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1.
Sensors (Basel) ; 23(9)2023 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-37177430

RESUMO

Following the COVID-19 outbreak, the health sector is undergoing a deep transformation that is increasingly pushing it towards the exploitation of technology, thus fostering the growth of digital health (eHealth). Cellular networks play a pivotal role in promoting the digitalization of healthcare, and researchers are banking on beyond fifth-generation (B5G) and sixth-generation (6G) technologies to reach the turning point, given that, according to forecasts, 5G will not be able to meet future expectations. Security is an aspect that definitely should not be overlooked for the success of eHealth to occur. This work aims to address the security issue from a poorly explored viewpoint, namely that of economics. In this paper, we first describe the main eHealth services, highlighting the key stakeholders involved. Then, we discuss how next-generation technologies could support these services to identify possible business relationships and, therefore, to realize an innovative business-oriented security analysis. A qualitative assessment of the impact of specific security breaches in diverse business conditions is provided. Moreover, we examine a case study in order to show the effects of security attacks in a definite scenario and discuss their impact on business dynamics.

2.
Neurobiol Dis ; 159: 105487, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34419621

RESUMO

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to α-synuclein accumulation and, hence, Parkinson's disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 α-synuclein inclusions were investigated in vivo. Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 α-synuclein inclusion in striatal and nigral neurons. No genotype differences in ALP markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/I ratio was reduced following administration of chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice. LC3B puncta accumulation and pSer129 α-synuclein inclusions were dissociated in striatal neurons of kinase-dead and G2019S knock-in mice. We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy.


Assuntos
Envelhecimento/genética , Autofagia/genética , Glucosilceramidase/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Neostriado/metabolismo , Neurônios/metabolismo , Doença de Parkinson/genética , alfa-Sinucleína/metabolismo , Envelhecimento/metabolismo , Animais , Técnicas de Introdução de Genes , Inativação Gênica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos , Camundongos , Camundongos Knockout , Doença de Parkinson/metabolismo
3.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502063

RESUMO

α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context.


Assuntos
Corpo Estriado/metabolismo , Substância Negra/metabolismo , Sinapses/metabolismo , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Corpo Estriado/ultraestrutura , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Substância Negra/ultraestrutura , Sinapses/ultraestrutura
4.
Int J Gynecol Pathol ; 39(4): 305-312, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31688243

RESUMO

Carcinosarcomas are rare, highly aggressive neoplasms composed of a combination of carcinomatous and sarcomatous elements. These tumors represent a paradigmatic field for the study of intratumor heterogeneity. A series of 8 tubo-ovarian carcinosarcomas was characterized for the following: (i) immunohistochemical expression of MNF116, epithelial membrane antigen, vimentin, S100, chromogranin, synaptophysin, desmin, myogenin (MYF4), and p53; (ii) mutational profiling of KRAS, BRAF, PIK3CA, NRAS, TP53, and DICER1 genes. Heterologous differentiation was present in 6 of 8 tumors. Cytokeratin MNF116 and epithelial membrane antigen were positive in all the carcinomatous components and in 87.5% and 50.0% of the sarcomatous components, respectively. The sarcomatous components showed positive staining for vimentin in all cases. Two cases demonstrated positivity for neuroendocrine markers in their carcinomatous components. All rhabdomyosarcomas were positive for desmin and MYF-4. Chondrosarcomas were positive for S100. All but one tumor showed similar p53 immunoreactivity in both the carcinomatous and sarcomatous components, and one case showed cytoplasmic p53 expression. Three of 8 cases (37.5%) showed TP53 mutations, and, in 2 cases, the TP53 mutation was shared by both epithelial and mesenchymal components. DICER1 mutation was found in all components of one case. Mutations in KRAS, NRAS, BRAF, and PIK3CA genes were not found in the study cohort. Our results highlight the heterogeneity of ovarian carcinosarcomas at the phenotypic level. A common mutational signature was observed in both components in 3 of 4 informative tumors. More studies are required to dissect different levels of ovarian carcinosarcomas' heterogeneity in order to define the best therapeutic approaches to these aggressive neoplasms.


Assuntos
Biomarcadores Tumorais/genética , Carcinossarcoma/genética , RNA Helicases DEAD-box/genética , Neoplasias das Tubas Uterinas/genética , Neoplasias Ovarianas/genética , Ribonuclease III/genética , Proteína Supressora de Tumor p53/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinossarcoma/metabolismo , Carcinossarcoma/patologia , Estudos de Coortes , RNA Helicases DEAD-box/metabolismo , Neoplasias das Tubas Uterinas/metabolismo , Neoplasias das Tubas Uterinas/patologia , Feminino , Heterogeneidade Genética , Humanos , Imuno-Histoquímica , Imunofenotipagem , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fenótipo , Ribonuclease III/metabolismo , Proteína Supressora de Tumor p53/metabolismo
5.
Sensors (Basel) ; 18(7)2018 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-29987221

RESUMO

The forthcoming fifth generation (5G) networks are claimed to deliver the large amount of traffic generated by the huge number of heterogeneous devices that constitute the Internet of Things (IoT). This unprecedented volume of both human- and machine-generated traffic to be managed imposes 5G network operators to move the focus from throughput-optimized to energy-efficiency-optimized resource allocation solutions. Device-to-device (D2D) communications are recognized as an effective offloading technique that the 5G network can exploit to boost the capacity and energy efficiency of future 5G networks. In this paper, we design a technique to efficiently deliver multicast traffic in a 5G New Radio (NR) network by exploiting the benefits of D2D communication and single-frequency operation in order to improve the overall network energy efficiency. In the designed solution, the subset of devices in better channel conditions are served through a conventional multicast transmission, while cell-edge devices receive the multicast service from relay nodes that simultaneously transmit in D2D mode the same content. The dimension of the multicast serving area and the set of D2D connections to establish are chosen in order to maximize the overall network energy efficiency. Performed simulation results show the effectiveness of the proposed solution under varying frame configurations and number of multicast devices.

6.
Hum Mol Genet ; 24(3): 649-58, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25274781

RESUMO

Aicardi-Goutières syndrome (AGS) is an inflammatory encephalopathy caused by defective nucleic acids metabolism. Over 50% of AGS mutations affect RNase H2 the only enzyme able to remove single ribonucleotide-monophosphates (rNMPs) embedded in DNA. Ribonucleotide triphosphates (rNTPs) are incorporated into genomic DNA with relatively high frequency during normal replication making DNA more susceptible to strand breakage and mutations. Here we demonstrate that human cells depleted of RNase H2 show impaired cell cycle progression associated with chronic activation of post-replication repair (PRR) and genome instability. We identify a similar phenotype in cells derived from AGS patients, which indeed accumulate rNMPs in genomic DNA and exhibit markers of constitutive PRR and checkpoint activation. Our data indicate that in human cells RNase H2 plays a crucial role in correcting rNMPs misincorporation, preventing DNA damage. Such protective function is compromised in AGS patients and may be linked to unscheduled immune responses. These findings may be relevant to shed further light on the mechanisms involved in AGS pathogenesis.


Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Dano ao DNA , DNA/química , Instabilidade Genômica , Malformações do Sistema Nervoso/genética , Ribonuclease H/metabolismo , Doenças Autoimunes do Sistema Nervoso/metabolismo , Doenças Autoimunes do Sistema Nervoso/patologia , Linhagem Celular , Proliferação de Células , DNA/genética , Reparo do DNA , Replicação do DNA , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/patologia , Ribonuclease H/genética , Ribonucleotídeos/metabolismo
7.
World J Surg Oncol ; 12: 105, 2014 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-24755359

RESUMO

Primary extra-gastrointestinal stromal tumor (EGISTs) arising in the pancreas is extremely rare: only 20 cases have previously been reported in the English literature from 2000 to 2013. We reported a case of EGIST of the pancreas in a 69-year-old woman who presented with abdominal pain and with a solid, heterogeneously enhancing neoplasm in the uncinate process of the pancreas, revealed preoperatively by an abdominal computed tomography scan. A diagnosis of neuroendocrine tumor was suggested. Positron emission tomography with 68Ga-DOTATOC did not show pathological accumulation of the tracer in the pancreas. The patient underwent enucleation, under ultrasonic guidance, of the pancreatic tumor that emerged to the surface of the pancreas. Histopathology and immunohistochemical examination confirmed the final diagnosis of EGIST of the pancreas (CD117+), with one mitosis per 50 high-power fields. Although rarely, GIST can involve the pancreas as a primary site, and this tumor should be considered in the differential diagnosis of pancreatic neoplasms.


Assuntos
Tumores do Estroma Gastrointestinal/patologia , Neoplasias Pancreáticas/patologia , Idoso , Feminino , Tumores do Estroma Gastrointestinal/cirurgia , Humanos , Neoplasias Pancreáticas/cirurgia , Prognóstico , Tomografia Computadorizada por Raios X
8.
Proc Natl Acad Sci U S A ; 108(33): 13647-52, 2011 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-21808022

RESUMO

UV light induces DNA lesions, which are removed by nucleotide excision repair (NER). Exonuclease 1 (EXO1) is highly conserved from yeast to human and is implicated in numerous DNA metabolic pathways, including repair, recombination, replication, and telomere maintenance. Here we show that hEXO1 is involved in the cellular response to UV irradiation in human cells. After local UV irradiation, fluorescent-tagged hEXO1 localizes, together with NER factors, at the sites of damage in nonreplicating cells. hEXO1 accumulation requires XPF-dependent processing of UV-induced lesions and is enhanced by inhibition of DNA repair synthesis. In nonreplicating cells, depletion of hEXO1 reduces unscheduled DNA synthesis after UV irradiation, prevents ubiquitylation of histone H2A, and impairs activation of the checkpoint signal transduction cascade in response to UV damage. These findings reveal a key role for hEXO1 in the UV-induced DNA damage response linking NER to checkpoint activation in human cells.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Raios Ultravioleta/efeitos adversos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Dano ao DNA , Histonas/metabolismo , Humanos , Ubiquitinação
9.
NPJ Parkinsons Dis ; 9(1): 44, 2023 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-36973269

RESUMO

In Parkinson's disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear, but they are hypothesized to involve the autophagy-lysosome pathway (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, and LRRK2 kinase activity has been shown to be involved in pS129-aSyn inclusion modulation. We observed selective downregulation of the novel PD risk factor RIT2 in vitro and in vivo. Rit2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of Rit2 operated neuroprotection against AAV-A53T-aSyn. Furthermore, Rit2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. On the other hand, reduction of Rit2 levels leads to defects in the ALP, similar to those induced by the G2019S-LRRK2 mutation. Our data indicate that Rit2 is required for correct lysosome function, inhibits overactive LRRK2 to ameliorate ALP impairment, and counteracts aSyn aggregation and related deficits. Targeting Rit2 could represent an effective strategy to combat neuropathology in familial and idiopathic PD.

10.
Viruses ; 15(1)2022 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-36680152

RESUMO

The present study aims to provide the sequential immunological, clinical and virological events occurring in a CMV-infected pregnant woman experiencing intrauterine CMV transmission. In brief, a case of primary CMV infection occurred in a 36-year-old pregnant woman. The patient exhibited early-sustained viremia and viruria, detectable presence of CMV in saliva concomitant with a strong CMV-specific cell-mediated response (427 EliSpots). CMV was detected in the amniotic fluid at 15 weeks of pregnancy (>1 × 106 CMV copies/mL). The pregnancy was deliberately interrupted at 16 weeks of gestation. Fetal histological and pathological examinations revealed placentitis and fetal brain alterations as microcephaly and cortical dysplasia. Interestingly, this clinical report shows: (1) there was a rapid and sustained CMV-specific cell mediated immune response (Th1) in association with low IgG avidity (Th2) correlated with fetal CMV transmission. (2) The levels of CMV-specific cell-mediated immune response persisted at high levels up to 200 weeks after infection despite clinical and viral clearance. (3) The histological and pathological evidence suggests that a potent pro-inflammatory condition at the placental level may lead to cCMV.


Assuntos
Doenças Transmissíveis , Infecções por Citomegalovirus , Doenças Fetais , Complicações Infecciosas na Gravidez , Gravidez , Feminino , Humanos , Adulto , Citomegalovirus , Seguimentos , Transmissão Vertical de Doenças Infecciosas , Placenta , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/diagnóstico , Líquido Amniótico
11.
J Clin Pathol ; 75(11): 751-758, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34083414

RESUMO

AIMS: This study aimed to identify any microscopic features associated with abnormal (membranous/velamentous or marginal) placental cord insertions and to analyse their adverse neonatal outcomes. METHODS: We retrospectively analysed the records-including pathological findings, clinical information and pregnancy outcomes-for 1060 singleton pregnancies, involving newborn delivered after 24 weeks of gestation. RESULTS: Marginal cord insertions were identified in 26.60% of cases and membranous cord insertions in 2.64%. Subchorionic vessel thrombus was more prevalent in marginal or membranous insertions (0.97%) than in normal cord insertions (0.27%) (p=0.129). Intervillous thrombi (13.73% vs 8.41%, p<0.05) and chorioamnionitis (8.53% vs 5.48%, p=0.089) were more prevalent in normal cord insertions. Premature rupture of membranes was significantly more commonly associated with abnormal (marginal 15.25% and membranous 17.86%) than with normal (9.87%) insertions (p<0.05). Pre-eclampsia was more common in the group with membranous cord insertions (7.14%) than in the other groups (marginal 0.35%; normal 0.80%) (p<0.05). Marginal and membranous placental cord insertions were associated with earlier gestational age at delivery and smaller fetuses than in the group with normal insertions. Intrauterine fetal demise, cardiac malformations and pregestational diabetes were also more common among cases of abnormal cord insertions. CONCLUSIONS: Subchorionic vessel thrombus and adverse pregnancy-related outcomes were more prevalent in cases of marginal/membranous cord insertion than for normal insertions.


Assuntos
Placenta , Cordão Umbilical , Recém-Nascido , Gravidez , Feminino , Humanos , Cordão Umbilical/anormalidades , Cordão Umbilical/patologia , Estudos Retrospectivos , Resultado da Gravidez , Idade Gestacional
12.
SLAS Discov ; 26(3): 460-469, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33334229

RESUMO

Voltage-gated ion channels produce rapid transmembrane currents responsible for action potential generation and propagation at the neuronal, muscular, and cardiac levels. They represent attractive clinical targets because their altered firing frequency is often the hallmark of pathological signaling leading to several neuromuscular disorders. Therefore, a method to study their functioning upon repeated triggers at different frequencies is desired to develop new drug molecules selectively targeting pathological phenotype. Optogenetics provides powerful tools for millisecond switch of cellular excitability in contactless, physiological, and low-cost settings. Nevertheless, its application to large-scale drug-screening operations is still limited by long processing time (due to sequential well read), rigid flashing pattern, lack of online compound addition, or high consumable costs of existing methods. Here, we developed a method that enables simultaneous analysis of 384-well plates with optical pacing, fluorescence recording, and liquid injection. We used our method to deliver programmable millisecond-switched depolarization through light-activated opsin in concomitance with continuous optical recording by a fluorescent indicator. We obtained 384-well pacing of recombinant voltage-activated sodium or calcium channels, as well as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, in all-optical parallel settings. Furthermore, we demonstrated the use-dependent behavior of known ion channel blockers by optogenetic pacing at normal or pathological firing frequencies, obtaining very good signal reproducibility and accordance with electrophysiology data. Our method provides a novel physiological approach to study frequency-dependent drug behavior using reversible programmable triggers. The all-optical parallel settings combined with contained operational costs make our method particularly suited for large-scale drug-screening campaigns as well as cardiac liability studies.


Assuntos
Bioensaio , Bloqueadores dos Canais de Cálcio/farmacologia , Optogenética/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Proteínas de Algas/antagonistas & inibidores , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Chlamydomonas reinhardtii , Corantes Fluorescentes/química , Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Imagem Óptica/métodos , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Rodopsina/antagonistas & inibidores , Rodopsina/genética , Rodopsina/metabolismo
13.
J Pediatr Gastroenterol Nutr ; 51(5): 610-7, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20706152

RESUMO

OBJECTIVES: Postrepair esophageal dysmotility and gastroesophageal reflux are well-known consequences in patients with congenital esophageal atresia (EA) with or without distal tracheoesophageal fistula (TEF). The interstitial cells of Cajal (ICC), considered the intestinal pacemaker, are altered in congenital diseases with abnormal peristalsis, but no data are available for EA. Therefore, presence and maturation of ICC was verified in EA-TEF newborns. PATIENTS AND METHODS: Fifteen full-term neonates underwent repair of EA-TEF. Control specimens were from 10 newborns who died of nonesophageal diseases. Specimens from upper pouch, fistula, proximal, and distal esophagus were processed for hematoxylin and eosin, c-kit immunohistochemistry for ICC identification, and transmission electron microscopy. Frequency of c-kit-positive cells was evaluated in 20 fields per slide using a visual score (absent, very low, low, medium, high, very high). Morphocytometry and statistical analysis were also performed. RESULTS: In the proximal normal esophagus, ICC frequency was very high (3 cases), high (5), and medium (2); distally, it was high (4) and medium (6). In EA-TEF upper pouch, it was high (2) and medium (13); in the fistula, it was medium (5), low (6), very low (3), and absent (1). Morphocytometry confirmed these results. Comparison between pouch and fistula versus proximal and lower esophagus, respectively, showed statistically significant differences. Transmission electron microscopy demonstrated ICC immaturity in EA-TEF. CONCLUSIONS: The significant lower ICC density in EA-TEF is in favor for the pathogenesis of esophageal dysmotility frequently observed in such patients.


Assuntos
Atresia Esofágica/patologia , Transtornos da Motilidade Esofágica/patologia , Esôfago/patologia , Células Intersticiais de Cajal/patologia , Fístula Traqueoesofágica/patologia , Estudos de Casos e Controles , Atresia Esofágica/complicações , Transtornos da Motilidade Esofágica/etiologia , Esôfago/citologia , Humanos , Recém-Nascido , Microscopia Eletrônica de Transmissão , Fístula Traqueoesofágica/complicações
14.
Cell Death Discov ; 6: 45, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32550012

RESUMO

The Parkinson's disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a crucial modulator of the autophagy-lysosome pathway, but unclarity exists on the precise mechanics of its role and the direction of this modulation. In particular, LRRK2 is involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and a significant proportion of LRRK2 patients presenting Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to obtain a comprehensive knowledge on LRRK2-associated pathology. Here, we investigated a G2019S-LRRK2 recombinant cell line exhibiting accumulation of endogenous, phosphorylated alpha-synuclein. We found that G2019S-LRRK2 leads to accumulation of LC3 and abnormalities in lysosome morphology and proteolytic activity in a kinase-dependent fashion, but independent from constitutively active Rab10. Notably, LRRK2 inhibition was ineffective upon upstream blockade of autophagosome-lysosome fusion events, highlighting this step as critical for alpha-synuclein clearance.

15.
Stem Cell Res ; 41: 101656, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31733438

RESUMO

Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.


Assuntos
Sistemas CRISPR-Cas , Neurônios Dopaminérgicos/metabolismo , Edição de Genes , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/biossíntese , Células-Tronco Pluripotentes Induzidas/metabolismo , Reação em Cadeia da Polimerase , Transgenes , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Microscopia de Fluorescência
16.
Fertil Steril ; 110(1): 103-112.e1, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29908776

RESUMO

OBJECTIVE: To evaluate the impact of antibiotic therapy for chronic endometritis (CE) on IVF outcome. DESIGN: Systematic review and meta-analysis. SETTING: Not applicable. PATIENT(S): Infertile women with history of recurrent implantation failure, defined as two or more failed ETs, undergoing one or more IVF cycle(s). INTERVENTION(S): The review was registered in PROSPERO (CRD42017062494) before the start of the literature search. Observational studies were identified by searching electronic databases. The following comparators were included: women with CE receiving antibiotics vs. untreated controls; women with cured CE vs. women with persistent CE; and women with cured CE vs. women with normal endometrial histology (negative for CE). The summary measures were reported as odds ratio (OR) with 95% confidence interval (CI). MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (CPR), ongoing pregnancy rate/live birth rate (OPR/LBR), implantation rate (IR), miscarriage rate. RESULT(S): A total of 796 patients (from five studies) were included. Women receiving antibiotic therapy (without the histologic confirmation of CE cure) did not show any advantage in comparison with untreated controls (OPR/LBR, CPR, and IR). Patients with cured CE showed higher OPR/LBR (OR 6.81), CPR (OR 4.02), and IR (OR 3.24) in comparison with patients with persistent CE. In vitro fertilization outcome was comparable between women with cured CE and those without CE (OPR/LBR, CPR, and IR). Miscarriage rate was not significantly different between groups. CONCLUSION(S): Chronic endometritis therapy may improve IVF outcome in patients suffering from recurrent implantation failure. A control biopsy should always confirm CE resolution before proceeding with IVF.


Assuntos
Aborto Habitual/epidemiologia , Aborto Habitual/terapia , Endometrite/epidemiologia , Endometrite/terapia , Fertilização in vitro , Infertilidade Feminina , Resultado da Gravidez , Aborto Habitual/etiologia , Doença Crônica , Implantação do Embrião/fisiologia , Endometrite/complicações , Feminino , Fertilização in vitro/métodos , Fertilização in vitro/estatística & dados numéricos , Humanos , Infertilidade Feminina/epidemiologia , Infertilidade Feminina/etiologia , Infertilidade Feminina/terapia , Gravidez , Resultado da Gravidez/epidemiologia , Taxa de Gravidez , Recidiva , Resultado do Tratamento
17.
J Clin Pathol ; 60(3): 278-82, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16644876

RESUMO

AIMS: To report three cases of serous cystadenoma and endocrine tumour in the same pancreas, to review the literature and to evaluate the clinicopathological features of the tumours. CASES: Three women (71, 57 and 31 years old) were admitted to hospital, two for diseases unrelated to the pancreas and the third for increasing obstructive jaundice in von Hippel-Lindau disease. Preoperative examination showed two distinct lesions in the first patient and only cystic lesions in the other two. RESULTS: Histological examination of the pancreas showed one serous oligocystic adenoma associated with a benign, well-differentiated endocrine tumour, one serous oligocystic adenoma associated with an endocrine microadenoma, and a von Hippel-Lindau-related cystic neoplasm with a well-differentiated endocrine carcinoma. CONCLUSIONS: Serous cystadenoma associated with endocrine tumour shows some clinicopathological differences with respect to the two tumours considered separately, and with respect to von Hippel-Lindau-related cases, although there is no convincing evidence at present to justify considering this association as a separate entity.


Assuntos
Cistadenoma Seroso/patologia , Neoplasias das Glândulas Endócrinas/patologia , Neoplasias Primárias Múltiplas/patologia , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Cistadenoma Seroso/metabolismo , Neoplasias das Glândulas Endócrinas/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Neoplasias Primárias Múltiplas/metabolismo , Neoplasias Pancreáticas/metabolismo , Doença de von Hippel-Lindau/metabolismo , Doença de von Hippel-Lindau/patologia
18.
Am J Clin Pathol ; 126(4): 593-601, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16938655

RESUMO

We ascertained the frequency of mitochondrial DNA (mtDNA) D-loop region somatic mutations in pancreatic cancer (PC) and verified whether polymorphisms were linked to diagnosis, prognosis, and PC-associated diabetes mellitus (DM) in 99 PC cases, 42 chronic pancreatitis (CP) cases, 18 pancreatobiliary tract tumors, and 87 healthy control subjects (CSs). Tissue samples were obtained from 19 patients with PC and 5 with CP. The D-loop region was sequenced from all tissue samples and from blood DNA of the same patients and 12 CSs. D-loop somatic mutations were found in 3 PC tissue samples (16%). Four single nucleotide polymorphisms (SNPs; T152C, T16189C, T16519C, A73G), more frequently found in PC than in CS, were analyzed by denaturing high-performance liquid chromatography-restriction fragment length polymorphism using blood DNA as the starting template in all cases. The T allele of 16519 SNP correlated with DM. The survival of patients with PC correlated with tumor stage and grade and with DM at diagnosis. When survival analysis was performed considering only patients with locally advanced disease, the T allele of mtDNA 16519 SNP correlated with shorter life expectancy. mtDNA D-loop somatic mutations, rarely found in PC, cannot be considered causative events for this tumor type and probably are epiphenomena; the mtDNA D-loop 16519 variant, which worsens PC prognosis, seems to be a predisposing genetic factor for DM.


Assuntos
Adenocarcinoma/genética , DNA Mitocondrial/genética , Mutação em Linhagem Germinativa , Neoplasias Pancreáticas/genética , Mutação Puntual , Adenocarcinoma/complicações , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/complicações , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Análise Mutacional de DNA , Diabetes Mellitus/etiologia , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Pancreatite Crônica/complicações , Pancreatite Crônica/genética , Pancreatite Crônica/metabolismo , Taxa de Sobrevida
20.
Cell Cycle ; 11(4): 668-74, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22373527

RESUMO

Genomic insults by endogenous or exogenous sources activate the DNA damage response (DDR). After the recognition of damaged DNA by specific factors, repair mechanisms process the lesions, and a surveillance mechanism, known as DNA damage checkpoint, is triggered by single-stranded (ss) DNA covered by RPA. UV light induces DNA lesions, mainly 6,4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD), which are removed by nucleotide excision repair (NER). Recent reports shed light onto the mechanism connecting NER and DDR after UV irradiation. How does UV-induced DNA damage activate checkpoint kinases? How is ssDNA generated at UV lesions? In yeast, UV lesions persisting during S phase represent a block for the advancing of replication forks, which temporarily stop and then reinitiate downstream of the damage, leaving a ssDNA region containing the lesion. Nonreplicating yeast and human cells with defects in NER are not able to properly activate the checkpoint cascade, indicating that processing of UV lesions is a prerequisite for checkpoint activation. This pathway also requires the function of exonuclease 1, which acts on NER intermediates generating long tracts of ssDNA. Here, we review the connections between NER processing of UV-induced lesions and checkpoint activation, discussing the role of recently identified players in this mechanism.


Assuntos
Dano ao DNA/genética , Reparo do DNA/fisiologia , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Quebras de DNA de Cadeia Simples/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Raios Ultravioleta/efeitos adversos
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