength range between 10 and 3 µm. In a proof-of-principle experiment carried out on EVs released from human colorectal adenocarcinoma (CRC) cells, the protein absorption bands (amide-I and amide-II between 5.9 and 6.4 µm) increase sharply within minutes when the EV solution is introduced in the fluidic chamber, indicating sensitivity to the EV proteins. A refractive index sensing curve is simultaneously provided by our sensor in the form of the redshift of a sharp spectral edge at wavelengths around 5 µm, where no vibrational absorption of organic molecules takes place: this permits to extract of the dynamics of EV capture by antibodies from the overall molecular layer deposition dynamics, which is typically measured by commercial surface plasmon resonance sensors. Additionally, the described metasurface is exploited to compare the spectral response of EVs derived from cancer cells with increasing invasiveness and metastatic potential, suggesting that the average secondary structure content in EVs can be correlated with cell malignancy. CONCLUSIONS: Thanks to the high protein sensitivity and the possibility to work with small sample volumes-two key features for ultrasensitive detection of extracellular vesicles- our lab-on-chip can positively impact the development of novel laboratory medicine methods for the molecular characterization of EVs.
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Vesículas Extracelulares , Neoplasias , Humanos , Vesículas Extracelulares/metabolismo , Biópsia Líquida , Neoplasias/metabolismo , Técnicas de Cultura de Células , Proteínas/análise , Amidas/análise , Amidas/metabolismoRESUMO
The aim of this work was to characterise three non-toxic ammunition (NTA) from the GECO and Fiocchi brands, which are available in the Italian market. Characterisation was carried out by considering both the elemental chemical composition and morphology, using scanning electron microscopy coupled with energy-dispersive X-ray analysis (SEM-EDS). Particles were collected from both the cartridge cases and the shooters' hands after shooting tests. Six volunteers fired two shots for each ammunition. Several elements, such as aluminium, potassium, silicon, sulphur, titanium and zinc were found in gunshot residue (GSR) particles from different ammunition. We also studied the persistence of these types of GSR on the hands of the shooters in a range between 1 and 6 h after shooting. The GSR particles from the three NTA tested were found on the hands of shooters until 6 h after the shots. The characterisations undertaken in this work will be useful for specialists in forensic science and legal medicine to evaluate trace evidence from these new NTA in casework, as such formulations are in growth.
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Armas de Fogo , Metaloides/análise , Metais Leves/análise , Pele/química , Zinco/análise , Balística Forense/métodos , Medicina Legal , Mãos , Humanos , Fatores de TempoRESUMO
In the medical field, extracellular vesicles (EVs) are gaining importance as they act as cells mediators. These are phospholipid bilayer vesicles and contain crucial biochemical information about their mother cells being carrier of different biomolecules such as small molecules, proteins, lipids, and nucleic acids. After release into the extracellular matrix, they enter the systemic circulation and can be found in all human biofluids. Since EVs reflect the state of the cell of origin, there is exponential attention as potential source of new circulating biomarkers for liquid biopsy. The use of EVs in clinical practice faces several challenges that need to be addressed: these include the standardization of lysis protocols, the availability of low-cost reagents and the development of analytical tools capable of detecting biomarkers. The process of lysis is a crucial step that can impact all subsequent analyses, towards the development of novel analytical strategies. To aid researchers to support the evolution of measurement science technology, this tutorial review evaluates and discuss the most commonly protocols used to characterize the contents of EVs, including their advantages and disadvantages in terms of experimental procedures, time and equipment. The purpose of this tutorial review is to offer practical guide to researchers which are intended to develop novel analytical approaches. Some of the most significant applications are considered, highlighting their main characteristics divided per mechanism of action. Finally, comprehensive tables which provide an overview at a glance are provided to readers.
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Vesículas Extracelulares , Ácidos Nucleicos , Humanos , Vesículas Extracelulares/química , Biópsia Líquida/métodos , Biomarcadores/análise , Ácidos Nucleicos/análise , Morte CelularRESUMO
The growth of liquid biopsy, i. e., the possibility of obtaining health information by analysing circulating species (nucleic acids, cells, proteins, and vesicles) in peripheric biofluids, is pushing the field of sensors and biosensors beyond the limit to provide decentralised solutions for nonspecialists. In particular, among all the circulating species that can be adopted in managing cancer evolution, both for diagnostic and prognostic applications, microRNAs have been highly studied and detected. The development of electrochemical devices is particularly relevant for liquid biopsy purposes, and the screen-printed electrodes (SPEs) represent one of the building blocks for producing novel portable devices. In this work, we have taken miR-2115-3p as model target (it is related to lung cancer), and we have developed a biosensor by exploiting the use of a complementary DNA probe modified with methylene blue as redox mediator. In particular, the chosen sensing architecture was applied to serum measurements of the selected miRNA, obtaining a detection limit within the low nanomolar range; in addition, various platforms were interrogated, namely commercial and hand-made SPEs, with the aim of providing the reader with some insights about the optimal platform to be used by considering both the cost and the analytical performance.
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Técnicas Biossensoriais , Técnicas Eletroquímicas , Eletrodos , MicroRNAs , Humanos , MicroRNAs/sangue , Biópsia Líquida , Técnicas Eletroquímicas/métodos , Neoplasias/diagnóstico , Limite de Detecção , Neoplasias Pulmonares/diagnóstico , Azul de Metileno/químicaRESUMO
Structural modification of different carbon-based nanomaterials is often necessary to improve their morphology and optical properties, particularly the incorporation of N-atoms in graphene quantum dots (GQDs). Here, a clean, simple, one-step, and eco-friendly method for N-doping of GQDs using gamma irradiation is reported. GQDs were irradiated in the presence of the different ethylenediamine (EDA) amounts (1 g, 5 g, and 10 g) and the highest % of N was detected in the presence of 10 g. N-doped GQDs emitted strong, blue photoluminescence (PL). Photoluminescence quantum yield was increased from 1.45, as obtained for non-irradiated dots, to 7.24% for those irradiated in the presence of 1 g of EDA. Modified GQDs were investigated as a PL probe for the detection of insecticide Carbofuran (2,2-Dimethyl-2,3-dihydro-1-benzofuran-7-yl methylcarbamate) and herbicide Amitrole (3-amino-1,2,4-triazole). The limit of detection was 5.4 µmol L-1 for Carbofuran. For the first time, Amitrole was detected by GQDs in a turn-off/turn-on mechanism using Pd(II) ions as a quenching agent. First, Pd(II) ions were quenched (turn-off) PL of GQDs, while after Amitrole addition, PL was recovered linearly with Amitrole concentration (turn-on). LOD was 2.03 µmol L-1. These results suggest that modified GQDs can be used as an efficient new material for Carbofuran and Amitrole detection. Furthermore, the phototoxicity of dots was investigated on both Gram-positive and Gram-negative bacterial strains. When bacterial cells were exposed to different GQD concentrations and illuminated with light of 470 nm wavelength, the toxic effects were not observed.
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Extracellular vesicles (EVs) are abundantly released into the systemic circulation, where they show remarkable stability and harbor molecular constituents that provide biochemical information about their cells of origin. Due to this characteristic, EVs are attracting increasing attention as a source of circulating biomarkers for cancer liquid biopsy and personalized medicine. Despite this potential, none of the discovered biomarkers has entered the clinical practice so far, and novel approaches for the label-free characterization of EVs are highly demanded. In this regard, Fourier Transform Infrared Spectroscopy (FTIR) has great potential as it provides a quick, reproducible, and informative biomolecular fingerprint of EVs. In this pilot study, we investigated, for the first time in the literature, the capability of FTIR spectroscopy to distinguish between EVs extracted from sera of cancer patients and controls based on their mid-IR spectral response. For this purpose, EV-enriched suspensions were obtained from the serum of patients diagnosed with Hepatocellular Carcinoma (HCC) of nonviral origin and noncancer subjects. Our data point out the presence of statistically significant differences in the integrated intensities of major mid-IR absorption bands, including the carbohydrate and nucleic acids band, the protein amide I and II bands, and the lipid CH stretching band. Additionally, we used Principal Component Analysis combined with Linear Discriminant Analysis (PCA-LDA) for the automated classification of spectral data according to the shape of specific mid-IR spectral signatures. The diagnostic performances of the proposed spectral biomarkers, alone and combined, were evaluated using multivariate logistic regression followed by a Receiving Operator Curve analysis, obtaining large Areas Under the Curve (AUC = 0.91, 95% CI 0.81-1.0). Very interestingly, our analyses suggest that the discussed spectral biomarkers can outperform the classification ability of two widely used circulating HCC markers measured on the same groups of subjects, namely alpha-fetoprotein (AFP), and protein induced by the absence of vitamin K or antagonist-II (PIVKA-II).
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Exosomes (EXOs) are considered an exceptionally promising source of cancer biomarkers for personalized medicine and liquid biopsy. Despite this potential, the EXOs translation process in diagnostics is still at its birth, and the development of reliable and reproducible methods for their characterization is highly demanded. Fourier Transform Infrared (FTIR) Spectroscopy is perfectly suited for this purpose, as it can provide a label-free biochemical profile of EXOs in terms of lipid, protein, and nucleic acid content. Here we evaluated the applicability of FTIR spectroscopy to the study of cancer-derived EXOs as a function of cell differentiation. For this purpose, we used N-acetyl-l-Cysteine (NAC) to induce a controlled differentiation in human colon carcinoma cells from a proliferative mesenchymal morphology to a less invasive epithelial phenotype, as measured with fluorescence and electron microscopy. EXOs derived from cells with different phenotypes showed significant variation in the relative intensity of the amide I-II and CH-stretching bands in the mid-IR range, indicating the spectroscopic lipid/protein ratio as an effective classification parameter. Additionally, we showed that different cell phenotypes are associated with a shape modification in these spectral bands that can be automatically detected by combining Principal Component Analysis (PCA) with Linear Discriminant Analysis (LDA). On the one hand, our study confirms that an FTIR analysis of EXOs allows scientists to precisely detect modifications occurring at the parental cell level; on the other hand, it unveils a set of effective spectral biomarkers able to monitoring cell changes from a mesenchymal to an epithelial phenotype, a clinically valuable piece of information considering that the epithelial-to-mesenchymal transition is a key step in the metastatic process.
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Exossomos , Neoplasias , Diferenciação Celular , Análise Discriminante , Humanos , Proteômica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Red blood cells (RBCs) are characterized by a remarkable elasticity, which allows them to undergo very large deformation when passing through small vessels and capillaries. This extreme deformability is altered in various clinical conditions, suggesting that the analysis of red blood cell (RBC) mechanics has potential applications in the search for non-invasive and cost-effective blood biomarkers. Here, we provide a comparative study of the mechanical response of RBCs in patients with Alzheimer's disease (AD) and healthy subjects. For this purpose, RBC viscoelastic response was investigated using atomic force microscopy (AFM) in the force spectroscopy mode. Two types of analyses were performed: (i) a conventional analysis of AFM force-distance (FD) curves, which allowed us to retrieve the apparent Young's modulus, E; and (ii) a more in-depth analysis of time-dependent relaxation curves in the framework of the standard linear solid (SLS) model, which allowed us to estimate cell viscosity and elasticity, independently. Our data demonstrate that, while conventional analysis of AFM FD curves fails in distinguishing the two groups, the mechanical parameters obtained with the SLS model show a very good classification ability. The diagnostic performance of mechanical parameters was assessed using receiving operator characteristic (ROC) curves, showing very large areas under the curves (AUC) for selected biomarkers (AUC > 0.9). Taken all together, the data presented here demonstrate that RBC mechanics are significantly altered in AD, also highlighting the key role played by viscous forces. These RBC abnormalities in AD, which include both a modified elasticity and viscosity, could be considered a potential source of plasmatic biomarkers in the field of liquid biopsy to be used in combination with more established indicators of the pathology.
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Drug adulterants containing contaminants have been known to cause lung disease by inhalation or intravenous intake. Talcosis due to intravenous talc injection has been widely described in the literature, whereas the hypothesis of granulomatosis due to asbestos related to adulterated cocaine injection has not yet been explored. Herein, a case of pulmonary granulomatosis due to asbestos fibres related to cocaine injection in a young woman is described. Inorganic material in the lung was first individuated by light microscopy and last was identified using the SEM-EDX method. This case is unique since the occupational and passive inhalation of asbestos was excluded with absolute certainty.
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Amianto/efeitos adversos , Estimulantes do Sistema Nervoso Central/efeitos adversos , Transtornos Relacionados ao Uso de Cocaína/complicações , Cocaína/efeitos adversos , Contaminação de Medicamentos , Granuloma de Corpo Estranho/etiologia , Granuloma do Sistema Respiratório/etiologia , Abuso de Substâncias por Via Intravenosa/complicações , Adulto , Amianto/administração & dosagem , Autopsia , Estimulantes do Sistema Nervoso Central/administração & dosagem , Cocaína/administração & dosagem , Usuários de Drogas , Evolução Fatal , Feminino , Granuloma de Corpo Estranho/patologia , Granuloma do Sistema Respiratório/patologia , HumanosRESUMO
Exosomes (EXOs) are nano-sized vesicles secreted by most cell types. They are abundant in bio-fluids and harbor specific molecular constituents from their parental cells. Due to these characteristics, EXOs have a great potential in cancer diagnostics for liquid biopsy and personalized medicine. Despite this unique potential, EXOs are not yet widely applied in clinical settings, with two main factors hindering their translational process in diagnostics. Firstly, conventional extraction methods are time-consuming, require large sample volumes and expensive equipment, and often do not provide high-purity samples. Secondly, characterization methods have some limitations, because they are often qualitative, need extensive labeling or complex sampling procedures that can induce artifacts. In this context, novel label-free approaches are rapidly emerging, and are holding potential to revolutionize EXO diagnostics. These methods include the use of nanodevices for EXO purification, and vibrational spectroscopies, scattering, and nanoindentation for characterization. In this progress report, we summarize recent key advances in label-free techniques for EXO purification and characterization. We point out that these methods contribute to reducing costs and processing times, provide complementary information compared to the conventional characterization techniques, and enhance flexibility, thus favoring the discovery of novel and unexplored EXO-based biomarkers. In this process, the impact of nanotechnology is systematically highlighted, showing how the effectiveness of these techniques can be enhanced using nanomaterials, such as plasmonic nanoparticles and nanostructured surfaces, which enable the exploitation of advanced physical phenomena occurring at the nanoscale level.
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Nowadays, a larger number of aggressive and corrosive chemical reagents as well as toxic solvents are used to achieve structural modification and cleaning of the final products. These lead to the production of residual, waste chemicals, which are often reactive, cancerogenic, and toxic to the environment. This study shows a new approach to the modification of graphene quantum dots (GQDs) using gamma irradiation where the usage of reagents was avoided. We achieved the incorporation of S and N atoms in the GQD structure by selecting an aqueous solution of L-cysteine as an irradiation medium. GQDs were exposed to gamma-irradiation at doses of 25, 50 and 200 kGy. After irradiation, the optical, structural, and morphological properties, as well as the possibility of their use as an agent in bioimaging and photodynamic therapy, were studied. We measured an enhanced quantum yield of photoluminescence with the highest dose of 25 kGy (21.60%). Both S- and N-functional groups were detected in all gamma-irradiated GQDs: amino, amide, thiol, and thione. Spin trap electron paramagnetic resonance showed that GQDs irradiated with 25 kGy can generate singlet oxygen upon illumination. Bioimaging on HeLa cells showed the best visibility for cells treated with GQDs irradiated with 25 kGy, while cytotoxicity was not detected after treatment of HeLa cells with gamma-irradiated GQDs.
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We report the case of a 38 year-old Caucasian man enrolled in a study aimed at investigating the physical properties of red blood cells (RBCs) using advanced microscopy techniques, including Atomic Force Microscopy (AFM). At the time of his first enrolment in the study, he had normal Fasting Plasma Glucose (FPG) values, a BMI of 24.1, and no other symptoms of diabetes, including fatigue, high triglycerides, low HDL cholesterol, and altered inflammatory and corpuscular RBC indices. The subject reported no family history of diabetes, obesity, and cardiovascular diseases. Despite his apparently healthy conditions, the biomechanics of his RBCs was altered, showing increased values of stiffness and viscosity. More than 1 year after the mechanical measurements, the subject was admitted to the Operational Unit of Diabetology of the Policlinico Gemelli Hospital with high blood glucose and glycosylated hemoglobin (HbA1c) levels and diagnosed with type 1 diabetes (T1DM). Here, we show these data, and we discuss the hypothesis that RBC mechanical properties could be sensitive to changes occurring during the pre-diabetic phase of T1DM.
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Exosomes possess great potential as cancer biomarkers in personalized medicine due to their easy accessibility and capability of representing their parental cells. To boost the translational process of exosomes in diagnostics, the development of novel and effective strategies for their label-free and automated characterization is highly desirable. In this context, Fourier Transform Infrared Spectroscopy (FTIR) has great potential as it provides direct access to specific biomolecular bands that give compositional information on exosomes in terms of their protein, lipid and genetic content. Here, we used FTIR spectroscopy in the mid-Infrared (mid-IR) range to study exosomes released from human colorectal adenocarcinoma HT-29 cancer cells cultured in different media. To this purpose, cells were studied in well-fed condition of growth, with 10% of exosome-depleted FBS (EVd-FBS), and under serum starvation with 0.5% EVd-FBS. Our data show the presence of statistically significant differences in the shape of the Amide I and II bands in the two conditions. Based on these differences, we showed the possibility to automatically classify cancer cell-derived exosomes using Principal Component Analysis combined with Linear Discriminant Analysis (PCA-LDA); we tested the effectiveness of the classifier with a cross-validation approach, obtaining very high accuracy, precision, and recall. Aside from classification purposes, our FTIR data provide hints on the underlying cellular mechanisms responsible for the compositional differences in exosomes, suggesting a possible role of starvation-induced autophagy.
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Adenocarcinoma , Exossomos , Análise Discriminante , Humanos , Análise de Componente Principal , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Vitreomacular traction (VMT) syndrome has only been surgically treated for a long time. Recently, enzymatic vitreolysis with ocriplasmin has emerged as a possible option to release VMT and, in some cases, close full thickness macular holes (FTMHs). Despite its clinical relevance, gathering information about the ocriplasmin-induced alterations of the Inner Limiting Membrane (ILM) of the retina in a clinical study is a complex task, mainly because of the inter-individual variability among patients. To obtain more insights into the mechanism underlying the drug action, we studied in-vitro the mechanical and morphological changes of the ILM using Atomic Force Microscopy (AFM). To this aim, we measured the ILM average Young's modulus (YM), hysteresis (H) and adhesion work (A) over time under ocriplasmin treatment. Our data unveil a time-dependent increase in the membrane YM of 19% of its initial value, along with changes in its adhesive and dissipative behavior. Such modifications well correlate with the morphological alterations detected in the AFM imaging mode. Taken all together, the results here presented provide more insights into the mechanism underlying the ocriplasmin action in-vivo, suggesting that it is only able to alter the top-most layer of the vitreal side of the membrane, not compromising the inner ILM structure.
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Descolamento do Vítreo , Fibrinolisina , Humanos , Injeções Intravítreas , Fragmentos de Peptídeos , Retina , Tomografia de Coerência Óptica , Acuidade Visual , Descolamento do Vítreo/tratamento farmacológicoRESUMO
Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.