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The skin is the largest organ in the body and the only one to come into contact with solar UV radiation (UVR). UVA (320-400 nm) is a significant contributor to UV-related skin damage. The UVA spectrum makes up over 95% of solar-UV energy reaching the earth's surface causing the majority of the visible signs of skin photoaging. Many consumer products also emit UVA, including nail dryers. There have been sporadic reports suggesting that these units may be contributing to skin cancer incidence. This notion was recently bolstered by a finding that nail dryer-irradiated mammalian skin cells develop a mutational signature consistent with UVA exposure. This report was surprising considering the comparatively low level of UVA to which the skin is exposed during nail treatments. In this research, we investigated how UVA-emitting devices caused cytotoxic/genotoxic impact after only low levels of UVA exposure. Our data showed that levels of UVA in the unit are highly variable and location dependent. We confirm previous reports that using prolonged exposure protocols could induce significant levels of DNA damage. It was also determined that UV-induced DNA damage only partially correlated with the level of UVA fluency. On investigation, we found that the unit had a rapid increase in internal temperature when in use. Exposing human cells to these elevated temperatures acted synergistically with UVA to magnify the cytotoxic and genotoxic impact of UV irradiation.
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Intracranial aneurysm (IA) is now a common term closely associated with subarachnoid hemorrhage. IA is the bulging of a blood vessel caused by a weakening of its wall. This bulge can rupture and, in most cases, cause internal bleeding. In most cases, internal bleeding leads to death or other fatal consequences. Therefore, the development of an automated system for detecting IA is needed to help physicians make more accurate diagnoses. For this reason, we have focused on this problem. In this paper, we propose a 2D Convolutional Neural Network (CNN) based on a network commonly used for data classification in medicine. In addition to our proposed network, we also tested ResNet 50, ResNet 101 and ResNet 152 on a publicly available dataset. In this case, ResNet 152 achieved better results than our proposed network, but our network was significantly smaller and the classifications took significantly less time. Our proposed network achieved an overall accuracy of 98%. This result was achieved on a dataset consisting of 611 images. In addition to the mentioned networks, we also experimented with the VGG network, but it was not suitable for this type of data and achieved only 20%. We compare the results in this work with neural networks that have been verified by the scientific community, and we believe that the results obtained by us can help in the creation of an automated system for the detection of IA.
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Aprendizado Profundo , Aneurisma Intracraniano , Redes Neurais de Computação , Aneurisma Intracraniano/classificação , Aneurisma Intracraniano/diagnóstico , Aneurisma Intracraniano/diagnóstico por imagem , Humanos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Hemorragia Subaracnóidea/classificação , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/diagnósticoRESUMO
CD133, a cancer stem cell (CSC) marker in tumors, including melanoma, is associated with tumor recurrence, chemoresistance, and metastasis. Patient-derived melanoma cell lines were transduced with a Tet-on vector expressing CD133, generating doxycycline (Dox)-inducible cell lines. Cells were exposed to Dox for 24 h to induce CD133 expression, followed by RNA-seq and bioinformatic analyses, revealing genes and pathways that are significantly up- or downregulated by CD133. The most significantly upregulated gene after CD133 was amphiregulin (AREG), validated by qRT-PCR and immunoblot analyses. Induced CD133 expression significantly increased cell growth, percentage of cells in S-phase, BrdU incorporation into nascent DNA, and PCNA levels, indicating that CD133 stimulates cell proliferation. CD133 induction also activated EGFR and the MAPK pathway. Potential mechanisms highlighting the role(s) of CD133 and AREG in melanoma CSC were further delineated using AREG/EGFR inhibitors or siRNA knockdown of AREG mRNA. Treatment with the EGFR inhibitor gefitinib blocked CD133-induced cell growth increase and MAPK pathway activation. Importantly, siRNA knockdown of AREG reversed the stimulatory effects of CD133 on cell growth, indicating that AREG mediates the effects of CD133 on cell proliferation, thus serving as an attractive target for novel combinatorial therapeutics in melanoma and cancers with overexpression of both CD133 and AREG.
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Antígeno AC133 , Anfirregulina , Proliferação de Células , Melanoma , Humanos , Antígeno AC133/metabolismo , Antígeno AC133/genética , Anfirregulina/metabolismo , Anfirregulina/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/patologia , Melanoma/metabolismo , Melanoma/genética , Regulação para Cima/efeitos dos fármacosRESUMO
The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, present a challenge for genome integrity maintenance as this requires the engagement of different DNA repair pathways. Specific 'repair controllers' that facilitate DNA repair pathway crosstalk between double strand break (DSB) repair and base excision repair (BER), and regulate BER protein trafficking at lesion sites, have yet to be identified. We find that DNA polymerase ß (Polß), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partners with UBR5 and restrains DSB repair signaling. Here we find that, TRIP12, but not UBR5, controls cellular levels and chromatin loading of Polß. Required for Polß foci formation, TRIP12 regulates Polß involvement after DNA damage. Notably, excessive TRIP12-mediated shuttling of Polß affects DSB formation and radiation sensitivity, underscoring its precedence for BER. We conclude that the herein discovered trafficking function at the nexus of DNA repair signaling pathways, towards Polß-directed BER, optimizes DNA repair pathway choice at complex lesion sites.
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Currently, three-dimensional convolutional neural networks (3DCNNs) are a popular approach in the field of human activity recognition. However, due to the variety of methods used for human activity recognition, we propose a new deep-learning model in this paper. The main objective of our work is to optimize the traditional 3DCNN and propose a new model that combines 3DCNN with Convolutional Long Short-Term Memory (ConvLSTM) layers. Our experimental results, which were obtained using the LoDVP Abnormal Activities dataset, UCF50 dataset, and MOD20 dataset, demonstrate the superiority of the 3DCNN + ConvLSTM combination for recognizing human activities. Furthermore, our proposed model is well-suited for real-time human activity recognition applications and can be further enhanced by incorporating additional sensor data. To provide a comprehensive comparison of our proposed 3DCNN + ConvLSTM architecture, we compared our experimental results on these datasets. We achieved a precision of 89.12% when using the LoDVP Abnormal Activities dataset. Meanwhile, the precision we obtained using the modified UCF50 dataset (UCF50mini) and MOD20 dataset was 83.89% and 87.76%, respectively. Overall, our work demonstrates that the combination of 3DCNN and ConvLSTM layers can improve the accuracy of human activity recognition tasks, and our proposed model shows promise for real-time applications.
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Aprendizado Profundo , Humanos , Redes Neurais de Computação , Atividades Humanas , Memória de Longo Prazo , Reconhecimento PsicológicoRESUMO
The targets of topical genotoxic agents are basal and stem cells of the skin. These cells may misrepair DNA lesions, resulting in deleterious mutations of tumor suppressors or oncogenes. However, the genotoxicity of many compounds has not as yet been determined and needs to be tested using a relevant skin model. To this end, we designed a new high-throughput assay for the detection of agents that create DNA damage in epidermal stem and basal cells and used it to test known DNA-damaging agents. We utilized either 2D epidermal cells or 3D skin equivalents and topically exposed them to different compounds. The Skin Immuno-CometChip assay uses arrays of microwells formed in a collagen/agarose mixture to capture single basal cells in each microwell by virtue of collagen binding to α2ß1 integrin, which is present only on basal and stem cells. The presence of ß1 integrin was verified by immunofluorescent labeling cells that were then subjected to an electrical field, allowing for the migration of nicked DNA out of the nucleoid in alkali, with the resulting DNA comets stained and imaged. Furthermore, using improved comet detection software allowed for the automated and rapid quantification of DNA damage. Our study indicates that we can accurately predict genotoxicity by using 3D skin cultures, as well as keratinocytes grown in 2D monolayers.
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Epiderme , Pele , Pele/metabolismo , Queratinócitos , Citocromos/metabolismo , DNA/metabolismoRESUMO
A clinical research requires a systematic approach with diligent planning, execution and sampling in order to obtain reliable and validated results, as well as an understanding of each research methodology is essential for researchers. Indeed, selecting an inappropriate study type, an error that cannot be corrected after the beginning of a study, results in flawed methodology. The results of clinical research studies enhance the repertoire of knowledge regarding a disease pathogenicity, an existing or newly discovered medication, surgical or diagnostic procedure or medical device. Medical research can be divided into primary and secondary research, where primary research involves conducting studies and collecting raw data, which is then analysed and evaluated in secondary research. The successful deployment of clinical research methodology depends upon several factors. These include the type of study, the objectives, the population, study design, methodology/techniques and the sampling and statistical procedures used. Among the different types of clinical studies, we can recognize descriptive or analytical studies, which can be further categorized in observational and experimental. Finally, also pre-clinical studies are of outmost importance, representing the steppingstone of clinical trials. It is therefore important to understand the types of method for clinical research. Thus, this review focused on various aspects of the methodology and describes the crucial steps of the conceptual and executive stages.
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Projetos de Pesquisa , HumanosRESUMO
Animal experimentation is widely used around the world for the identification of the root causes of various diseases in humans and animals and for exploring treatment options. Among the several animal species, rats, mice and purpose-bred birds comprise almost 90% of the animals that are used for research purpose. However, growing awareness of the sentience of animals and their experience of pain and suffering has led to strong opposition to animal research among many scientists and the general public. In addition, the usefulness of extrapolating animal data to humans has been questioned. This has led to Ethical Committees' adoption of the 'four Rs' principles (Reduction, Refinement, Replacement and Responsibility) as a guide when making decisions regarding animal experimentation. Some of the essential considerations for humane animal experimentation are presented in this review along with the requirement for investigator training. Due to the ethical issues surrounding the use of animals in experimentation, their use is declining in those research areas where alternative in vitro or in silico methods are available. However, so far it has not been possible to dispense with experimental animals completely and further research is needed to provide a road map to robust alternatives before their use can be fully discontinued.
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Experimentação Animal , Humanos , Ratos , Camundongos , Animais , Projetos de PesquisaRESUMO
Recognizing various abnormal human activities from video is very challenging. This problem is also greatly influenced by the lack of datasets containing various abnormal human activities. The available datasets contain various human activities, but only a few of them contain non-standard human behavior such as theft, harassment, etc. There are datasets such as KTH that focus on abnormal activities such as sudden behavioral changes, as well as on various changes in interpersonal interactions. The UCF-crime dataset contains categories such as fighting, abuse, explosions, robberies, etc. However, this dataset is very time consuming. The events in the videos occur in a few seconds. This may affect the overall results of the neural networks that are used to detect the incident. In this article, we create a dataset that deals with abnormal activities, containing categories such as Begging, Drunkenness, Fight, Harassment, Hijack, Knife Hazard, Normal Videos, Pollution, Property Damage, Robbery, and Terrorism. We use the created dataset for the training and testing of the ConvLSTM (convolutional long short-term memory) neural network, which we designed. However, we also test the created dataset using other architectures. We use ConvLSTM architectures and 3D Resnet50, 3D Resnet101, and 3D Resnet152. With the created dataset and the architecture we designed, we obtained an accuracy of classification of 96.19% and a precision of 96.50%.
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Atividades Humanas , Redes Neurais de Computação , Humanos , Memória de Longo Prazo , Reconhecimento PsicológicoRESUMO
Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and is characterized by the elevated expression of stem cell markers, including CD133. The siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor trametinib in melanoma cells. This study investigates the underlying mechanisms of CD133's anti-apoptotic activity in patient-derived BAKP and POT cells, harboring difficult-to-treat NRASQ61K and NRASQ61R drivers, after CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. MACS-sorted CD133(+) BAKP cells were conditionally reprogrammed to derive BAKR cells with sustained CD133 expression and MIC features. Compared to BAKP, CD133(+) BAKR exhibit increased cell survival and reduced apoptosis in response to trametinib or the chemotherapeutic dacarbazine (DTIC). CRISPR-Cas9-mediated CD133 knockout in BAKR cells (BAKR-KO) re-sensitized cells to trametinib. CD133 knockout in BAKP and POT cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in both trametinib-treated cell lines, coincident with elevated p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. AKT1/2 siRNA knockdown or inhibition of BCL-2 family members with navitoclax (ABT-263) in BAKP-KO cells further enhanced caspase-mediated apoptotic PARP cleavage. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the CD133, AKT, or BCL-2 survival pathways with trametinib highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma.
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Melanoma , Proteínas Proto-Oncogênicas c-akt , Apoptose/genética , Sistemas CRISPR-Cas/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/farmacologia , Neoplasias Cutâneas , Células-Tronco/metabolismo , Proteína X Associada a bcl-2/metabolismo , Melanoma Maligno CutâneoRESUMO
Changes in nicotinamide adenine dinucleotide (NAD+) levels that compromise mitochondrial function trigger release of DNA damaging reactive oxygen species. NAD+ levels also affect DNA repair capacity as NAD+ is a substrate for PARP-enzymes (mono/poly-ADP-ribosylation) and sirtuins (deacetylation). The ecto-5'-nucleotidase CD73, an ectoenzyme highly expressed in cancer, is suggested to regulate intracellular NAD+ levels by processing NAD+ and its bio-precursor, nicotinamide mononucleotide (NMN), from tumor microenvironments, thereby enhancing tumor DNA repair capacity and chemotherapy resistance. We therefore investigated whether expression of CD73 impacts intracellular NAD+ content and NAD+-dependent DNA repair capacity. Reduced intracellular NAD+ levels suppressed recruitment of the DNA repair protein XRCC1 to sites of genomic DNA damage and impacted the amount of accumulated DNA damage. Further, decreased NAD+ reduced the capacity to repair DNA damage induced by DNA alkylating agents. Overall, reversal of these outcomes through NAD+ or NMN supplementation was independent of CD73. In opposition to its proposed role in extracellular NAD+ bioprocessing, we found that recombinant human CD73 only poorly processes NMN but not NAD+. A positive correlation between CD73 expression and intracellular NAD+ content could not be made as CD73 knockout human cells were efficient in generating intracellular NAD+ when supplemented with NAD+ or NMN.
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5'-Nucleotidase/metabolismo , 5'-Nucleotidase/fisiologia , Dano ao DNA , Reparo do DNA , NAD/metabolismo , NAD/fisiologia , Poli ADP Ribosilação , Poli(ADP-Ribose) Polimerases/fisiologia , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologia , 5'-Nucleotidase/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Sirtuínas , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismoRESUMO
CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably a cancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+) 'melanoma-initiating cells' are associated with chemoresistance, contributing to poor patient outcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magnetic-activated cell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealed that CD133(+) cells are significantly more invasive than CD133(-) cells. Conditional reprogramming of BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stem cell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cells showed upregulated CD133 expression, and consequently were more invasive and metastatic than BAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAK-R-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAK-R-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA or CRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels, doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9 concentrations. CD133 may therefore play an essential role in invasion and metastasis via upregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target for intervention in melanoma.
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Bisphenol A (BPA) is used heavily in the production of polycarbonate plastics, thermal receipt paper, and epoxies. Ubiquitous exposure to BPA has been linked to obesity, diabetes, and breast and reproductive system cancers. Resistance to chemotherapeutic agents has also been shown in cancer cell models. Here, we investigated BPA's ability to confer resistance to camptothecin (CPT) in mouse embryonic fibroblasts (MEFs). MEFs are sensitive to CPT; however, co-exposure of BPA with CPT improved cell survival. Co-exposure significantly reduced Top1-DNA adducts, decreasing chromosomal aberrations and DNA strand break formation. This decrease occurs despite BPA treatment increasing the protein levels of Top1. By examining chromatin structure after BPA exposure, we determined that widespread compaction and loss of nuclear volume occurs. Therefore, BPA reduced CPT activity by reducing the accessibility of DNA to Top1, inhibiting DNA adduct formation, the generation of toxic DNA strand breaks, and improving cell survival.
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Antineoplásicos Fitogênicos/farmacologia , Compostos Benzidrílicos/farmacologia , Camptotecina/farmacologia , Fibroblastos/efeitos dos fármacos , Fenóis/farmacologia , Inibidores da Topoisomerase I/farmacologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Adutos de DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fibroblastos/citologia , Instabilidade Genômica , CamundongosRESUMO
DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage. We previously validated a novel, high through-put approach to measure levels of DNA damage in cultured mammalian cells. This new CometChip Platform builds on the classical single cell gel electrophoresis or comet methodology used extensively in environmental toxicology and molecular biology. We asked whether the CometChip Platform could be used to measure DNA damage in samples derived from environmental field studies. To this end, we determined that nucleated erythrocytes from multiple species of turtle could be successfully evaluated in the CometChip Platform to quantify levels of DNA damage. In total, we compared levels of DNA damage in 40 animals from two species: the box turtle (Terrapene carolina) and the red-eared slider (Trachemys scripta elegans). Endogenous levels of DNA damage were identical between the two species, yet we did discover some sex-linked differences and changes in DNA damage accumulation. Based on these results, we confirm that the CometChip Platform allows for the measurement of DNA damage in a large number of samples quickly and accurately, and is particularly adaptable to environmental studies using field-collected samples. Environ. Mol. Mutagen. 59:322-333, 2018. © 2018 Wiley Periodicals, Inc.
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Bioensaio/instrumentação , Dano ao DNA/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Eritrócitos/efeitos dos fármacos , Animais , Coleta de Amostras Sanguíneas , Feminino , Masculino , Fatores Sexuais , TartarugasRESUMO
Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair.
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Ensaio Cometa/métodos , Dano ao DNA , DNA/química , Ensaios de Triagem em Larga Escala , Mutagênicos/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Humanos , Células Jurkat , Leucócitos Mononucleares/efeitos dos fármacos , Mutagênicos/química , Mutagênicos/farmacologia , Reprodutibilidade dos TestesRESUMO
Alzheimer's disease (AD) involves the progressive degeneration of neurons critical for learning and memory. In addition, patients with AD typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD, we determined whether inefficient BER due to reduced DNA polymerase-ß (Polß) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild-type and 3xTgAD mice with normal or reduced Polß levels. Compared to wild-type control mice, Polß heterozygous (Polß+/- ), and 3xTgAD mice, 3xTgAD/Polß+/- mice exhibited impaired performance in a buried food test of olfaction. Polß deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However, numbers of newly generated neurons were reduced by approximately 25% in Polß+/- and 3xTgAD mice, and by over 60% in the 3xTgAD/Polß+/- mice compared to wild-type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Polß+/- mice compared to 3xTgAD mice. Levels of amyloid ß-peptide (Aß) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Polß+/- mice, and cultured Polß-deficient neurons exhibited increased vulnerability to Aß-induced death. Olfactory deficit is an early sign in human AD, but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons, and suggest a mechanism underlying early olfactory impairment in AD.
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Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , DNA Polimerase beta/metabolismo , Bulbo Olfatório/enzimologia , Bulbo Olfatório/patologia , Olfato , Peptídeos beta-Amiloides/metabolismo , Animais , Apoptose , Morte Celular , Diferenciação Celular , Respiração Celular , Dano ao DNA , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Haploinsuficiência , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Neurogênese , Neurônios/metabolismo , Neurônios/patologiaRESUMO
The aging process and several age-related neurodegenerative disorders have been linked to elevated levels of DNA damage induced by ROS and deficiency in DNA repair mechanisms. DNA damage induced by ROS is a byproduct of cellular respiration and accumulation of damage over time, is a fundamental aspect of a main theory of aging. Mitochondria have a pivotal role in generating cellular oxidative stress, and mitochondrial dysfunction has been associated with several diseases. DNA base excision repair is considered the major pathway for repair of oxidized bases in DNA both in the nuclei and in mitochondria, and in neurons this mechanism is particularly important because non-diving cells have limited back-up DNA repair mechanisms. An association between elevated oxidative stress and a decrease in BER is strongly related to the aging process and has special relevance in age-related neurodegenerative diseases. Here, we review the role of DNA repair in aging, focusing on the implications of the DNA base excision repair pathways and how alterations in expression of these DNA repair proteins are related to the aging process and to age-related neurodegenerative diseases.
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Envelhecimento/metabolismo , Reparo do DNA , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo , Envelhecimento/genética , Envelhecimento/patologia , Animais , DNA Mitocondrial/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Aborted DNA ligation events in eukaryotic cells can generate 5'-adenylated (5'-AMP) DNA termini that can be removed from DNA by aprataxin (APTX). Mutations in APTX cause an inherited human disease syndrome characterized by early-onset progressive ataxia with ocular motor apraxia (AOA1). APTX is found in the nuclei and mitochondria of eukaryotic cells. Depletion of APTX causes mitochondrial dysfunction and renders the mitochondrial genome, but not the nuclear genome susceptible to damage. The biochemical processes that link APTX deficiency to mitochondrial dysfunction have not been well elucidated. Here, we monitored the repair of 5'-AMP DNA damage in nuclear and mitochondrial extracts from human APTX(+/+) and APTX(-/-) cells. The efficiency of repair of 5'-AMP DNA was much lower in mitochondrial than in nuclear protein extracts, and resulted in persistent DNA repair intermediates in APTX deficient cells. Moreover, the removal of 5'-AMP from DNA was significantly slower in the mitochondrial extracts from human cell lines and mouse tissues compared with their corresponding nuclear extracts. These results suggest that, contrary to nuclear DNA repair, mitochondrial DNA repair is not able to compensate for APTX deficiency resulting in the accumulation of mitochondrial DNA damage.