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1.
Proc Natl Acad Sci U S A ; 117(5): 2560-2569, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964835

RESUMO

De novo mutations (DNMs), or mutations that appear in an individual despite not being seen in their parents, are an important source of genetic variation whose impact is relevant to studies of human evolution, genetics, and disease. Utilizing high-coverage whole-genome sequencing data as part of the Trans-Omics for Precision Medicine (TOPMed) Program, we called 93,325 single-nucleotide DNMs across 1,465 trios from an array of diverse human populations, and used them to directly estimate and analyze DNM counts, rates, and spectra. We find a significant positive correlation between local recombination rate and local DNM rate, and that DNM rate explains a substantial portion (8.98 to 34.92%, depending on the model) of the genome-wide variation in population-level genetic variation from 41K unrelated TOPMed samples. Genome-wide heterozygosity does correlate with DNM rate, but only explains <1% of variation. While we are underpowered to see small differences, we do not find significant differences in DNM rate between individuals of European, African, and Latino ancestry, nor across ancestrally distinct segments within admixed individuals. However, we did find significantly fewer DNMs in Amish individuals, even when compared with other Europeans, and even after accounting for parental age and sequencing center. Specifically, we found significant reductions in the number of C→A and T→C mutations in the Amish, which seem to underpin their overall reduction in DNMs. Finally, we calculated near-zero estimates of narrow sense heritability (h 2), which suggest that variation in DNM rate is significantly shaped by nonadditive genetic effects and the environment.

2.
Opt Express ; 27(19): 26355-26368, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31674519

RESUMO

Imaging in poorly illuminated environments using three-dimensional (3D) imaging with passive imaging sensors that operate in the visible spectrum is a formidable task due to the low number of photons detected. 3D integral imaging, which integrates multiple two-dimensional perspectives, is expected to perform well in the presence of noise, as well as statistical fluctuation in the detected number of photons. In this paper, we present an investigation of 3D integral imaging in low-light-level conditions, where as low as a few photons and as high as several tens of photons are detected on average per pixel. In the experimental verification, we use an electron multiplying charge-coupled device (EM-CCD) and a scientific complementary metal-oxide-semiconductor (sCMOS) camera. For the EM-CCD, a theoretical model for the probability distribution of the pixel values is derived, then fitted with the experimental data to determine the camera parameters. Likewise, pixelwise calibration is performed on the sCMOS to determine the camera parameters for further analysis. Theoretical derivation of the expected signal-to-noise-ratio is provided for each image sensor and corroborated by the experimental findings. Further comparison between the cameras includes analysis of the contrast-to-noise ratio (CNR) as well as the perception-based image quality estimator (PIQE). Improvement of image quality metrics in the 3D reconstructed images is successfully confirmed compared with those of the 2D images. To the best of our knowledge, this is the first experimental report of low-light-level 3D integral imaging with as little as a few photons detected per pixel on average to improve scene visualization including occlusion removal from the scene.

3.
FASEB J ; 33(12): 13747-13761, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31585510

RESUMO

Folates are vital cofactors for the regeneration of S-adenosyl methionine, which is the methyl source for DNA methylation, protein methylation, and other aspects of one-carbon (C1) metabolism. Thus, folates are critical for establishing and preserving epigenetic programming. Folypolyglutamate synthetase (FPGS) is known to play a crucial role in the maintenance of intracellular folate levels. Therefore, any modulation in FPGS is expected to alter DNA methylation and numerous other metabolic pathways. To explore the role of polyglutamylation of folate, we eliminated both isoforms of FPGS in human cells (293T), producing FPGS knockout (FPGSko) cells. The elimination of FPGS significantly decreased cell proliferation, with a major effect on oxidative phosphorylation and a lesser effect on glycolysis. We found a substantial reduction in global DNA methylation and noteworthy changes in gene expression related to C1 metabolism, cell division, DNA methylation, pluripotency, Glu metabolism, neurogenesis, and cardiogenesis. The expression levels of NANOG, octamer-binding transcription factor 4, and sex-determining region Y-box 2 levels were increased in the mutant, consistent with the transition to a stem cell-like state. Gene expression and metabolite data also indicate a major change in Glu and GABA metabolism. In the appropriate medium, FPGSko cells can differentiate to produce mainly cells with characteristics of either neural stem cells or cardiomyocytes.-Srivastava, A. C., Thompson, Y. G., Singhal, J., Stellern, J., Srivastava, A., Du, J., O'Connor, T. R., Riggs, A. D. Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells.

4.
J Clin Imaging Sci ; 9: 35, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31538033

RESUMO

Objective: Pneumonia is the leading cause of pediatric mortality worldwide among children 0-5 years old. Lung ultrasound can be used to diagnose pneumonia in rural areas as it is a portable and relatively economic imaging modality with ~95% sensitivity and specificity for pneumonia in children. Lack of trained sonographers is the current limiting factor to its deployment in rural areas. In this study, we piloted training of a volume sweep imaging (VSI) ultrasound protocol for pneumonia detection in Peru with rural health workers. VSI may be taught to individuals with limited medical/ultrasound experience as it requires minimal anatomical knowledge and technical skill. In VSI, the target organ is imaged with a series of sweeps and arcs of the ultrasound probe in relation to external body landmarks. Methods: Rural health workers in Peru were trained on a VSI ultrasound protocol for pneumonia detection. Subjects were given a brief didactic session followed by hands-on practice with the protocol. Each attempt was timed and mistakes were recorded. Participants performed the protocol until they demonstrated two mistake-free attempts. Results: It took participants a median number of three attempts (range 1-6) to perform the VSI protocol correctly. Time to mastery took 51.4 ± 17.7 min. There were no significant differences among doctors, nurses, and technicians in total training time (P = 0.43) or number of attempts to success (P = 0.72). Trainee age was not found to be significantly correlated with training time (P = 0.50) or number of attempts to success (P = 0.40). Conclusion: Rural health workers learned a VSI protocol for pneumonia detection with relative ease in a short amount of time. Future studies should investigate the clinical efficacy of this VSI protocol for pneumonia detection. Key Message: A volume sweep imaging (VSI) protocol for pneumonia detection can be taught with minimal difficulty to rural health workers without prior ultrasound experience. No difference was found in training performance related to education level or age. VSI involves no significant knowledge of anatomy or technical skill.

5.
J Biogeogr ; 46(3): 597-610, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31534296

RESUMO

Aim: Whole-genome duplication (polyploidy) can influence the biogeography and ecology of plants that differ in ploidy level (cytotype). Here, we address how two consequences of plant polyploidy (parapatry of cytotypes and altered species interactions) shape the biogeography of herbivorous insects. Location: Warm deserts of North America. Taxa: Gall midges (Asphondylia auripila group, Diptera: Cecidomyiidae) that attack three parapatric cytotypes of creosote bush (Larrea tridentata, Zygophyllaceae). Methods: We surveyed Asphondylia species diversity at 177 sites across a 2300-km extent. After noting a correspondence between the distributions of eight Asphondylia species and L. tridentata cytotypes, we fine-mapped Asphondylia species range limits with transects spanning cytotype contact zones. We then tested whether plant-insect interactions and/or abiotic factors explain this coincidence by (1) comparing attack rates and gall midge communities on alternative cytotypes in a narrow zone of sympatry and (2) using species distribution models (SDMs) to determine if climatically suitable habitat for each midge species extended beyond cytotype contact zones. Results: The range limits of 6/17 Asphondylia species (including two novel putative species confirmed with COI sequencing) perfectly coincided with the contact zone of diploid and tetraploid L. tridentata. One midge species was restricted to diploid host plants while five were restricted to tetraploid and hexaploid host plants. Where diploid and tetraploid L. tridentata are sympatric, cytotype-restricted midge species more frequently attacked their typical host and Asphondylia community structure differed markedly between cytotypes. SDMs predicted that distributions of cytotype-restricted midge species were not constrained by climatic conditions near cytotype contact zones. Main conclusions: Contact zones between plant cytotypes are dispersal barriers for many Asphondylia species due to plant-insect interactions. The distribution of L. tridentata cytotypes therefore shapes herbivore species ranges and herbivore community structure across North American deserts. Our results demonstrate that polyploidy in plants can affect the biogeography of ecological communities.

7.
J Immunol ; 203(7): 1882-1896, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31492741

RESUMO

Comparative analyses suggest that the MHC was derived from a prevertebrate "primordial immune complex" (PIC). PIC duplicated twice in the well-studied two rounds of genome-wide duplications (2R) early in vertebrate evolution, generating four MHC paralogous regions (predominantly on human chromosomes [chr] 1, 6, 9, 19). Examining chiefly the amphibian Xenopus laevis, but also other vertebrates, we identified their MHC paralogues and mapped MHC class I, AgR, and "framework" genes. Most class I genes mapped to MHC paralogues, but a cluster of Xenopus MHC class Ib genes (xnc), which previously was mapped outside of the MHC paralogues, was surrounded by genes syntenic to mammalian CD1 genes, a region previously proposed as an MHC paralogue on human chr 1. Thus, this gene block is instead the result of a translocation that we call the translocated part of the MHC paralogous region (MHCtrans) Analyses of Xenopus class I genes, as well as MHCtrans, suggest that class I arose at 1R on the chr 6/19 ancestor. Of great interest are nonrearranging AgR-like genes mapping to three MHC paralogues; thus, PIC clearly contained several AgR precursor loci, predating MHC class I/II. However, all rearranging AgR genes were found on paralogues derived from the chr 19 precursor, suggesting that invasion of a variable (V) exon by the RAG transposon occurred after 2R. We propose models for the evolutionary history of MHC/TCR/Ig and speculate on the dichotomy between the jawless (lamprey and hagfish) and jawed vertebrate adaptive immune systems, as we found genes related to variable lymphocyte receptors also map to MHC paralogues.

8.
Artigo em Inglês | MEDLINE | ID: mdl-31342070

RESUMO

Robot-assisted pedicle screw insertion has been slowly gaining popularity in the spine surgery community. In previous studies, robotics has been shown to increase accuracy and reduce complication rates compared to other navigation technologies, although those studies have been conducted using traditional trajectories for pedicle screw insertion. We present a surgical video in which a robotics system (Mazor X; Mazor Robotics Ltd, Caesarea, Israel) was used to create cortical bone trajectories for the insertion of the screws. The patient in this case is a 52-yr-old woman with severe L4-5 disc herniation requiring a transforaminal interbody fusion with the insertion of pedicle screws. The robotic system's scan-and-plan technique was utilized, in which an intraoperative computed tomography (CT) scan generates a real-time operative plan. Other techniques for inserting pedicle screws using cortical bone trajectories include CT navigation and fluoroscopic guidance. These techniques allow the surgeon to manually direct the screw under precise guidance in multiple planes, although the surgeon is still using all 6 degrees of freedom the human hand provides. With robotic guidance, a pilot hole is drilled, which eliminates 4 of 6 degrees of freedom, which can potentially reduce the risk of misplaced screws. To our knowledge, this is the first video demonstrating pedicle screw insertion through cortical bone trajectories using robotic guidance. Future studies are warranted to compare cortical bone trajectory insertion using different navigation techniques to determine the long-term efficacy of each technique. The patient gave informed consent for surgery and video recording. Institutional review board approval was deemed unnecessary.

9.
Nat Commun ; 10(1): 2665, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209259

RESUMO

Estimates of Plasmodium falciparum migration may inform strategies for malaria elimination. Here we elucidate fine-scale parasite population structure and infer recent migration across Southeast Asia using identity-by-descent (IBD) approaches based on genome-wide single nucleotide polymorphisms called in 1722 samples from 54 districts. IBD estimates are consistent with isolation-by-distance. We observe greater sharing of larger IBD segments between artemisinin-resistant parasites versus sensitive parasites, which is consistent with the recent spread of drug resistance. Our IBD analyses reveal actionable patterns, including isolated parasite populations, which may be prioritized for malaria elimination, as well as asymmetrical migration identifying potential sources and sinks of migrating parasites.


Assuntos
Resistência a Medicamentos/genética , Monitoramento Epidemiológico , Genoma de Protozoário/genética , Malária Falciparum/microbiologia , Plasmodium falciparum/genética , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artemisininas/farmacologia , Artemisininas/uso terapêutico , Ásia Sudeste , Biodiversidade , Genótipo , Geografia Médica , Malária Falciparum/tratamento farmacológico , Malária Falciparum/prevenção & controle , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/isolamento & purificação , Polimorfismo de Nucleotídeo Único
10.
Opt Lett ; 44(9): 2326-2329, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31042221

RESUMO

A compact and field-portable three-dimensional (3D)-printed structured illumination (SI) digital holographic microscope based on shearing geometry is presented. By illuminating the sample using a SI pattern, the lateral resolution in both reconstructed phase and amplitude images can be improved up to twice the resolution provided by conventional illumination. The use of a 3D-printed system and shearing geometry reduces the complexity of the system, while providing high temporal stability. The experimental results for the USAF resolution target show a resolution improvement of a factor of two which corroborates the theoretical prediction. Resolution enhancement in phase imaging is also demonstrated by imaging a biological sample. To the best of our knowledge, this is the first report of a compact and field-portable SI digital holographic system based on shearing geometry.

12.
Genome Biol Evol ; 11(5): 1417-1430, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30942856

RESUMO

The metabolic conversion of dietary omega-3 and omega-6 18 carbon (18C) to long chain (>20 carbon) polyunsaturated fatty acids (LC-PUFAs) is vital for human life. The rate-limiting steps of this process are catalyzed by fatty acid desaturase (FADS) 1 and 2. Therefore, understanding the evolutionary history of the FADS genes is essential to our understanding of hominin evolution. The FADS genes have two haplogroups, ancestral and derived, with the derived haplogroup being associated with more efficient LC-PUFA biosynthesis than the ancestral haplogroup. In addition, there is a complex global distribution of these haplogroups that is suggestive of Neanderthal introgression. We confirm that Native American ancestry is nearly fixed for the ancestral haplogroup, and replicate a positive selection signal in Native Americans. This positive selection potentially continued after the founding of the Americas, although simulations suggest that the timing is dependent on the allele frequency of the ancestral Beringian population. We also find that the Neanderthal FADS haplotype is more closely related to the derived haplogroup and the Denisovan clusters closer to the ancestral haplogroup. Furthermore, the derived haplogroup has a time to the most recent common ancestor of 688,474 years before present. These results support an ancient polymorphism, as opposed to Neanderthal introgression, forming in the FADS region during the Pleistocene with possibly differential selection pressures on both haplogroups. The near fixation of the ancestral haplogroup in Native American ancestry calls for future studies to explore the potential health risk of associated low LC-PUFA levels in these populations.


Assuntos
Evolução Molecular , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Insaturados/metabolismo , Hominidae/genética , Animais , Ácidos Graxos Dessaturases/metabolismo , Hominidae/metabolismo , Humanos , Índios Norte-Americanos/genética , Seleção Genética , Sibéria
13.
Cancer ; 125(12): 2076-2088, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30865299

RESUMO

BACKGROUND: Although cell lines are an essential resource for studying cancer biology, many are of unknown ancestral origin, and their use may not be optimal for evaluating the biology of all patient populations. METHODS: An admixture analysis was performed using genome-wide chip data from the Catalogue of Somatic Mutations in Cancer (COSMIC) Cell Lines Project to calculate genetic ancestry estimates for 1018 cancer cell lines. After stratifying the analyses by tissue and histology types, linear models were used to evaluate the influence of ancestry on gene expression and somatic mutation frequency. RESULTS: For the 701 cell lines with unreported ancestry, 215 were of East Asian origin, 30 were of African or African American origin, and 453 were of European origin. Notable imbalances were observed in ancestral representation across tissue type, with the majority of analyzed tissue types having few cell lines of African American ancestral origin, and with Hispanic and South Asian ancestry being almost entirely absent across all cell lines. In evaluating gene expression across these cell lines, expression levels of the genes neurobeachin line 1 (NBEAL1), solute carrier family 6 member 19 (SLC6A19), HEAT repeat containing 6 (HEATR6), and epithelial cell transforming 2 like (ECT2L) were associated with ancestry. Significant differences were also observed in the proportions of somatic mutation types across cell lines with varying ancestral proportions. CONCLUSIONS: By estimating genetic ancestry for 1018 cancer cell lines, the authors have produced a resource that cancer researchers can use to ensure that their cell lines are ancestrally representative of the populations they intend to affect. Furthermore, the novel ancestry-specific signal identified underscores the importance of ancestral awareness when studying cancer.

14.
Nat Commun ; 10(1): 880, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30787307

RESUMO

Asthma is a complex disease with striking disparities across racial and ethnic groups. Despite its relatively high burden, representation of individuals of African ancestry in asthma genome-wide association studies (GWAS) has been inadequate, and true associations in these underrepresented minority groups have been inconclusive. We report the results of a genome-wide meta-analysis from the Consortium on Asthma among African Ancestry Populations (CAAPA; 7009 asthma cases, 7645 controls). We find strong evidence for association at four previously reported asthma loci whose discovery was driven largely by non-African populations, including the chromosome 17q12-q21 locus and the chr12q13 region, a novel (and not previously replicated) asthma locus recently identified by the Trans-National Asthma Genetic Consortium (TAGC). An additional seven loci reported by TAGC show marginal evidence for association in CAAPA. We also identify two novel loci (8p23 and 8q24) that may be specific to asthma risk in African ancestry populations.


Assuntos
Afro-Americanos/genética , Asma/genética , Predisposição Genética para Doença/genética , Asma/epidemiologia , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 8/genética , Loci Gênicos , Estudo de Associação Genômica Ampla , Hispano-Americanos/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Estados Unidos/epidemiologia
15.
J Vasc Surg ; 69(2): 507-515, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30683197

RESUMO

OBJECTIVE: Arteriovenous fistulas created in patients with chronic kidney disease often lose patency and fail to become usable. This prospective trial evaluated the efficacy of vonapanitase, a recombinant human elastase, in promoting radiocephalic fistula patency and use for hemodialysis. METHODS: PATENCY-1 was a double-blind, placebo-controlled trial that enrolled 349 patients on or approaching hemodialysis and being evaluated for radiocephalic arteriovenous fistula creation. Of these, 313 were randomized and 311 treated. Patients were assigned to vonapanitase (n = 210) or placebo (n = 103). The study drug solution was applied topically to the artery and vein for 10 minutes immediately after fistula creation. The primary and secondary end points were primary patency (time to first thrombosis or corrective procedure) and secondary patency (time to abandonment). Tertiary end points included use of the fistula for hemodialysis, fistula maturation by ultrasound, and procedure rates. RESULTS: The Kaplan-Meier estimates of 12-month primary patency were 42% (95% confidence interval [CI], 35-49) and 31% (95% CI, 21-42) for vonapanitase and placebo (P = .25). The Kaplan-Meier estimates of 12-month secondary patency were 74% (95% CI, 68-80) and 61% (95% CI, 51-71) for vonapanitase and placebo (P = .048). The proportions of vonapanitase and placebo patients were 39% and 25% (P = .035) with unassisted use for hemodialysis and 64% and 44% (P = .006) with unassisted plus assisted use. CONCLUSIONS: Vonapanitase treatment did not significantly improve primary patency but was associated with increased secondary patency and use for hemodialysis. Further research is needed to evaluate these end points.


Assuntos
Derivação Arteriovenosa Cirúrgica , Proteínas de Transporte/administração & dosagem , Oclusão de Enxerto Vascular/prevenção & controle , Elastase Pancreática/administração & dosagem , Artéria Radial/cirurgia , Diálise Renal , Trombose/prevenção & controle , Extremidade Superior/irrigação sanguínea , Grau de Desobstrução Vascular/efeitos dos fármacos , Veias/cirurgia , Administração Tópica , Adulto , Idoso , Derivação Arteriovenosa Cirúrgica/efeitos adversos , Proteínas de Transporte/efeitos adversos , Método Duplo-Cego , Feminino , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Elastase Pancreática/efeitos adversos , Estudos Prospectivos , Artéria Radial/diagnóstico por imagem , Artéria Radial/fisiopatologia , Trombose/etiologia , Trombose/fisiopatologia , Fatores de Tempo , Resultado do Tratamento , Estados Unidos , Veias/diagnóstico por imagem , Veias/fisiopatologia
16.
Cell ; 176(1-2): 405-406, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30633908
17.
World Neurosurg ; 123: e474-e481, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30500593

RESUMO

OBJECTIVE: To study the differences between robot-guided (Mazor X, Mazor Robotics Ltd., Caesarea, Israel) and 3-dimensional (3D) computed tomography (CT) navigation (O-arm Surgical Imaging System, Medtronic, Minneapolis, Minnesota, USA) for the insertion of pedicle screws. METHODS: We reviewed the charts of 50 patients who underwent robot-guided pedicle screw insertion (between May 2017-October 2017), and 49 patients who underwent 3D-CT navigation pedicle screw insertion (between September 2015-August 2016). Variables included were age, sex, body mass index, blood loss, length of stay, lumbar level(s), operation time, fluoroscopy time, radiation dose, accuracy, and time-per-screw placement. RESULTS: Mean ages were 59.3 years in the robotic group and 58.2 years in the 3D-CT navigation group. Mean was 30.7 kg/m2 in the robotic group and 32.1 kg/m2 in the 3D-CT navigation group. Mean time-per-screw placement was 3.7 minutes for the robotic group and 6.8 minutes for the 3D-CT navigation group, P < 0.001. In the robotic group, 189 of 190 screws were placed with Ravi grade I accuracy, and 1 was grade II. In the 3D-CT navigation group, 157 of 165 screws were Ravi grade I, and 8 were grade II (P = 0.11). Fluoroscopy time (P < 0.001), time-per-screw placement (P < 0.001), and length of stay (P < 0.001) were significantly lower in the robotic group. CONCLUSIONS: Both technologies are safe and accurate. Robotic technology exposed patients to less fluoroscopy time, decreased time-per-screw placement and shorter hospital stay than 3D-CT navigation. Further studies are warranted to verify our results.


Assuntos
Parafusos Pediculares , Procedimentos Cirúrgicos Robóticos/instrumentação , Espondilolistese/cirurgia , Adulto , Idoso , Fios Ortopédicos , Feminino , Humanos , Imagem Tridimensional , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Estudos Retrospectivos , Tomografia Computadorizada por Raios X
19.
Cell ; 175(5): 1173-1174, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445034

RESUMO

Ancient DNA is a powerful tool to understand the evolutionary dynamics of both current and ancestral populations. Posth et al. use ancient DNA to elucidate important questions surrounding the peopling of Central and South America, giving us greater insights into the ancestry of genetically understudied populations.


Assuntos
DNA Antigo , Índios Norte-Americanos , Genética Populacional , Genômica , Humanos , América do Sul
20.
Angew Chem Int Ed Engl ; 57(39): 12896-12900, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30098084

RESUMO

The DNA repair enzyme ALKBH2 is implicated in both tumorigenesis as well as resistance to chemotherapy in certain cancers. It is currently under study as a potential diagnostic marker and has been proposed as a therapeutic target. To date, however, there exist no direct methods for measuring the repair activity of ALKBH2 in vitro or in biological samples. Herein, we report a highly specific, fluorogenic probe design based on an oligonucleotide scaffold that reports directly on ALKBH2 activity both in vitro and in cell lysates. Importantly, the probe enables the monitoring of cellular regulation of ALKBH2 activity in response to treatment with the chemotherapy drug temozolomide through a simple fluorescence assay, which has only previously been observed through indirect means such as qPCR and western blots. Furthermore, the probe provides a viable high-throughput assay for drug discovery.


Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/química , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Corantes Fluorescentes/química , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Alquilação , Antineoplásicos Alquilantes/uso terapêutico , Corantes Fluorescentes/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Cinética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Espectrometria de Fluorescência , Temozolomida/uso terapêutico
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