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1.
Insect Mol Biol ; 33(4): 362-371, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38450861

RESUMO

Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha-mannosidase-2 (αMan2) gene. Here we use CRISPR-Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia-infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use of Wolbachia-based strategies to control vector-borne pathogens.


Assuntos
Aedes , Wolbachia , alfa-Manosidase , Animais , Aedes/microbiologia , Aedes/virologia , Aedes/genética , Wolbachia/fisiologia , alfa-Manosidase/metabolismo , alfa-Manosidase/genética , Vírus da Dengue/fisiologia , Arbovírus/fisiologia , Mosquitos Vetores/microbiologia , Mosquitos Vetores/virologia , Mosquitos Vetores/genética , Feminino , Infecções por Arbovirus/transmissão , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Sistemas CRISPR-Cas
2.
Insect Mol Biol ; 31(3): 356-368, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35112745

RESUMO

One approach to control dengue virus transmission is the symbiont Wolbachia, which limits viral infection in mosquitoes. Despite plans for its widespread use in Aedes aegypti, Wolbachia's mode of action remains poorly understood. Many studies suggest that the mechanism is likely multifaceted, involving aspects of immunity, cellular stress and nutritional competition. A previous study from our group used artificial selection to identify a new mosquito candidate gene related to viral blocking; alpha-mannosidase-2a (alpha-Mann-2a) with a predicted role in protein glycosylation. Protein glycosylation pathways tend to be involved in complex host-viral interactions; however, the function of alpha-mannosidases has not been described in mosquito-virus interactions. We examined alpha-Mann-2a expression in response to virus and Wolbachia infections and whether reduced gene expression, caused by RNA interference, affected viral loads. We show that dengue virus (DENV) infection affects the expression of alpha-Mann-2a in a tissue- and time-dependent manner, whereas Wolbachia infection had no effect. In the midgut, DENV prevalence increased following knockdown of alpha-Mann-2a expression in Wolbachia-free mosquitoes, suggesting that alpha-Mann-2a interferes with infection. Expression knockdown had the same effect on the togavirus chikungunya virus, indicating that alpha-Mann-2a may have broad antivirus effects in the midgut. Interestingly, we were unable to knockdown the expression in Wolbachia-infected mosquitoes. We also provide evidence that alpha-Mann-2a may affect the transcriptional level of another gene predicted to be involved in viral blocking and cell adhesion; cadherin87a. These data support the hypothesis that glycosylation and adhesion pathways may broadly be involved in viral infection in Ae. aegypti.


Assuntos
Aedes , Vírus Chikungunya , Vírus da Dengue , Viroses , Wolbachia , Aedes/genética , Animais , Vírus da Dengue/genética , Mosquitos Vetores/genética , Wolbachia/fisiologia
3.
J Musculoskelet Neuronal Interact ; 20(2): 291-296, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32481245

RESUMO

Vitamin D (vitD) deficiency and bone loss may occur after bariatric surgery and hence, supplementation with high oral doses of vitD may be required. Alternatively, intramuscular depot ergocalciferol, which slowly releases vitD and bypasses the gastrointestinal tract, could be administrated. We present a case of severe vitD deficiency-osteomalacia after gastric bypass operation for morbid obesity, treated with ergocalciferol intramuscularly. A 45-year-old woman was presented with hip pain and muscle weakness, which led ultimately to immobilization in a wheelchair. Fifteen years ago, she underwent roux-en-Y gastric by-pass for morbid obesity. Occasionally, she was treated with multivitamin supplements. On admission, iron deficiency anaemia, vitD deficiency (25OHD: 3.7 ng/ml) and secondary hyperparathyroidism were revealed. Bone turnover markers (BTM) were elevated. Radiological evaluation demonstrated insufficiency fractures on the pubic and left femur and reduced BMD. Osteomalacia due to vitD deficiency and calcium malabsorption were diagnosed. Calcium citrate 500 mg qid and intramuscular ergocalciferol 600,000 IU every 20 days were initiated. One month later, musculoskeletal pain and weakness were resolved and the patient was mobilized. Few months later, vitD, BTM and BMD showed substantial improvement. Intramuscular ergocalciferol administration can improve the clinical and biochemical status and thus, is suggested to prevent and/or treat osteomalacia in such patients.


Assuntos
Conservadores da Densidade Óssea/administração & dosagem , Ergocalciferóis/administração & dosagem , Fraturas de Estresse/etiologia , Derivação Gástrica/efeitos adversos , Osteomalacia/tratamento farmacológico , Deficiência de Vitamina D/tratamento farmacológico , Preparações de Ação Retardada/administração & dosagem , Feminino , Humanos , Injeções Intramusculares , Pessoa de Meia-Idade , Obesidade Mórbida/cirurgia , Osteomalacia/etiologia , Deficiência de Vitamina D/etiologia
4.
PLoS Comput Biol ; 13(9): e1005779, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28945754

RESUMO

Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR) trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system.


Assuntos
Transporte Biológico/fisiologia , Modelos Biológicos , Receptores de Superfície Celular/metabolismo , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Biologia Computacional , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Cinética , Microscopia Confocal , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/química , Receptores da Eritropoetina
5.
Anal Chem ; 82(19): 8186-93, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20815338

RESUMO

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.


Assuntos
Corantes Fluorescentes/química , Guanina/análogos & derivados , Microscopia de Fluorescência/métodos , Proteínas Recombinantes de Fusão/química , Animais , Escherichia coli/metabolismo , Guanina/química , Guanina/farmacologia , Humanos , Camundongos , Células NIH 3T3 , O(6)-Metilguanina-DNA Metiltransferase/química , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT5/química , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo
6.
Chemphyschem ; 11(1): 43-53, 2010 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-19960557

RESUMO

The recent explosion in papers utilising single-molecule experiments pushes the envelope further for increased spatial and temporal resolution. In order to achieve this, a combination of novel fluorescent probes and spectroscopy techniques are required. Herein, we provide an overview on our contribution to developments in the field of fluorescent probes along with a palette of alternative delivery methods for introducing the probes into living cells. We discuss probe requirements arising from the use of single-molecule spectroscopy methods and the customisation of probes that depends on the target molecule, the chemical state of the molecule as well as the distance and the type of interaction between sensor and ligand. We explain how Förster resonance energy transfer (FRET) and photon-induced electron transfer (PET) can increase the probe customisation. We also discuss additional requirements that arise when performing experiments in living cells like toxicity and cell permeability. Regarding the latter, we devote a special paragraph on the different ways to introduce the desired probe into the cell and how the different properties of each probe and cell type may require different delivery methods. We offer insights based on our experience working with a variety of single-molecule methods, fluorescent probes and delivery systems. Overall, we encompass the latest developments on probe design and delivery and illustrate that the wealth of information provided by single-molecule studies goes along with increased complexity.


Assuntos
Citofotometria/métodos , Corantes Fluorescentes/farmacocinética , Animais , Células/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Análise Espectral/métodos
7.
Methods Mol Biol ; 543: 503-21, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19378183

RESUMO

We present a single-molecule method for studying protein-DNA interactions based on fluorescence resonance energy transfer (FRET) and alternating-laser excitation (ALEX) of single diffusing molecules. An application of this method to the study of a bacterial transcription initiation complex is presented.


Assuntos
DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/metabolismo , Soluções Tampão , Cromatografia Líquida de Alta Pressão , DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/metabolismo , Lasers , Espectrofotometria Ultravioleta , Coloração e Rotulagem , Transcrição Gênica
8.
Insect Biochem Mol Biol ; 111: 103169, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31103782

RESUMO

The yellow fever mosquito, Aedes aegypti, serves as the primary vector for epidemic transmission of yellow fever, dengue, Zika (ZIKV), and chikungunya viruses to humans. Control of Ae. aegypti is currently limited to insecticide applications and larval habitat management; however, to combat growing challenges with insecticide resistance, novel genetic approaches for vector population reduction or transmission interruption are being aggressively pursued. The objectives of this study were to assess the ability of the Ae. aegypti antiviral exogenous-small interfering RNA (exo-siRNA) response to inhibit ZIKV infection and transmission, and to identify the optimal RNA interference (RNAi) target region in the ZIKV genome. We accomplished these objectives by in vitro transcription of five long double-stranded RNAs (dsRNAs) from the genome region spanning the NS2B-NS3-NS4A genes, which were the most highly conserved among ZIKV RNA sequences representing both East and West African and Asian-American clades, and evaluation of the ability of these dsRNAs to trigger an effective antiviral exo-siRNA response after intrathoracic injection into Ae. aegypti. In a pilot study, five ZIKV dsRNAs were tested by intrathoracic inoculation of 250 ng dsRNA into groups of approximately 5-day-old mosquitoes. Three days post-inoculation, mosquitoes were provided an infectious blood-meal containing ZIKV strain PRVABC59 (Puerto Rico), MR766 (Uganda), or 41525 (Senegal). On days 7 and 14 post-infection individual whole mosquito bodies were assessed for ZIKV infectious titer by plaque assays. Based on the results of this initial assessment, three dsRNAs were selected for further evaluation of viral loads of matched body and saliva expectorants using a standardized infectious dose of 1 × 107 PFU/mL of each ZIKV strain. Fourteen days post-exposure to ZIKV, paired saliva and carcass samples were harvested from individual mosquitoes and assessed for ZIKV RNA load by qRT-PCR. Injection of each of the three dsRNAs resulted in significant inhibition of replication of all three strains of ZIKV in mosquito bodies and saliva. This study lays critical groundwork for pursuing ZIKV transmission-blocking strategies that exploit the Ae. aegypti exo-siRNA response for arbovirus suppression in natural populations.


Assuntos
Aedes/virologia , Interferência de RNA , Infecção por Zika virus/transmissão , Zika virus/genética , Animais , Bovinos , Chlorocebus aethiops , Mosquitos Vetores/virologia , Projetos Piloto , RNA de Cadeia Dupla , RNA Interferente Pequeno , Saliva/virologia , Análise de Sequência de RNA , Células Vero , Carga Viral , Replicação Viral , Zika virus/fisiologia , Infecção por Zika virus/virologia
9.
Chest ; 154(4): e101-e105, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30290950

RESUMO

CASE PRESENTATION: A 38-year-old man of Indian origin, who migrated to Greece 13 years prior to presentation, was admitted to our hospital with a 2-month history of nonprogressive, intermittent (mostly evening), low-grade (up to 38.5°C) fever, accompanied by night sweats, dry cough, mild dyspnea on exertion (modified Medical Research Council Dyspnea Scale grade 1), anorexia, fatigue, and weight loss of 10 kg. He also experienced continuous palpitations, which were regular, not associated with chest pain or dizziness, and aggravated on exertion. He had not taken any medication for his condition, except for antipyretic agents, nor had he sought medical advice. He was a nonsmoker, had a history of past alcohol dependence, and had been hospitalized twice for acute pancreatitis due to hypertriglyceridemia. He had also been diagnosed with diabetes mellitus, presumably poorly controlled because he mentioned not taking any medication or having regular follow-up.


Assuntos
Cardiomiopatia Dilatada/microbiologia , Miocardite/microbiologia , Tuberculose Cardiovascular/diagnóstico , Adulto , Antituberculosos/uso terapêutico , Arritmias Cardíacas/microbiologia , Tosse/microbiologia , Quimioterapia Combinada , Dispneia/microbiologia , Febre/microbiologia , Humanos , Masculino , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Cardiovascular/tratamento farmacológico , Redução de Peso
11.
Hormones (Athens) ; 15(2): 291-296, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27376431

RESUMO

UNLABELLED: Primary hypophysitis (PH) is a rare clinical entity characterized by inflammatory infiltration of the pituitary gland with various degrees of pituitary dysfunction. OBJECTIVE: To present a complicated case of aggressive PH with bilateral cavernous sinuses infiltration, successfully treated with azathioprine after failure of corticosteroid treatment. METHODS AND RESULTS: A 48-year-old woman presented with episodes of recurrent headache and progressively worsening muscle weakness. Magnetic resonance imaging (MRI) identified an intrasellar pituitary lesion with thickened pituitary stalk extending to the cavernous sinuses and causing asymptomatic occlusion of both internal carotid arteries (ICAs). Hormonal investigation showed severe anterior pituitary deficiency. The diagnosis of PH, and more specifically of lymphocytic hypophysitis (LYH), was suspected and glucocorticoid treatment was initiated. Because of the patient's susceptibility to infections, the attempt to gradually reduce glucocorticoid dosage induced a relapse of PH. Immunosuppressive therapy with azathioprine was administered. Significant pituitary mass reduction with regression of the inflammation to the cavernous sinuses was documented. At follow-up the pituitary function was normal, while the patient was on the minimum dose of azathioprine. Thereafter, azathioprine was discontinued without any clinical/biochemical or radiological evidence of PH except for the permanent ICA occlusion. CONCLUSIONS: Despite its rarity, PH should be included in the differential diagnosis of pituitary masses and involvement of ICAs occlusion should not be underestimated. Azathioprine, applied as an alternative treatment, was shown to result in remarkable PH improvement.


Assuntos
Hipofisite Autoimune/complicações , Artéria Carótida Interna , Estenose das Carótidas/etiologia , Corticosteroides/uso terapêutico , Hipofisite Autoimune/diagnóstico por imagem , Hipofisite Autoimune/tratamento farmacológico , Azatioprina/uso terapêutico , Artéria Carótida Interna/diagnóstico por imagem , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/tratamento farmacológico , Angiografia por Tomografia Computadorizada , Substituição de Medicamentos , Feminino , Humanos , Imunossupressores/uso terapêutico , Imageamento por Ressonância Magnética , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Testes de Função Hipofisária , Fatores de Risco , Resultado do Tratamento
13.
Biophys Chem ; 184: 1-7, 2013 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-24012911

RESUMO

Biosensors have recognized a rapid development the last years in both industry and science. Recently, a single-molecule assay based on alternating laser excitation has been established for the quantitative detection of transcription factors. These proteins specifically recognize and bind DNA and play an important role in controlling gene expression. We implemented this assay format on a total internal reflection fluorescence microscope to detect transcription factors with immobilized single-molecule DNA biosensors. We quantify transcription factors via colocalization of the two halves of their binding site with immobilized single molecules of a two-color DNA biosensor. We could detect a model transcription factor, the bacterial lactose repressor, at different concentrations down to 150pM. We found that robust modeling of stoichiometry derived TIRF data is achieved with Student's t-distributions and nonlinear least-squares estimation with weights equal to the inverse of the expected number of bin entries. This significantly improved transcription factor concentration estimates with respect to distribution modeling with Gaussians without adding notable computational effort. The proposed model may enhance the precision of other single-molecule assays quantifying molecular distributions. Our measurements reliably confirm that the immobilized biosensor format is more sensitive than a previously published solution based approach.


Assuntos
Técnicas Biossensoriais , DNA/química , Fatores de Transcrição/análise , Propriedades de Superfície , Fatores de Transcrição/química
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