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1.
PLoS One ; 15(4): e0231450, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302342

RESUMO

RNA-binding proteins (RBPs) control many types of post-transcriptional regulation, including mRNA splicing, mRNA stability, and translational efficiency, by directly binding to their target RNAs and their mutation and dysfunction are often associated with several human neurological diseases and tumorigenesis. Crosslinking immunoprecipitation (CLIP), coupled with high-throughput sequencing (HITS-CLIP), is a powerful technique for investigating the molecular mechanisms underlying disease pathogenesis by comprehensive identification of RBP target sequences at the transcriptome level. However, HITS-CLIP protocol is still required for some optimization due to experimental complication, low efficiency and time-consuming, whose library has to be generated from very small amounts of RNAs. Here we improved a more efficient, rapid, and reproducible CLIP method by optimizing BrdU-CLIP. Our protocol produced a 10-fold greater yield of pre-amplified CLIP library, which resulted in a low duplicate rate of CLIP-tag reads because the number of PCR cycles required for library amplification was reduced. Variance of the yields was also reduced, and the experimental period was shortened by 2 days. Using this, we validated IL-6 expression by a nuclear RBP, HNRNPU, which directly binds the 3'-UTR of IL-6 mRNA in HeLa cells. Importantly, this interaction was only observed in the cytoplasmic fraction, suggesting a role of cytoplasmic HNRNPU in mRNA stability control. This optimized method enables us to accurately identify target genes and provides a snapshot of the protein-RNA interactions of nucleocytoplasmic shuttling RBPs.

2.
J Perinat Med ; 2020 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-32284451

RESUMO

Background There are numerous significant physiological changes occurring in circulation during labor. To detect these rapid hemodynamic changes, invasive and intermittent measurement techniques are not reliable. To suggest a suitable delivery method for pregnancy with cardiac disease, this study analyzed how each delivery method influences cardiac function using a noninvasive and continuous measurement technique. Methods A prospective study was accomplished at the National Cerebral and Cardiovascular Center in Japan from October 1, 2014, to November 30, 2018. The classification of the healthy heart pregnant women was according to the delivery method: vaginal delivery (VD) without epidural anesthesia, VD with epidural anesthesia, and caesarean section (CS). The hemodynamic parameters cardiac index (CI), stroke volume index (SI), and heart rate (HR) were evaluated regularly throughout delivery by noninvasive electrical cardiometry monitor. Results Ten cases were examined for each group. CI and HR were significantly increased before VD, while the increase in CI and HR was mild in the epidural group in comparison to the nonepidural group. SI was increased toward the delivery in the epidural group, and it was constant in the nonepidural group. However, there was no alteration in the level of outcomes of the two groups. In CS, SI increased and HR decreased before delivery. After delivery, SI continued to increase, while HR did not change but CI increased. Conclusion In VD, the increase in venous circulation according to the autotransfusion is managed by increasing HR. By epidural anesthesia, the increase in HR was suppressed and SI was increased. However, as epidural anesthesia increases the vascular capacity, the level of SI outcome was comparable. In CS, the HR was decreased because of the spinal anesthesia and the SI was increased because of many factors like hydration. As there are many factors to control in CS, VD with epidural anesthesia will be the first preference for most cardiac patients.

3.
Biochem Biophys Res Commun ; 523(3): 795-801, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-31954521

RESUMO

The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNA-RNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From 102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1, which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity of compounds.

4.
Heredity (Edinb) ; 124(1): 170-181, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31485029

RESUMO

Bird pollination can vary spatially in response to spatial fluctuations in flowering even within plant populations. In this study, we examined the hypothesis that the spatial variation in bird pollination may induce mitigating effects, which maintains or increases genetic diversity of pollen pools at local sites with low flowering densities. To test this hypothesis, we analyzed the landscape-level genetic effects within a population of Camellia japonica on the pollen pools accepted by individuals in two reproductive years by using genotypes at eight microsatellite loci of 1323 seeds from 19 seed parents. Regression analyses using the quadratic models of correlated paternity between pollen pools against spatial distances between the seed-parent pairs revealed not only local pollination but also some amount of long-distance pollen dispersal. The genetic diversity of pollen pools accepted by seed parents tended to be negatively related to the densities of flowering individuals near the seed parents during winter (when the effective pollination of C. japonica is mediated mostly by Zosterops japonica). We show that the low density of flowering individuals may induce the expansion of the foraging areas of Z. japonica and consequently increase the genetic diversity of pollen pools. This spatial variation in bird pollination may induce the mitigating effects on the C. japonica population. The comparisons between the two study years indicate that the overall pattern of bird pollination and the genetic effects described here, including the mitigating effects, may be stable over time.

5.
New Phytol ; 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31424559

RESUMO

Introgression from one species in a specific environment to another may facilitate colonization of the environment by the recipient species. However, such environment-dependent introgression has been clarified in limited plant taxa. In northern Japan, there are two interfertile oak species: Quercus dentata (Qd) in coastal areas and Q. mongolica var. crispula (Qc) in inland areas. However, at higher latitudes where Qd is rare, a coastal Qc ecotype with Qd-like traits is distributed in the coastal areas. We distinguished inland Qc, coastal Qc, and coastal Qd populations based on genome-wide genotypes and multitrait phenotypes and verified introgression from coastal Qd to coastal Qc using reduced library sequencing. Genotypes and phenotypes differed among the populations, and coastal Qc was intermediate between inland Qc and coastal Qd. The ABBA-BABA test showed introgression from coastal Qd to coastal Qc. In coastal Qc, we found various stages of introgression after the first generation of backcross but detected no genomic regions where introgression was enhanced. Overall, we show evidence for introgression from a coastal species to an ecotype of an inland species, which has colonized the coastal environment. It remains unclear whether introgressed alleles are selected in the coastal environment.

6.
Pediatr Cardiol ; 40(6): 1134-1143, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31087143

RESUMO

The effect of pregnancy on aortic root in women with repaired conotruncal anomalies (CTA) has not been clarified. This study examined aortic diameters during and after pregnancy in women with repaired CTA. A retrospective review of consecutive pregnant women with repaired CTA was performed for results of echocardiography from 1 year before pregnancy to 3 years after delivery and compared with findings from healthy pregnant volunteers. Participants comprised 42 subjects and 49 deliveries with repaired CTA (CTA group), and 47 healthy pregnant women (control group). Although no maternal aortic events were encountered, aortic diameters during pregnancy increased by 1.0 ± 2.2 mm (maximum, 7.0 mm) in the CTA group and 0.6 ± 1.3 mm (maximum, 3.4 mm) in the control group (p = 0.13). The CTA subgroup with increase in aortic diameter ≥ 3.5 mm during pregnancy showed no reversion to baseline diameter at follow-up > 6 months after delivery. Significant risk factors for increased aortic diameter and no reversal included pulmonary atresia, history of aortopulmonary shunt, older age at repair, and smaller left ventricular end-diastolic diameter pre-pregnancy. Women with repaired CTA tolerated pregnancy and delivery well. However, the aortic root progressively dilated during pregnancy and 1/4 of them, especially those with longstanding high aortic blood flow before repair, showed an increase of aortic root size of ≥ 3.5 mm during the peripartum period; this dilatation tended not to revert to the pre-pregnancy diameter. Therefore, serial measurement of the aortic root during pregnancy and after delivery is recommended in these women.


Assuntos
Aorta/patologia , Valva Aórtica/patologia , Seio Aórtico/patologia , Adulto , Fatores Etários , Aorta/anormalidades , Aorta/diagnóstico por imagem , Valva Aórtica/anormalidades , Valva Aórtica/diagnóstico por imagem , Estudos de Casos e Controles , Angiografia por Tomografia Computadorizada , Parto Obstétrico/efeitos adversos , Dilatação Patológica/etiologia , Ecocardiografia , Feminino , Cardiopatias Congênitas/cirurgia , Humanos , Imageamento Tridimensional , Imagem por Ressonância Magnética , Masculino , Gravidez , Atresia Pulmonar/etiologia , Estudos Retrospectivos , Fatores de Risco , Seio Aórtico/diagnóstico por imagem , Fatores de Tempo
7.
Commun Biol ; 2: 165, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069274

RESUMO

The RNA helicase EIF4A3 regulates the exon junction complex and nonsense-mediated mRNA decay functions in RNA transcript processing. However, a transcriptome-wide network definition of these functions has been lacking, in part due to the lack of suitable pharmacological inhibitors. Here we employ short-duration graded EIF4A3 inhibition using small molecule allosteric inhibitors to define the transcriptome-wide dependencies of EIF4A3. We thus define conserved cellular functions, such as cell cycle control, that are EIF4A3 dependent. We show that EIF4A3-dependent splicing reactions have a distinct genome-wide pattern of associated RNA-binding protein motifs. We also uncover an unanticipated role of EIF4A3 in the biology of RNA stress granules, which sequester and silence the translation of most mRNAs under stress conditions and are implicated in cell survival and tumour progression. We show that stress granule induction and maintenance is suppressed on the inhibition of EIF4A3, in part through EIF4A3-associated regulation of G3BP1 and TIA1 scaffold protein expression.

8.
Br J Radiol ; 92(1097): 20180825, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30835500

RESUMO

OBJECTIVE: The purpose of the study was to investigate variation in the use of in-hospital CT for venous thromboembolism (VTE) detection after total knee or hip replacement (TKR/THR) among surgical patients, using a nationwide Japanese in-hospital administrative database. METHODS: This retrospective study using a national administrative database (4/2012-3/2013) extracted patients who underwent TKR/THR surgeries at hospitals meeting the annual case-volume threshold of ≥ 30. Hospitals were categorized into three equally sized groups by frequency of postoperative CT use (low, middle, and high CT use group) to compare baseline patient-level and hospital-level characteristics. To further investigate between-hospital variation in CT usage, we fitted a hierarchical logistic regression model including hospital-specific random intercepts and fixed patient- and hospital-level effects. The intra class correlation coefficient was used to measure the amount of variability in CT use attributable to between-hospital variation. RESULTS: A total of 39,127 patients discharged from 447 hospitals met the inclusion criteria. The median hospital stay was 25 days (interquartile range, 20 - 32) and 7,599 (19.4%) patients underwent CT for VTE. CT utilization varied greatly among the hospitals; the crude frequency ranged from 0 to 100 % (median, 7.3 %; interquartile range, 1.8 - 18.3 %). After adjustment for known hospital- and patient-level factors related to CT use, 47 % of the variation in CT use remained attributable to the behavior of individual hospitals. CONCLUSION: We observed large inter hospital variability in the utilization of post-procedure CT for VTE detection in this Japanese TKR/THR cohort, suggesting that CT utilization is not optimized across the nation. ADVANCES IN KNOWLEDGE: We observed significant variability in the utilization of post-procedure CT for VTE detection among inpatients who underwent TKR/THR surgeries in a large sample of Japanese hospitals. The large variation suggests that CT utilization is not optimized across the nation, and that there may be potential overutilization of the technology in the highest CT use hospitals.


Assuntos
Artroplastia de Quadril/efeitos adversos , Artroplastia do Joelho/efeitos adversos , Hospitais/estatística & dados numéricos , Utilização de Procedimentos e Técnicas , Embolia Pulmonar/diagnóstico por imagem , Tomografia Computadorizada por Raios X/estatística & dados numéricos , Trombose Venosa/diagnóstico por imagem , Adulto , Idoso , Bases de Dados Factuais , Feminino , Mortalidade Hospitalar , Humanos , Japão , Tempo de Internação , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico por imagem , Estudos Retrospectivos
9.
Pediatr Cardiol ; 40(4): 865-870, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30830281

RESUMO

The frequency of newborns with congenital heart disease (CHD) is approximately 1% in the general population; however, the recurrence rate of CHD in mothers with CHD differs in ethnicity and reports. We therefore aimed to determine the prevalence of CHD among neonates born to mothers with CHD in our institute in Japan. We reviewed the medical charts of 803 neonates delivered by 529 women with CHD at the National Cerebral and Cardiovascular Center from 1982 to 2016. They included isolated ventricular septal defect (VSD,31.4%), isolated atrial septal defect (ASD, 23.3%), tetralogy of Fallot (TOF,10.6%). We defined CHD in neonates as being diagnosed within 1 month of birth. We estimated that the average rate of the CHD recurrence was 3.1%. The recurrence ratios in each maternal CHD were 8.6%, 7.1%, 6.2%, 4.8%, 3.6%, and 1.5% for PS, CoA, TOF, atrioventricular septal defect, VSD, and ASD, respectively. The rate of CHD in offsprings whose mothers have CHD was 3 times greater than that of mothers with healthy hearts. Almost half of neonates with CHD had the same phenotype as their mother in our series. Especially, PS and CoA were closely related to the type of maternal CHD.


Assuntos
Cardiopatias Congênitas/epidemiologia , Adolescente , Adulto , Criança , Feminino , Cardiopatias Congênitas/etiologia , Humanos , Recém-Nascido , Japão , Masculino , Pessoa de Meia-Idade , Mães/estatística & dados numéricos , Prevalência , Recidiva , Estudos Retrospectivos , Adulto Jovem
10.
Opt Express ; 27(3): 1884-1893, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30732235

RESUMO

We demonstrate high-quality non-destructive imaging using a broadband terahertz quantum cascade laser source based on Cerenkov difference-frequency generation. The source exhibited ultra-broadband terahertz emission spectra, as well as a single-lobed Gaussian-like far-field pattern at -30 °C. These features allowed us to build a compact imaging system with a high spatial resolution, from which a nearly theoretical minimum beam spot size was obtained. As a result, we achieve well-resolved, high-contrast images of objects obscured by opaque materials. We also achieved terahertz imaging with the THz DFG-QCL operated at room temperature.

11.
Ann Nucl Med ; 33(4): 288-294, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30707349

RESUMO

OBJECTIVE: Whole-body dynamic imaging using positron emission tomography (PET) facilitates the quantification of tracer kinetics. It is potentially valuable for the differential diagnosis of tumors and for the evaluation of therapeutic efficacy. In whole-body dynamic PET with continuous bed motion (CBM) (WBDCBM-PET), the pass number and bed velocity are key considerations. In the present study, we aimed to investigate the effect of a combination of pass number and bed velocity on the quantitative accuracy and quality of WBDCBM-PET images. METHODS: In this study, WBDCBM-PET imaging was performed at a body phantom using seven bed velocity settings in combination with pass numbers. The resulting image quality was evaluated. For comparing different acquisition settings, the dynamic index (DI) was obtained using the following formula: [P/S], where P represents the pass number, and S represents the bed velocity (mm/s). The following physical parameters were evaluated: noise equivalent count at phantom (NECphantom), percent background variability (N10 mm), percent contrast of the 10 mm hot sphere (QH, 10 mm), the QH, 10 mm/N10 mm ratio, and the maximum standardized uptake value (SUVmax). Furthermore, visual evaluation was performed. RESULTS: The NECphantom was equivalent for the same DI settings regardless of the bed velocity. The N10 mm exhibited an inverse correlation (r < - 0.89) with the DI. QH,10 mm was not affected by DI, and a correlation between QH,10 mm/N10 mm ratio and DI was found at all the velocities (r > 0.93). The SUVmax of the spheres was not influenced by the DI. The coefficient of variations caused by bed velocity decreased in larger spheres. There was no significant difference between the bed velocities on visual evaluation. CONCLUSION: The quantitative accuracy and image quality achieved with WBDCBM-PET was comparable to that achieved with non-dynamic CBM, regardless of the pass number and bed velocity used during imaging for a given acquisition time.


Assuntos
Movimento (Física) , Tomografia por Emissão de Pósitrons/instrumentação , Imagem Corporal Total/instrumentação , Artefatos , Fluordesoxiglucose F18 , Processamento de Imagem Assistida por Computador , Imagens de Fantasmas , Controle de Qualidade , Razão Sinal-Ruído
12.
J Plant Res ; 132(2): 211-222, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30604174

RESUMO

In northern Japan, coastal oak forests consist of Quercus dentata (Qd) on the coastal side and Q. mongolica var. crispula (Qc) on the inland side. In the forests of northern Hokkaido, Qd is rare, and a coastal ecotype of Qc with some Qd-like traits grows on the coastal side. To reveal the genetic background of this ecotype, nuclear microsatellite genotypes in closely related oak taxa were obtained from the Eurasian continent, Sakhalin, and Hokkaido. The clustering of these genotypes suggests an admixture of Qd in the coastal ecotype of Qc. Next, we evaluated the effects of admixture and coastal stress on the leaf and shoot traits of Qc and Qd along coastal-inland gradients in northern Hokkaido. The admixture of Qd in Qc was quantified by the Qd ancestry proportions. Coastal stress causes bud mortality in the upper parts of shoots and was quantified by the survival patterns of buds in shoots. The genetic and environmental effects on the traits at Qd-abundant and Qd-rare sites were estimated using linear mixed models. The genetic effect was detected in all traits. Both genetic and environmental effects were detected in most traits. Some traits differed between Qd-abundant and Qd-rare sites in addition to these effects, indicating more Qd-like traits at Qd-rare sites. The findings suggest that an admixture of Qd characterizes the genetic background of the coastal ecotype of Qc and that not only the coastal stress but also the genetic background is responsible for the leaf and shoot traits of Qc and Qd in northern Hokkaido.


Assuntos
Ecótipo , Quercus/genética , Florestas , Japão , Repetições de Microssatélites , Estresse Fisiológico
13.
Magn Reson Med Sci ; 18(3): 219-224, 2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-30504639

RESUMO

PURPOSE: Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) are representative disorders of dementia of the elderly and the neuroimaging has contributed to early diagnosis by estimation of alterations of brain volume, blood flow and metabolism. A brain network analysis by MR imaging (MR connectome) is a recently developed technique and can estimate the dysfunction of the brain network in AD and DLB. A graph theory which is a major technique of network analysis is useful for a group study to extract the feature of disorders, but is not necessarily suitable for the disorder differentiation at the individual level. In this investigation, we propose a deep learning technique as an alternative method of the graph analysis for recognition and classification of AD and DLB at the individual subject level. MATERIALS AND METHODS: Forty-eight brain structural connectivity data of 18 AD, 8 DLB and 22 healthy controls were applied to the machine learning consisting of a six-layer convolution neural network (CNN) model. Estimation of the deep learning model to classify AD, DLB and non-AD/DLB was performed using the 4-fold cross-validation method. RESULTS: The accuracy, average precision and recall of our CNN model were 0.73, 0.78 and 0.73, and the specificity precision and recall were 0.68 and 0.79 in AD, 0.94 and 0.65 in DLB and 0.73 and 0.75 in non-AD/DLB. The triangular probability map of the MR connectome revealed the probability of AD, DLB and non-AD/DLB in each subject. CONCLUSION: Our preliminary investigation revealed the adaptation of deep learning to the MR connectome and proposed its utility in the differentiation of dementia disorders at the individual subject level.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Demência/diagnóstico por imagem , Demência/patologia , Doença por Corpos de Lewy/diagnóstico por imagem , Doença por Corpos de Lewy/patologia , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade
14.
Front Genet ; 9: 490, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405697

RESUMO

Conifers in northern forests, such as fir and spruce, preferably regenerate on coarse woody debris, including fallen logs, stumps, and snags. In northern Japan, the sub-boreal conifer species Picea jezoensis is completely dependent on coarse woody debris for seedling establishment. To understand the fine-scale spatial genetic structure (FSGS) of this species, a 5-ha plot was established in central Hokkaido, and 531 individual trees were categorized into four life-stages (seedling, sapling, juvenile, and mature) on the basis of age and size. The FSGS of the established seedlings and later growth stages was investigated using 11 nuclear simple sequence repeat loci. A STRUCTURE analysis of seedlings and saplings established on fallen logs revealed that genetically related individuals were spatially localized between adjacent logs. We also found a significant FSGS in early life-stages based on a decline in the kinship coefficient calculated between individuals over shorter to longer spatial distances. Furthermore, the estimation of dispersal kernels indicated the frequent occurrence of short-distance seed dispersal. These results indicated that genetically related seedlings and saplings regenerated on the same or nearby fallen logs. In contrast to the results for the early stages, mature-stage trees showed no significant FSGS. We ran a simulation to examine the hypothesis that the FSGS could be eliminated by demographic thinning during life history processes. We calculated values for simulated offspring generated under three sets of conditions; i.e., by removing (i) inbred individuals, (ii) randomly chosen individuals, and (iii) all individuals on the specific fallen logs. However, the results for the FSGS were significant for all simulated data sets. This indicated that inbreeding depression, stochastic loss, or eradication of establishment sites by local disturbances alone could not explain the lack of FSGS among mature-stage trees. Therefore, it is possible that the colonization history of mature trees present on the study site might differ from that of the current offspring.

15.
Cell Chem Biol ; 25(12): 1470-1484.e5, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30293940

RESUMO

Alternative polyadenylation (APA) plays a critical role in regulating gene expression. However, the balance between genome-encoded APA processing and autoregulation by APA modulating RNA binding protein (RBP) factors is not well understood. We discovered two potent small-molecule modulators of APA (T4 and T5) that promote distal-to-proximal (DtoP) APA usage in multiple transcripts. Monotonically responsive APA events, induced by short exposure to T4 or T5, were defined in the transcriptome, allowing clear isolation of the genomic sequence features and RBP motifs associated with DtoP regulation. We found that longer vulnerable introns, enriched with distinctive A-rich motifs, were preferentially affected by DtoP APA, thus defining a core set of genes with genomically encoded DtoP regulation. Through APA response pattern and compound-small interfering RNA epistasis analysis of APA-associated RBP factors, we further demonstrated that DtoP APA usage is partly modulated by altered autoregulation of polyadenylate binding nuclear protein-1 signaling.


Assuntos
Poliadenilação/efeitos dos fármacos , Poliadenilação/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Transcriptoma/efeitos dos fármacos , Linhagem Celular , Feminino , Homeostase/efeitos dos fármacos , Humanos , Bibliotecas de Moléculas Pequenas/química , Transcriptoma/genética
16.
J Med Chem ; 61(17): 7710-7728, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30067358

RESUMO

Cyclin-dependent kinase 12 (CDK12) plays a key role in the coordination of transcription with elongation and mRNA processing. CDK12 mutations found in tumors and CDK12 inhibition sensitize cancer cells to DNA-damaging reagents and DNA-repair inhibitors. This suggests that CDK12 inhibitors are potential therapeutics for cancer that may cause synthetic lethality. Here, we report the discovery of 3-benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea derivatives as novel and selective CDK12 inhibitors. Structure-activity relationship studies of a HTS hit, structure-based drug design, and conformation-oriented design using the Cambridge Structural Database afforded the optimized compound 2, which exhibited not only potent CDK12 (and CDK13) inhibitory activity and excellent selectivity but also good physicochemical properties. Furthermore, 2 inhibited the phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and induced growth inhibition in SK-BR-3 cells. Therefore, 2 represents an excellent chemical probe for functional studies of CDK12 and could be a promising lead compound for drug discovery.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Sobrevivência Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Inibidores Enzimáticos/química , Feminino , Humanos , Fosforilação , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
17.
Int Heart J ; 59(4): 862-867, 2018 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-29794382

RESUMO

Little is known about pregnancies of left ventricular noncompaction cardiomyopathy (LVNC), much less cases in which LVNC was definitively diagnosed prepregnancy. We report the cases of three pregnant Japanese women definitively diagnosed with LVNC prepregnancy. Case 1 presented LVNC with restrictive phenotype. Her pregnancy was terminated due to exacerbated pulmonary hypertension and low output status at 30 weeks' gestation. Case 2 presented isolated LVNC with nonsustained ventricle tachycardia. A cesarean section was performed at 36 weeks' gestation because of placenta previa. Case 3 presented dilated LVNC. Labor induction was performed because of decreased left ventricular ejection fraction, leading to a vaginal delivery at 37 weeks' gestation. In all cases, no thromboembolic event was identified during pregnancy; two patients received anticoagulants. We reviewed all English-literature cases of pregnant women definitively diagnosed with LVNC prepregnancy to analyze causes of adverse pregnancy outcomes and the necessity of anticoagulation. Four of the six pregnancies identified were terminated due to exacerbated cardiomyopathy phenotypes and not complications due to noncompaction itself, resulting in three cases' preterm deliveries. No thromboembolic event was identified by maintenance of the anticoagulation strategy determined prepregnancy. In pregnancies with LVNC, the possibility of a severe cardiac event and the indications for termination of the pregnancy can depend on the cardiomyopathy phenotypes, not noncompaction itself. Anticoagulation only because of the pregnancy itself may be redundant. In the management of LVNC during pregnancy, close monitoring of the condition of different phenotypes and reassessment of the necessity of anticoagulation can contribute to the pregnancy outcome.


Assuntos
Anticoagulantes/administração & dosagem , Miocárdio Ventricular não Compactado Isolado , Complicações Cardiovasculares na Gravidez , Tromboembolia/prevenção & controle , Adulto , Cesárea/métodos , Feminino , Humanos , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/etiologia , Miocárdio Ventricular não Compactado Isolado/complicações , Miocárdio Ventricular não Compactado Isolado/diagnóstico , Miocárdio Ventricular não Compactado Isolado/fisiopatologia , Trabalho de Parto Induzido/métodos , Administração dos Cuidados ao Paciente/métodos , Gravidez , Complicações Cardiovasculares na Gravidez/diagnóstico , Complicações Cardiovasculares na Gravidez/fisiopatologia , Complicações Cardiovasculares na Gravidez/terapia , Resultado da Gravidez , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/etiologia , Tromboembolia/etiologia , Disfunção Ventricular Esquerda/diagnóstico , Disfunção Ventricular Esquerda/etiologia
18.
Eur Radiol ; 28(4): 1600-1608, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29063252

RESUMO

PURPOSE: To test the hypothesis that "acute-period" lumbar MRI in non-elderly patients with low back pain is less frequently performed at clinics/hospitals with greater involvement of full-time radiologists in the imaging workflow. METHODS: In a national-level claims database, we identified 14,819 non-elderly patients (mean age: 38.7±8.0 years) who visited clinics/hospitals for low back pain in 2013-2015. We classified the clinics/hospitals into four groups based on the level of full-time radiologist involvement and MRI ownership, and compared the frequency of acute-period lumbar MRI using hierarchical logistic regression analysis. RESULTS: Patients visiting facilities without a full-time radiologist (n=2105) were significantly (p<0.001) more likely to undergo acute-period MRI than those visiting facilities with ≥1 radiologist partially managing imaging workflow (level-1, n=491) or ≥1 radiologist intensively involved in imaging workflow (level-2, n=1190) (15.7% vs. 6.9% and 7.3%; adjusted odds ratio of no-radiologist versus level-2: 2.93, p=0.018). No difference was observed between level-1 and level-2 involvement. CONCLUSIONS: Facilities with no full-time radiologist were more likely to perform acute-period MRI to assess for low back pain, while no difference was seen between facilities with varying levels of radiologist involvement in the imaging workflow. Radiologist involvement may contribute to optimal utilisation of medical imaging. KEY POINTS: • Lumbar MRI was more frequently performed at facilities without full-time radiologists. • Full-time radiologists may play an important role in appropriate utilisation of imaging. • Frequency of MRI was similar between moderate and intensive radiologist involvement.


Assuntos
Dor Lombar/diagnóstico por imagem , Imagem por Ressonância Magnética/métodos , Imagem por Ressonância Magnética/estatística & dados numéricos , Radiologistas/estatística & dados numéricos , Doença Aguda , Adolescente , Adulto , Feminino , Humanos , Vértebras Lombares/diagnóstico por imagem , Região Lombossacral/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Razão de Chances , Adulto Jovem
19.
Genes Dev ; 31(18): 1910-1925, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29021239

RESUMO

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.


Assuntos
Encéfalo/embriologia , Adesão Celular/fisiologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Neurais/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo/fisiologia , Animais , Comunicação Celular , Regulação para Baixo , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Neurogênese/genética , Neurogênese/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais
20.
Bioorg Med Chem ; 25(17): 4753-4767, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28751196

RESUMO

Bad response to refrigeration 2 (Brr2) is a member of the Ski2-like RNA helicases, and an essential component of the U5 small nuclear ribonucleoprotein (snRNP). A particularly important role of Brr2 is the ATP-dependent unwinding of the U4/U6 RNA duplex, which is a critical step in spliceosomal activation. Despite its biological importance, selective inhibitor for Brr2 had not been reported until our recent report. Here, we describe novel and structurally distinct spiro[indole-3,2'-pyrrolidin]-2(1H)-one based Brr2 inhibitors with superior activity to the previously reported 4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione series. Using an RNA dependent ATPase assay as a guide, high-throughput screening, hit validation by structure-activity relationship (SAR) study, and subsequent chemical optimization to increase the ATPase inhibitory activity were performed. Thereafter, selectivity and helicase inhibitory activity of optimized compounds were confirmed. In the course of the study, compounds were synthesized using a three-component reaction, which accelerated the optimization process. All these efforts finally culminated in the discovery of the potent and selective Brr2 inhibitors (32a and 33a) exhibiting helicase inhibitory activity at submicromolar concentrations. Thus, compounds 32a and 33a could be valuable molecular probes to study the functions of Brr2 and molecular machinery of RNA splicing.


Assuntos
Ribonucleoproteínas Nucleares Pequenas/antagonistas & inibidores , Compostos de Espiro/química , Humanos , Indóis/química , Concentração Inibidora 50 , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Compostos de Espiro/metabolismo , Relação Estrutura-Atividade
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