RESUMO
DNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1-, and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision-making process during DSB repair.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Proteínas Mad2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Fase G2/genética , Células HEK293 , Humanos , Proteínas Mad2/genética , Fase S/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
The homologous recombination repair (HRR) pathway repairs DNA double-strand breaks in an error-free manner. Mutations in HRR genes can result in increased mutation rate and genomic rearrangements, and are associated with numerous genetic disorders and cancer. Despite intensive research, the HRR pathway is not yet fully mapped. Phylogenetic profiling analysis, which detects functional linkage between genes using coevolution, is a powerful approach to identify factors in many pathways. Nevertheless, phylogenetic profiling has limited predictive power when analyzing pathways with complex evolutionary dynamics such as the HRR. To map novel HRR genes systematically, we developed clade phylogenetic profiling (CladePP). CladePP detects local coevolution across hundreds of genomes and points to the evolutionary scale (e.g., mammals, vertebrates, animals, plants) at which coevolution occurred. We found that multiscale coevolution analysis is significantly more biologically relevant and sensitive to detect gene function. By using CladePP, we identified dozens of unrecognized genes that coevolved with the HRR pathway, either globally across all eukaryotes or locally in different clades. We validated eight genes in functional biological assays to have a role in DNA repair at both the cellular and organismal levels. These genes are expected to play a role in the HRR pathway and might lead to a better understanding of missing heredity in HRR-associated cancers (e.g., heredity breast and ovarian cancer). Our platform presents an innovative approach to predict gene function, identify novel factors related to different diseases and pathways, and characterize gene evolution.
Assuntos
Evolução Molecular , Reparo de DNA por Recombinação , Software , Animais , Enzimas Reparadoras do DNA/genética , Loci Gênicos , Filogenia , Plantas/genéticaRESUMO
BACKGROUND: The optimal treatment approach for low-risk prostate cancer (LRPC) remains controversial. While active surveillance is an increasingly popular option, definitive local treatments, including radical prostatectomy (RP), external beam radiotherapy (EBRT), and prostate seed implantation (PSI), are also commonly used. This study aimed to evaluate the survival outcomes of patients with LRPC using a large patient population from the National Cancer Database (NCDB). METHODS: We analyzed data from 195,452 patients diagnosed with LRPC between 2004 and 2015 using the NCDB. Patients were classified based on their treatment modalities, including RP, EBRT, PSI, or no local treatment (NLT). Only patients with Charlson-Deyo comorbidity scores of 0 or 1 were included to ensure comparability. Propensity score analysis was used to balance the treatment groups, and the accelerated failure time model was used to analyze the survival rates of the treatment groups. RESULTS: After a median follow-up of 70.8 months, 24,545 deaths occurred, resulting in an all-cause mortality rate of 13%. RP demonstrated a survival benefit compared with NLT, particularly in patients younger than 74 years of age. In contrast, radiation treatments (EBRT and PSI) did not improve survival in the younger age groups, except for patients older than 70 years for EBRT and older than 65 years for PSI. Notably, EBRT in patients younger than 65 years was associated with inferior outcomes. CONCLUSION: This study highlights the differences in survival outcomes among LRPC treatment modalities. RP was associated with improved survival compared to NLT, especially in younger patients. In contrast, EBRT and PSI showed survival benefits primarily in the older age groups. NLT is a reasonable choice, particularly in younger patients when RP is not chosen. These findings emphasize the importance of individualized treatment decisions for LRPC management.
Assuntos
Prostatectomia , Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/terapia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/radioterapia , Idoso , Pessoa de Meia-Idade , Bases de Dados Factuais , Taxa de Sobrevida , Resultado do Tratamento , Braquiterapia , Fatores Etários , Estados Unidos/epidemiologiaRESUMO
Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a stress-responsive hub that inhibits the translation elongation factor eEF2, and consequently mRNA translation elongation, in response to hypoxia and nutrient deprivation. EEF2K is also involved in the response to DNA damage but its role in response to DNA crosslinks, as induced by cisplatin, is not known. Here we found that eEF2K is critical to mediate the cellular response to cisplatin. We uncovered that eEF2K deficient cells are more resistant to cisplatin treatment. Mechanistically, eEF2K deficiency blunts the activation of the DNA damage response associated ATM and ATR pathways, in turn preventing p53 activation and therefore compromising induction of cisplatin-induced apoptosis. We also report that loss of eEF2K delays the resolution of DNA damage triggered by cisplatin, suggesting that eEF2K contributes to DNA damage repair in response to cisplatin. In support of this, our data shows that eEF2K promotes the expression of the DNA repair protein ERCC1, critical for the repair of cisplatin-caused DNA damage. Finally, using Caenorhabditis elegans as an in vivo model, we find that deletion of efk-1, the worm eEF2K ortholog, mitigates the induction of germ cell death in response to cisplatin. Together, our data highlight that eEF2K represents an evolutionary conserved mediator of the DNA damage response to cisplatin which promotes p53 activation to induce cell death, or alternatively facilitates DNA repair, depending on the extent of DNA damage.
Assuntos
Caenorhabditis elegans , Cisplatino , Dano ao DNA , Quinase do Fator 2 de Elongação , Proteína Supressora de Tumor p53 , Cisplatino/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Quinase do Fator 2 de Elongação/metabolismo , Quinase do Fator 2 de Elongação/genética , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Humanos , Reparo do DNA/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Apoptose/efeitos dos fármacosRESUMO
BACKGROUND: Urachal carcinoma (UrC) is a rare, aggressive cancer with a poor prognosis that is frequently diagnosed in advanced stages. Due to its rarity, the current staging systems, namely Sheldon, Mayo, and Ontario were established based on relatively small patient cohorts, necessitating further validation. We used a large patient population from the National Cancer Database to model a novel staging system based on the Tumor (T), Node(N), and Metastasis (M) (TNM) staging system and compared it to established staging systems. METHODS: We identified patients diagnosed with UrC between the years of 2004-2016. To determine median overall survival (OS), a Kaplan-Meier (KM) curve was generated using the Sheldon, Mayo, Ontario, and TNM staging system. A cox proportional-hazards regression model was developed to highlight predictors of overall survival. RESULTS: A total of 626 patients were included in the analysis. The OS for the entire cohort was 58.2 months (50.1-67.8) with survival rates at 12, 24, and 60 months of 83%, 70%, and 49%, respectively (p < 0.0001). As compared to the Sheldon, Mayo, and Ontario staging system, our TNM staging system had a more balanced sample and survival distribution per stage and no overlap among stages on KM survival curves. The Mayo, Ontario, and TNM staging systems were more accurate in terms of stage-survival correlation than the Sheldon staging system (p < 0.05 for all stages). CONCLUSIONS: The proposed novel TNM staging system for UrC has a more balanced sample distribution and a more accurate stage-survival correlation than the traditional Mayo, Sheldon, and Ontario staging systems. It is clinically applicable and enables better risk stratification, prognosis, and therapeutic decision-making.
Assuntos
Neoplasias da Bexiga Urinária , Humanos , Neoplasias da Bexiga Urinária/patologia , Prognóstico , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Estudos RetrospectivosRESUMO
DNA repair by homologous recombination (HR) is critical for the maintenance of genome stability. Germline and somatic mutations in HR genes have been associated with an increased risk of developing breast (BC) and ovarian cancers (OvC). However, the extent of factors and pathways that are functionally linked to HR with clinical relevance for BC and OvC remains unclear. To gain a broader understanding of this pathway, we used multi-omics datasets coupled with machine learning to identify genes that are associated with HR and to predict their sub-function. Specifically, we integrated our phylogenetic-based co-evolution approach (CladePP) with 23 distinct genetic and proteomic screens that monitored, directly or indirectly, DNA repair by HR. This omics data integration analysis yielded a new database (HRbase) that contains a list of 464 predictions, including 76 gold standard HR genes. Interestingly, the spliceosome machinery emerged as one major pathway with significant cross-platform interactions with the HR pathway. We functionally validated 6 spliceosome factors, including the RNA helicase SNRNP200 and its co-factor SNW1. Importantly, their RNA expression correlated with BC/OvC patient outcome. Altogether, we identified novel clinically relevant DNA repair factors and delineated their specific sub-function by machine learning. Our results, supported by evolutionary and multi-omics analyses, suggest that the spliceosome machinery plays an important role during the repair of DNA double-strand breaks (DSBs).
RESUMO
SIGNIFICANCE: Peripheral pitting edema is a clinician-administered measure for grading edema. Peripheral edema is graded 0, 1 + , 2 + , 3 + , or 4 + , but subjectivity is a major limitation of this technique. A pilot clinical study for short-wave infrared (SWIR) molecular chemical imaging (MCI) effectiveness as an objective, non-contact quantitative peripheral edema measure is underway. AIM: We explore if SWIR MCI can differentiate populations with and without peripheral edema. Further, we evaluate the technology for correctly stratifying subjects with peripheral edema. APPROACH: SWIR MCI of shins from healthy subjects and heart failure (HF) patients was performed. Partial least squares discriminant analysis (PLS-DA) was used to discriminate the two populations. PLS regression (PLSR) was applied to assess the ability of MCI to grade edema. RESULTS: Average spectra from edema exhibited higher water absorption than non-edema spectra. SWIR MCI differentiated healthy volunteers from a population representing all pitting edema grades with 97.1% accuracy (N = 103 shins). Additionally, SWIR MCI correctly classified shin pitting edema levels in patients with 81.6% accuracy. CONCLUSIONS: Our study successfully achieved the two primary endpoints. Application of SWIR MCI to monitor patients while actively receiving HF treatment is necessary to validate SWIR MCI as an HF monitoring technology.
Assuntos
Insuficiência Cardíaca , Imagem Molecular , Análise Discriminante , Edema/diagnóstico por imagem , Insuficiência Cardíaca/diagnóstico por imagem , Humanos , Análise dos Mínimos QuadradosRESUMO
SIGNIFICANCE: A key risk faced by oncological surgeons continues to be complete removal of tumor. Currently, there is no intraoperative imaging device to detect kidney tumors during excision. AIM: We are evaluating molecular chemical imaging (MCI) as a technology for real-time tumor detection and margin assessment during tumor removal surgeries. APPROACH: In exploratory studies, we evaluate visible near infrared (Vis-NIR) MCI for differentiating tumor from adjacent tissue in ex vivo human kidney specimens, and in anaesthetized mice with breast or lung tumor xenografts. Differentiation of tumor from nontumor tissues is made possible with diffuse reflectance spectroscopic signatures and hyperspectral imaging technology. Tumor detection is achieved by score image generation to localize the tumor, followed by application of computer vision algorithms to define tumor border. RESULTS: Performance of a partial least squares discriminant analysis (PLS-DA) model for kidney tumor in a 22-patient study is 0.96 for area under the receiver operating characteristic curve. A PLS-DA model for in vivo breast and lung tumor xenografts performs with 100% sensitivity, 83% specificity, and 89% accuracy. CONCLUSION: Detection of cancer in surgically resected human kidney tissues is demonstrated ex vivo with Vis-NIR MCI, and in vivo on mice with breast or lung xenografts.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Modelos Animais de Doenças , Imageamento Hiperespectral/métodos , Neoplasias Renais/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Adenocarcinoma/diagnóstico por imagem , Animais , Carcinoma Ductal de Mama/diagnóstico por imagem , Carcinoma de Células Renais/diagnóstico por imagem , Carcinoma de Células de Transição/diagnóstico por imagem , Sistemas Computacionais , Análise Discriminante , Xenoenxertos , Humanos , Processamento de Imagem Assistida por Computador , Raios Infravermelhos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Curva ROC , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Pediatric obstructive sleep apnea (OSA) is associated with cognitive dysfunction, suggesting altered neurotransmitter function. We explored overnight changes in neurotransmitters in the urine of children with and without OSA. METHODS: Urine samples were collected from children with OSA and from control subjects before and after sleep studies. A neurocognitive battery assessing general cognitive ability (GCA) was administered to a subset of children with OSA. Samples were subjected to multiple enzyme-linked immunosorbent assays for 12 neurotransmitters, and adjusted for creatinine concentrations. RESULTS: The study comprised 50 children with OSA and 20 control subjects. Of the children with OSA, 20 had normal GCA score (mean ± SD) (101.2 ± 14.5) and 16 had a reduced GCA score (87.3 ± 13.9; P < .001). Overnight increases in epinephrine, norepinephrine, and γ-aminobutyric acid (GABA) levels emerged in children with OSA; taurine levels decreased. Using combinatorial approaches and cutoff values for overnight changes of these four neurotransmitters enabled prediction of OSA (area under the curve [AUC]: 0.923; P < .0001). Furthermore, GABA and taurine alterations, as well as overnight reductions in phenylethylamine, were more prominent in children with OSA and low GCA than in children with OSA and normal GCA (P < .001), and they reliably discriminated GCA status (AUC: 0.977; P < .0001). CONCLUSIONS: Pediatric OSA is associated with overnight increases in urinary concentrations of catecholamines indicative of heightened sympathetic outflow. Increases in GABA levels and decreases in taurine levels could underlie mechanisms of neuronal excitotoxicity and dysfunction. Combinatorial approaches using defined cutoffs in overnight changes in concentrations of selected neurotransmitters in urine may not only predict OSA but also the presence of cognitive deficits. Larger cohort studies appear warranted to confirm these findings.
Assuntos
Transtornos Cognitivos/urina , Neurotransmissores/urina , Apneia Obstrutiva do Sono/urina , Análise de Variância , Área Sob a Curva , Estudos de Casos e Controles , Criança , Pré-Escolar , Transtornos Cognitivos/diagnóstico , Creatinina/urina , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Testes Neuropsicológicos , Polissonografia , Curva ROC , Sensibilidade e Especificidade , Apneia Obstrutiva do Sono/diagnósticoRESUMO
BACKGROUND: Obstructive sleep apnea (OSA) is a low-grade inflammatory disease affecting the cardiovascular and metabolic systems. Increasing OSA severity reduces T-regulatory lymphocytes (Tregs) in OSA children. Since Tregs modulate endothelial activation, and attenuate insulin resistance, we hypothesized that Tregs are associated with endothelial and metabolic dysfunction in pediatric OSA. METHODS: 50 consecutively recruited children (ages 4.8-12 years) underwent overnight polysomnography and fasting homeostatic model (HOMA) of insulin resistance was assessed. Percentage of Tregs using flow cytometry, and endothelial function, expressed as the time to peak occlusive hyperemia (Tmax), were examined. In a subgroup of children (nâ=â21), in vitro Treg suppression tests were performed. RESULTS: Circulating Tregs were not significantly associated with either BMI z score or HOMA. However, a significant inverse correlation between percentage of Tregs and Tmax emerged (p<0.0001, râ=â-0.56). A significant negative correlation between Tregs suppression and the sleep pressure score (SPS), a surrogate measure of sleep fragmentation emerged (pâ=â0.02, râ=â-0.51) emerged, but was not present with AHI. CONCLUSIONS: Endothelial function, but not insulin resistance, in OSA children is strongly associated with circulating Tregs and their suppressive function, and appears to correlate with sleep fragmentation. Thus, alterations in T cell lymphocytes may contribute to cardiovascular morbidity in pediatric OSA.
Assuntos
Endotélio Vascular/metabolismo , Apneia Obstrutiva do Sono/imunologia , Apneia Obstrutiva do Sono/metabolismo , Linfócitos T Reguladores/imunologia , Índice de Massa Corporal , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Resistência à Insulina , Contagem de Linfócitos , Masculino , Polissonografia , Apneia Obstrutiva do Sono/fisiopatologiaRESUMO
BACKGROUND: Restorative sleep is expected to promote improved endothelial function (EF) in the morning compared to the evening. However, in adults with obstructive sleep apnea (OSA) EF is not only adversely affected, but it worsens during the night. Data in pediatric OSA are scarce, and overnight changes have not been explored. Therefore, we sought to examine potential associations between pediatric OSA and overnight changes in EF. METHODS: 59 habitually snoring children with various degrees of sleep-disordered breathing (age range, 4-16 years) underwent EF assessment (reactive hyperemia test by EndoPAT, Itamar Medical, Israel) in the evening before and the morning after an overnight polysomnography (PSG). Two brachial occlusion periods (1 min and 5 min) also were tested. Potential associations between evening-to-morning changes in EF and polysomnographic parameters were explored. RESULTS: Evening-to-morning changes in children with OSA displayed severity-dependent deterioration of EF, and occlusions lasting 1 or 5 min during the reactive hyperemia test yielded similar findings. CONCLUSIONS: In children deterioration in EF during the night significantly correlated with the severity of OSA. Furthermore, the reactive hyperemia test can be reliably performed with only 60 seconds of arterial flow occlusion in children. These findings support our hypothesis that similarly to adults, sleep apnea in children results in endothelial dysfunction (ED). We speculate that pediatric OSA is less commonly associated with cardiovascular complications possibly due to the shorter duration of the syndrome.