RESUMO
Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. Here, we describe the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodeling by Vasa facilitates transfer of 5' sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. Our results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons.
Assuntos
Bombyx/metabolismo , Proteínas de Insetos/metabolismo , RNA Interferente Pequeno/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bombyx/genética , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Proteínas de Insetos/genética , Mutação , Ovário/citologia , Ovário/metabolismoRESUMO
INTRODUCTION: Aim of the study was to evaluate perioperative, postoperative and mid-term functional outcomes of Florence intracorporeal neobladder (FloRIN) configuration technique performed with stentless procedure. MATERIALS AND METHODS: This single institution randomized 1:1 prospective series included consecutive patients treated with Robot-Assisted Radical Cystectomy (RARC) and FloRIN reconfiguration from January 2021 to February 2022. Postoperative complications were graded according to Clavien Dindo classification and divided in early (<30 days from discharge) and delayed (>30 days). RESULTS: Overall, 63 patients were included in the analysis. Among these 32 (50.8 %) were treated with RARC + stentless FloRIN while 31 (49.2 %) underwent stent placement procedure. No differences were found in terms of baseline characteristics between the two groups. Stentless procedure was associated with significant shorter console time 328 vs 374 min (p = 0.04) and lower estimated blood loss (EBL) 330 vs 350 ml (p = 0.04) comparing to stent group. As regards perioperative features, no significant differences were recorded in terms of canalization (p = 0.58) and time to drainage removal (p = 0.11) while a shorter length of hospital stay was found in case of stentless procedure (p = 0.04). Early postoperative complications Clavien ≥ 3a occurred in 9.3 % and 12.9 % of patients while delayed major complications were recorded in the 3.1 % and 9.6 % of patients treated with stentless and stent FloRIN, respectively (p = 0.09). As regards the mid-term functional outcomes, no differences were found in terms of kidney function loss in both 3rd and 6th month assessment (p = 0.13 and p = 0.14, respectively). CONCLUSIONS: In conclusion, Stentless FloRIN is a feasible and safe IntraCorporeal Neobladder technique, as confirmed by the worthy functional and perioperative outcomes achieved in comparison with the standard FloRIN ureteral management strategy.
Assuntos
Procedimentos Cirúrgicos Robóticos , Robótica , Neoplasias da Bexiga Urinária , Derivação Urinária , Humanos , Procedimentos Cirúrgicos Robóticos/métodos , Neoplasias da Bexiga Urinária/cirurgia , Neoplasias da Bexiga Urinária/complicações , Estudos de Viabilidade , Resultado do Tratamento , Cistectomia/métodos , Complicações Pós-Operatórias/etiologia , Derivação Urinária/métodosRESUMO
OBJECTIVE: To describe a case of acute nonherpetic limbic encephalitis (LE) with negative testing for antibodies directed against onconeuronal and cell membrane antigens, including voltage-gated potassium channels and N-methyl-D-aspartate receptor, that showed a dramatic response to immune therapy. MATERIALS AND METHODS: A 30-year-old woman manifested generalized seizures, altered consciousness, and memory impairment shortly after a prodromal viral illness. Few days later the patient developed a drug-resistant epileptic status. RESULTS: Electroencephalograph showed bitemporal slowing and paroxysmal slow wave bursts. Brain magnetic resonance imaging showed bilateral swelling in the medial temporal lobes. Cerebrospinal fluid analysis ruled out viral etiologies. A diagnostic search for cancer, including serum testing for known onconeuronal antibodies proved negative. Screening for cell membrane antigen antibodies, including voltage-gated potassium channels and N-methyl-D-aspartate receptor, was also negative. Suspecting an autoimmune etiology, we started an immunomodulatory treatment with intravenous immunoglobulin followed by a short course of oral prednisone, obtaining a full clinical recovery. CONCLUSIONS: Our report confirms previous observations of "seronegative" autoimmune LE, suggesting the presence of other, still unknown central nervous system antigens representing a target of a postinfectious, autoimmune response in these patients. Moreover, it emphasizes the importance of early recognition and treatment of acute autoimmune LE, to reduce the risk of intensive care unit-related complications and the occurrence of permanent cognitive or behavioral defects.
Assuntos
Doenças Autoimunes/terapia , Imunoglobulinas Intravenosas/uso terapêutico , Encefalite Límbica/terapia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/imunologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/imunologia , Doença Aguda , Adulto , Anti-Inflamatórios/uso terapêutico , Anticonvulsivantes/uso terapêutico , Doenças Autoimunes/imunologia , Doenças Autoimunes/psicologia , Membrana Celular/imunologia , Eletroencefalografia , Feminino , Humanos , Encefalite Límbica/imunologia , Encefalite Límbica/psicologia , Imageamento por Ressonância Magnética , Transtornos da Memória/etiologia , Transtornos da Memória/psicologia , Testes Neuropsicológicos , Prednisona/uso terapêutico , Convulsões/tratamento farmacológico , Convulsões/etiologiaRESUMO
Duchenne's muscular dystrophy is an X-linked neuromuscular disease that manifests as muscle atrophy and cardiomyopathy in young boys. However, a considerable percentage of carrier females are often diagnosed with cardiomyopathy at an advanced stage. Existing therapy is not disease-specific and has limited effect, thus many patients and symptomatic carrier females prematurely die due to heart failure. Early detection is one of the major challenges that muscular dystrophy patients, carrier females, family members and, research and medical teams face in the complex course of dystrophic cardiomyopathy management. Despite the widespread adoption of advanced imaging modalities such as cardiac magnetic resonance, there is much scope for refining the diagnosis and treatment of dystrophic cardiomyopathy. This comprehensive review will focus on the pertinent clinical aspects of cardiac disease in muscular dystrophy while also providing a detailed consideration of the known and developing concepts in the pathophysiology of muscular dystrophy and forthcoming therapeutic options.
RESUMO
Becker muscular dystrophy (BMD) is a dystrophinopathy caused by mutations in the dystrophin gene on chromosome Xp21. BMD mutations result in truncated semi-functional dystrophin isoforms. Consequently, less severe clinical symptoms become apparent later in life compared to Duchenne muscular dystrophy. Dermal fibroblasts from a BMD patient were electroporated with episomal plasmids containing reprogramming factors to create the induced pluripotent stem cell line: CCMi002BMD-A-9 that showed pluripotent markers, were karyotypically normal and capable of trilineage differentiation. MLPA analyses performed on DNA extracted from CCMi002BMD-A-9 showed an in-frame deletion of exons 45 to 55 (CCMi002BMD-A-9 Δ45-55).
Assuntos
Técnicas de Cultura de Células/métodos , Distrofina/genética , Éxons/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Deleção de Sequência/genética , Adulto , Humanos , MasculinoRESUMO
Small RNAs called PIWI-interacting RNAs (piRNAs) act as an immune system to suppress transposable elements in the animal gonads. A poorly understood adaptive pathway links cytoplasmic slicing of target RNA by the PIWI protein MILI to loading of target-derived piRNAs into nuclear MIWI2. Here we demonstrate that MILI slicing generates a 16-nt by-product that is discarded and a pre-piRNA intermediate that is used for phased piRNA production. The ATPase activity of Mouse Vasa Homolog (MVH) is essential for processing the intermediate into piRNAs, ensuring transposon silencing and male fertility. The ATPase activity controls dissociation of an MVH complex containing PIWI proteins, piRNAs, and slicer products, allowing safe handover of the intermediate. In contrast, ATPase activity of TDRD9 is dispensable for piRNA biogenesis but is essential for transposon silencing and male fertility. Our work implicates distinct RNA helicases in specific steps along the nuclear piRNA pathway.
Assuntos
Proteínas Argonautas/metabolismo , DNA Helicases/metabolismo , Inativação Gênica/fisiologia , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/genética , Elementos de DNA Transponíveis/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Camundongos , RNA Interferente Pequeno/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is caused by abnormalities in the dystrophin gene and is clinically characterised by childhood muscle degeneration and cardiomyopathy. We produced an induced pluripotent stem cell line from a DMD patient's dermal fibroblasts by electroporation with episomal vectors containing: hL-MYC, hLIN28, hSOX2, hKLF4, hOCT3/4. The resultant DMD iPSC line (CCMi001DMD-A-3) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal. MLPA analyses performed on DNA extracted from CCMi001DMD-A-3 showed a deletion of exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50).
Assuntos
Éxons/genética , Células-Tronco Pluripotentes Induzidas/citologia , Distrofia Muscular de Duchenne/enzimologia , Adulto , Células Cultivadas , Reprogramação Celular/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , MasculinoRESUMO
Semantic and, to a lesser extent, phonological verbal fluency tasks are impaired in Alzheimer's disease (AD) and in amnesic mild cognitive impairment (aMCI). Furthermore, both fluency tasks have been considered as possible markers of conversion from aMCI to AD. Up to recent years, the use of fluency tasks has been limited to word count, but, more recently, linguistic variables, such as word frequency, age of acquisition, familiarity, and typicality, have also been considered. In particular, attention has been focused on typicality of words produced on semantic verbal fluency tasks, because the tendency to produce only the more typical members of various categories points to an impoverishment of semantic memory. The aim of our study was to compare in aMCI, AD, and control subjects a lexical (word frequency) and a lexical-semantic variable (item typicality) in a semantic verbal fluency task, and to evaluate the possible value of these variables in predicting conversion from aMCI to AD during a 2 years follow-up period. We found no difference in mean typicality of words produced by aMCI and AD subjects whereas both groups produced words of higher mean typicality than control subjects. Furthermore, to assess the relationship between typicality values and risk of conversion to AD, the aMCI group was split in two subgroups, including subjects who obtained a mean typicality value lower or higher than the median value of the whole aMCI group. Consistent with our hypothesis, conversion to AD was significantly more frequent in high typicality than in low typicality subjects.
Assuntos
Doença de Alzheimer/psicologia , Disfunção Cognitiva/psicologia , Semântica , Adulto , Idoso , Doença de Alzheimer/diagnóstico , Disfunção Cognitiva/diagnóstico , Progressão da Doença , Feminino , Seguimentos , Humanos , Testes de Linguagem , Masculino , Testes Neuropsicológicos , Prognóstico , Risco , VocabulárioRESUMO
Subjects with Mild Cognitive Impairment (MCI) are normally classified according to the presence of episodic memory deficits associated or not to disturbances of other cognitive domains. The present study had two aims: to identify discrete subtypes of amnestic MCI (a-MCI) with hippocampal atrophy; and to assess if the identified subtypes show different rates of progression to dementia. Sixty-seven a-MCI subjects were enrolled, all showing significant hippocampal atrophy on MRI. The subjects underwent at baseline and at follow-up a comprehensive neuropsychological examination, and were followed-up for five years to detect the conversion to dementia. An exploratory factor analysis on neuropsychological performances at baseline identified three main factors that were subsequently used to perform a k-means cluster analysis. Three cluster of a-MCI subjects were identified: "pure amnestic" (N=29), "multiple domain"(N=16), and "amnestic/semantic"(N=22). The successive discriminant functions were able to correctly classify 88% of the subjects. During the follow-up, 33 subjects converted to dementia (49.2%), 14 "pure amnestic" (48.3%), 11 "multiple domain" (68.5%) and 8 "amnestic/semantic" (36.4%; log-rank: p=0.016); median survival was respectively 36, 22, and 39 months. On Cox proportional hazard model, baseline MMSE (HR=0,709; p=0.006), education (HR=1,115; p=0.011) and belonging to the "multiple domain" subgroup (HR=2,706; p=0.013) were significantly associated to higher rate of conversion to dementia. Our findings confirm the tendency to worst outcome of subjects with multiple domain MCI, and show that the association of episodic and semantic memory deficits, without other cognitive disturbances, could identify a specific cognitive pattern associated to slower cognitive decline, as previously reported in Alzheimer's Disease.
Assuntos
Disfunção Cognitiva/patologia , Disfunção Cognitiva/fisiopatologia , Hipocampo/patologia , Transtornos da Memória/fisiopatologia , Idoso , Atrofia , Análise por Conglomerados , Disfunção Cognitiva/psicologia , Demência/patologia , Demência/fisiopatologia , Demência/psicologia , Análise Discriminante , Progressão da Doença , Feminino , Seguimentos , Humanos , Imageamento por Ressonância Magnética , Masculino , Transtornos da Memória/patologia , Transtornos da Memória/psicologia , Testes Neuropsicológicos , Tamanho do Órgão , Análise de Componente Principal , Modelos de Riscos Proporcionais , Análise de SobrevidaRESUMO
According to the "embodied cognition" theory and the "sensory-motor model of semantic knowledge": (a) concepts are represented in the brain in the same format in which they are constructed by the sensory-motor system and (b) various conceptual categories differ according to the weight of different kinds of information in their representation. In this study, we tried to check the second assumption by asking normal elderly subjects to subjectively evaluate the role of various perceptual, motor and language-mediated sources of knowledge in the construction of different semantic categories. Our first aim was to rate the influence of different sources of knowledge in the representation of animals, plant life and artifact categories, rather than in living and non-living beings, as many previous studies on this subject have done. We also tried to check the influence of age and stimulus modality on these evaluations of the "sources of knowledge" underlying different conceptual categories. The influence of age was checked by comparing results obtained in our group of elderly subjects with those obtained in a previous study, conducted with a similar methodology on a sample of young students. And the influence of stimulus modality was assessed by presenting the stimuli in the verbal modality to 50 subjects and in the pictorial modality to 50 other subjects. The distinction between "animals" and "plant life" in the "living" categories was confirmed by analyzing their prevalent sources of knowledge and by a cluster analysis, which allowed us to distinguish "plant life" items from animals. Furthermore, results of the study showed: (a) that our subjects considered the visual modality as the main source of knowledge for all categories taken into account; and (b) that in biological categories the next most important source of information was represented by other perceptual modalities, whereas in artifacts it was represented by the actions performed with them. Finally, age and stimulus modality did not significantly influence judgment of relevance of the sources of knowledge involved in the construction of different conceptual categories.
RESUMO
The mechanisms subsuming the brain organization of categories and the corresponding gender related asymmetries are controversial. Some authors believe that the brain organization of categories is innate, whereas other authors maintain that it is shaped by experience. According to these interpretations, gender-related asymmetries should respectively be inborn or result from the influence of social roles. In a previous study, assessing the familiarity of young students with different 'biological' and 'artefact' categories, we had observed no gender-related difference on any of these categories. Since these data could be due to the fact that our students belonged to a generation in which the traditional social roles have almost completely disappeared, we predicted that gender-related asymmetries should be found in older men and women. The familiarity of young and elderly men and women with various semantic categories was, therefore, studied presenting in the verbal and pictorial modality different kinds of living and artefact categories. Results confirmed the hypothesis, because elderly women showed a greater familiarity for flowers and elderly men for animals. These findings are consistent with the hypothesis assuming that gender-related asymmetries for different semantic categories is due to the influence of gender-related social roles.
Assuntos
Formação de Conceito/fisiologia , Identidade de Gênero , Reconhecimento Psicológico/fisiologia , Caracteres Sexuais , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Estimulação Luminosa , SemânticaRESUMO
A sensitive, simple and reproducible protocol for nanoparticle-assisted laser desorption/ionization mass spectrometry imaging technique is described. The use of commercially available TiO2 nanoparticles abolishes heterogeneous crystallization, matrix background interferences and enhances signal detection, especially in the low mass range. Molecular image normalization was based on internal standard deposition on tissues, allowing direct comparison of drug penetration and distribution between different organs and tissues. The method was applied to analyze the distribution of the anticancer drug paclitaxel, inside normal and neoplastic mouse tissue sections. Spatial resolution was good, with a linear response between different in vivo treatments and molecular imaging intensity using therapeutic drug doses. This technique distinguishes the different intensity of paclitaxel distribution in control organs of mice, such as liver and kidney, in relation to the dose. Animals treated with 30 mg/kg of paclitaxel had half of the concentration of those treated with 60 mg/kg. We investigated the spatial distribution of paclitaxel in human melanoma mouse xenografts, following different dosage schedules and found a more homogeneous drug distribution in tumors of mice given repeated doses (5×8 mg/kg) plus a 60 mg/kg dose than in those assigned only a single 60 mg/kg dose. The protocol can be readily applied to investigate anticancer drug distribution in neoplastic lesions and to develop strategies to optimize and enhance drug penetration through different tumor tissues.
Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Espectrometria de Massas/métodos , Melanoma/metabolismo , Nanopartículas , Paclitaxel/farmacocinética , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Calibragem , Feminino , Humanos , Melanoma/tratamento farmacológico , Camundongos , Camundongos Nus , Paclitaxel/uso terapêuticoRESUMO
Some previous studies in brain-damaged patients suggested that neural systems in the left temporal lobe might be crucial in the production and comprehension of nouns, while analogous systems in posterior frontal cortical areas might be involved in the production and comprehension of verbs. We assessed performance on neuropsychological tasks of production and comprehension of nouns and action-verbs in 10 patients with progressive supranuclear palsy (PSP) and in 10 age-matched healthy controls. PSP patients also underwent measurements of regional cerebral blood flow by means of single photon emission computed tomography (SPECT), using 99mTc-Ethyl Cysteinate Dimer. In all PSP patients, SPECT showed a significant hypoperfusion in the inferior frontal gyrus (IFG). PSP patients performed significantly worse than controls on all lexical-semantic tasks, except for the auditory lexical decision task on nouns. Within PSP patients, however, a significantly lower performance was observed on action-verbs as compared to nouns on various lexical-semantic tasks (oral and written confrontation naming, auditory and visual single-word comprehension). Analysis of individual performance revealed heterogeneous patterns of neuropsychological impairment in different PSP patients. Despite some difficulty in drawing clear-cut conclusions about the locus of functional damage, we hypothesise that in most of our PSP patients such selective impairment in the production and in the comprehension of action-verbs could be due to semantic deficits affecting the conceptual category of actions. These findings are consistent with the hypothesis that in PSP a dysfunction of neural systems in posterior frontal cortical areas (mainly involving the IFG) critical for processing the conceptual category of actions might result in a selective impairment of production and comprehension of action-verbs.