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1.
Artigo em Inglês | MEDLINE | ID: mdl-32449858

RESUMO

The development of membranes with low fuel crossover and high fuel efficiency is a key issue for direct borohydride fuel cells (DBFC). In previous work, we produced a polyvinyl alcohol (PVA) - anion exchange resin (AER) membrane with low fuel crossover and low fuel efficiency by introducing Co ions. In this work, a bilayer membrane was designed to improve fuel efficiency and cell performance. The bilayer membrane was prepared by casting a PVA-AER wet gel onto the partially desiccated Co-PVA-AER gel. The bilayer membrane showed a borohydride permeability of 1.34 106 cm2s-1, which was even lower than that of the Co-PVA-AER membrane (1.98 106 cm2s-1) and the PVA-AER membrane (2.80 106 cm2s-1). The DBFC using the bilayer membrane exhibited higher fuel efficiency (37.4%) and output power (1.73 Wh) than the DBFCs using the Co-PVA-AER membrane (33.3%, 1.27 Wh) and the PVA-AER membrane (34.3%, 1.2 Wh). Furthermore, the DBFC using the bilayer membrane achieved a peak power density of 327 mWcm-2, which was 2.14 times of that of the DBFC using the PVA-AER membrane (153 mWcm-2). The drastic improvement benefited from the bilayer design, which introduced an interphase to suppress fuel crossover and avoided unnecessary borohydride hydrolysis.

2.
Cell Rep ; 31(3): 107529, 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32320660

RESUMO

The asymmetric distribution of auxin leads to the bending growth of hypocotyls during gravitropic and phototropic responses, but the signaling events downstream of auxin remain unclear. Here, we identify many SAUR genes showing asymmetric expression in soybean hypocotyls during gravistimulation and then study their homologs in Arabidopsis. SAUR19 subfamily genes have asymmetric expression in Arabidopsis hypocotyls during gravitropic and phototropic responses, induced by the lateral redistribution of auxin. Both the mutation of SAUR19 subfamily genes and the ectopic expression of SAUR19 weaken these tropic responses, indicating the critical role of their asymmetric expression. The auxin-responsive transcription factor ARF7 may directly bind the SAUR19 promoter and activate SAUR19 expression asymmetrically in tropic responses. Taken together, our results reveal that a gravity- or light-triggered asymmetric auxin distribution induces the asymmetric expression of SAUR19 subfamily genes by ARF7 and ARF19 in the hypocotyls, which leads to bending growth during gravitropic and phototropic responses.

3.
Int J Surg ; 77: 120-127, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32234578

RESUMO

BACKGROUND: Currently, 310 million patients undergo surgery every year worldwide, and there is still controversy over which anesthetic technique to choose for a considerable of surgeries.This study evaluates the association of the anesthetic technique with thirty-day mortality after noncardiac- and nonneurosurgery. METHODS: Electronic medical records of 90,785 patients who underwent non-cardiac- and nonneurosurgery at the *** General Hospital from January 1, 2012 to October 31, 2016, were subject to secondary retrospective analysis. The principal exposure was regional versus general anesthesia. Outcome measures were death, intensive care unit (ICU) admission and blood transfusion requirement within 30 days after surgery. Propensity-score matching was used to assemble a cohort of patients with similar baseline characteristics. RESULTS: We identified 90,785 patients, of whom 76,442 received regional anesthesia and 14,343 received general anesthesia. A total of 11,351 patients in the general anesthesia group had propensity scores similar to those of patients who received regional anesthesia and were included in the analyses. In the propensity-score matched cohort, the postoperative 30-day mortality rate was 0.75% (n = 85) in the regional anesthesia group (Odds Ratio, 0.567; 95% CI, 0.434 to 0.741; P = 0.00003) compared with 1.31% (n = 149) in the general anesthesia group. Regional anesthesia was also associated with a reduced rate of ICU admission compared with that of patients who received general anesthesia (0.44% vs. 2.68%; OR, 0.161; 95% CI, 0.119 to 0.217, P < 0.00001). There was a nonsignificant relationship between the anesthetic technique and postoperative blood transfusion (P = 0.082). CONCLUSIONS: The results of this observational, propensity score-matched cohort study suggest a significant association between regional anesthesia and low thirty-day mortality and a worse postoperative prognosis in patients who underwent noncardiac- and nonneurosurgery, which provides information for anesthetic technique decision making in the clinical setting.

4.
J Cell Mol Med ; 24(10): 5420-5427, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32283573

RESUMO

Breast cancer is the most common cancer diagnosed in women. Breast cancer research is currently based mainly on animal models and traditional cell culture. However, the inherent species gap between humans and animals, as well as differences in organization between organs and cells, limits research advances. The breast cancer organoid can reproduce many of the key features of human breast cancer, thereby providing a new platform for investigating the mechanisms underlying the development, progression, metastasis and drug resistance of breast cancer. The application of organoid technology can also promote drug discovery and the design of individualized treatment strategies. Here, we discuss the latest advances in the use of organoid technology for breast cancer research.

5.
Plant Physiol ; 182(1): 159-166, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31690709

RESUMO

The phytochrome B (phyB) photoreceptor stimulates light responses in plants in part by inactivating repressors of light responses, such as PHYTOCHROME-INTERACTING FACTOR3 (PIF3). Activated phyB inhibits PIF3 by rapid protein degradation and decreased transcription. PIF3 protein degradation is mediated by EIN3-BINDING F-BOX PROTEIN (EBF) and LIGHT-RESPONSE BTB (LRB) E3 ligases, the latter of which simultaneously targets phyB for degradation. In this study, we show that PIF3 levels are additionally regulated by alternative splicing and protein translation in Arabidopsis (Arabidopsis thaliana). Overaccumulation of photo-activated phyB, which occurs in the mutant defective for LRB genes under continuous red light, induces a specific alternative splicing of PIF3 that results in retention of an intron in the 5' untranslated region of PIF3 mRNA. In turn, the upstream open reading frames contained within this intron inhibit PIF3 protein synthesis. The phyB-dependent alternative splicing of PIF3 is diurnally regulated under the short-day light cycle. We hypothesize that this reversible regulatory mechanism may be utilized to fine tune the level of PIF3 protein in light-grown plants and may contribute to the oscillation of PIF3 protein abundance under the short-day environment.

6.
Stem Cell Reports ; 13(6): 960-969, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31708475

RESUMO

With extended stays aboard the International Space Station (ISS) becoming commonplace, there is a need to better understand the effects of microgravity on cardiac function. We utilized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of microgravity on cell-level cardiac function and gene expression. The hiPSC-CMs were cultured aboard the ISS for 5.5 weeks and their gene expression, structure, and functions were compared with ground control hiPSC-CMs. Exposure to microgravity on the ISS caused alterations in hiPSC-CM calcium handling. RNA-sequencing analysis demonstrated that 2,635 genes were differentially expressed among flight, post-flight, and ground control samples, including genes involved in mitochondrial metabolism. This study represents the first use of hiPSC technology to model the effects of spaceflight on human cardiomyocyte structure and function.

7.
Proc Natl Acad Sci U S A ; 116(40): 20218-20225, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31527236

RESUMO

The PHYTOCHROME-INTERACTING FACTORs (PIFs) play a central role in repressing photomorphogenesis, and phosphorylation mediates the stability of PIF proteins. Although the kinases responsible for PIF phosphorylation have been extensively studied, the phosphatases that dephosphorylate PIFs remain largely unknown. Here, we report that seedlings with mutations in FyPP1 and FyPP3, 2 genes encoding the catalytic subunits of protein phosphatase 6 (PP6), exhibited short hypocotyls and opened cotyledons in the dark, which resembled the photomorphogenic development of dark-grown pifq mutants. The hypocotyls of dark-grown sextuple mutant fypp1 fypp3 (f1 f3) pifq were shorter than those of parental mutants f1 f3 and pifq, indicating that PP6 phosphatases and PIFs function synergistically to repress photomorphogenesis in the dark. We showed that FyPPs directly interacted with PIF3 and PIF4, and PIF3 and PIF4 proteins exhibited mobility shifts in f1 f3 mutants, consistent with their hyperphosphorylation. Moreover, PIF4 was more rapidly degraded in f1 f3 mutants than in wild type after light exposure. Whole-genome transcriptomic analyses indicated that PP6 and PIFs coregulated many genes, and PP6 proteins may positively regulate PIF transcriptional activity. These data suggest that PP6 phosphatases may repress photomorphogenesis by controlling the stability and transcriptional activity of PIF proteins via regulating PIF phosphorylation.

8.
ACS Nano ; 13(9): 9880-9894, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31149806

RESUMO

A major challenge in myocardial infarction (MI)-related heart failure treatment using microRNA is the efficient and sustainable delivery of miRNAs into myocardium to achieve functional improvement through stimulation of intrinsic myocardial restoration. In this study, we established an in vivo delivery system using polymeric nanoparticles to carry miRNA (miNPs) for localized delivery within a shear-thinning injectable hydrogel. The miNPs triggered proliferation of human embryonic stem cell-derived cardiomyocytes and endothelial cells (hESC-CMs and hESC-ECs) and promoted angiogenesis in hypoxic conditions, showing significantly lower cytotoxicity than Lipofectamine. Furthermore, one injected dose of hydrogel/miNP in MI rats demonstrated significantly improved cardiac functions: increased ejection fraction from 45% to 64%, reduced scar size from 20% to 10%, and doubled capillary density in the border zone compared to the control group at 4 weeks. As such, our results indicate that this injectable hydrogel/miNP composite can deliver miRNA to restore injured myocardium efficiently and safely.

9.
Circ Res ; 125(1): 90-103, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31104567

RESUMO

RATIONALE: The immature presentation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is currently a challenge for their application in disease modeling, drug screening, and regenerative medicine. Long-term culture is known to achieve partial maturation of iPSC-CMs. However, little is known about the molecular signaling circuitries that govern functional changes, metabolic output, and cellular homeostasis during long-term culture of iPSC-CMs. OBJECTIVE: We aimed to identify and characterize critical signaling events that control functional and metabolic transitions of cardiac cells during developmental progression, as recapitulated by long-term culture of iPSC-CMs. METHODS AND RESULTS: We combined transcriptomic sequencing with pathway network mapping in iPSC-CMs that were cultured until a late time point, day 200, in comparison to a medium time point, day 90, and an early time point, day 30. Transcriptomic landscapes of long-term cultured iPSC-CMs allowed mapping of distinct metabolic stages during development of maturing iPSC-CMs. Temporally divergent control of mitochondrial metabolism was found to be regulated by cAMP/PKA (protein kinase A)- and proteasome-dependent signaling events. The PKA/proteasome-dependent signaling cascade was mediated downstream by Hsp90 (heat shock protein 90), which in turn modulated mitochondrial respiratory chain proteins and their metabolic output. During long-term culture, this circuitry was found to initiate upregulation of iPSC-CM metabolism, resulting in increased cell contractility that reached a maximum at the day 200 time point. CONCLUSIONS: Our results reveal a PKA/proteasome- and Hsp90-dependent signaling pathway that regulates mitochondrial respiratory chain proteins and determines cardiomyocyte energy production and functional output. These findings provide deeper insight into signaling circuitries governing metabolic homeostasis in iPSC-CMs during developmental progression.

10.
Mol Plant ; 12(6): 847-862, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31009752

RESUMO

Light serves as the source of energy as well as an information signal for photosynthetic plants. During evolution, plants have acquired the ability to monitor environmental light radiation and adjust their developmental patterns to optimally utilize light energy for photosynthesis. The mechanisms of light perception and signal transduction have been comprehensively studied in past decades, mostly in a few model plants, including Arabidopsis thaliana. However, systematic analyses of the origin and evolution of core components involved in light perception and signaling are still lacking. In this study, we took advantage of the recently sequenced genomes and transcriptomes covering all the main Archaeplastida clades in the public domain to identify orthologous genes of core components involved in light perception and signaling and to reconstruct their evolutionary history. Our analyses suggested that acclimation to different distribution of light quality in new environments led to the origination of specific light signaling pathways in plants. The UVR8 (UV Resistance Locus 8) signaling pathway originated during the movement of plants from the deeper sea to shallow water and enabled plants to deal with ultraviolet B light (UV-B). After acquisition of UV-B adaptation, origination of the phytochrome signaling pathway helped plants to colonize water surface where red light became the prominent light energy source. The seedling emergence pathway, which is mediated by a combination of light and phytohormone signals that orchestrate plant growth pattern transitions, originated before the emergence of seed plants. Although cryptochromes and some key components of E3 ubiquitin ligase systems already existed before the divergence of the plant and animal kingdoms, the coevolution and optimization of light perception and downstream signal transduction components, including key transcription factors and E3 ubiquitin ligase systems, are evident during plant terrestrialization.

11.
Plant Cell ; 31(5): 1155-1170, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30914467

RESUMO

Light elicits different growth responses in different organs of plants. These organ-specific responses are prominently displayed during de-etiolation. While major light-responsive components and early signaling pathways in this process have been identified, this information has yet to explain how organ-specific light responses are achieved. Here, we report that members of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factor family participate in photomorphogenesis and facilitate light-induced cotyledon opening in Arabidopsis (Arabidopsis thaliana). Chromatin immunoprecipitation sequencing and RNA sequencing analyses indicated that TCP4 targets a number of SMALL AUXIN UPREGULATED RNA (SAUR) genes that have previously been shown to exhibit organ-specific, light-responsive expression. We demonstrate that TCP4-like transcription factors, which are predominantly expressed in the cotyledons of both light- and dark-grown seedlings, activate SAUR16 and SAUR50 expression in response to light. Light regulates the binding of TCP4 to the promoters of SAUR14, SAUR16, and SAUR50 through PHYTOCHROME-INTERACTING FACTORs (PIFs). PIF3, which accumulates in etiolated seedlings and its levels rapidly decline upon light exposure, also binds to the SAUR16 and SAUR50 promoters, while suppressing the binding of TCP4 to these promoters in the dark. Our study reveals that the interplay between light-responsive factors PIFs and the developmental regulator TCP4 determines the cotyledon-specific light regulation of SAUR16 and SAUR50, which contributes to cotyledon closure and opening before and after de-etiolation.

12.
J Hazard Mater ; 369: 268-278, 2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-30780023

RESUMO

Lithium-ion batteries are the most popular used portable energy storage technology due to the relatively high energy density. While thermal instability induced safety concerns impede the pace of developing large scale applications, the practical applications have no tolerance for the catastrophic failure. To learn more about the characteristics of battery failure, the criticality of battery thermal runaway is studied in this paper. Semenov and Thomas models are employed to analyze the criticality of battery thermal runaway in uniform and nonuniform temperature distribution situations. In order to improve accuracy of prediction, the critical parameters of overall reaction are taken as a weighted average of four exothermic reactions and the critical criteria are revised by the consumption of reactants. Results from revised model are consistence with oven model. According to the revised thermal abuse models, the critical criterion (ψcr,δcr) and critical temperature distribution (θcr) are analyzed in different composite materials, convective heat transfer coefficients and cell deformations. Results give the variation of critical criteria and critical temperature with these factors.

13.
Sci Rep ; 9(1): 86, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30643161

RESUMO

In recent years, heavy metal pollution has become a more serious global problem, and all countries are actively engaged in finding methods to remediate heavy metal-contaminated soil. We conducted transcriptome sequencing of the roots of cotton grown under three different cadmium concentrations, and analysed the potential strategies for coping with cadmium stress. Through Gene Ontology analysis, we found that most of the genes differentially regulated under cadmium stress were associated with catalytic activity and binding action, especially metal iron binding, and specific metabolic and cellular processes. The genes responsive to cadmium stress were mainly related to membrane and response to stimulus. The KEGG pathways enriched differentially expressed genes were associated with secondary metabolite production, Starch and sucrose metabolism, flavonoid biosynthesis, phenylalanina metalism and biosynthesis, in order to improve the activity of antioxidant system, repair systems and transport system and reduction of cadmium toxicity. There are three main mechanisms by which cotton responds to cadmium stress: thickening of physical barriers, oxidation resistance and detoxification complexation. Meanwhile, identified a potential cotton-specific stress response pathway involving brassinolide, and ethylene signaling pathways. Further investigation is needed to define the specific molecular mechanisms underlying cotton tolerance to cadmium stress. In this study potential coping strategies of cotton root under cadmium stress were revealed. Our findings can guide the selection of cotton breeds that absorb high levels of cadmium.


Assuntos
Cádmio/toxicidade , Gossypium/efeitos dos fármacos , Poluentes do Solo/toxicidade , Estresse Fisiológico , Perfilação da Expressão Gênica , Gossypium/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética
14.
J Hazard Mater ; 364: 539-547, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30388638

RESUMO

Ammonium nitrate (AN) is a commonly-used industrial raw material in industrial explosives and fertilizers areas. However, as an energetic material, its danger exists during the production, transportation, and storage, resulting in a large number of accidents involving personal injury and property loss. To obtain the accurate kinetic triplet parameters of AN thermal decomposition, a series of thermogravimetry analysis (TGA) experiments was conducted with four different heating rates. Activation energies were calculated by different isoconversional methods Then the kinetic triplet of AN pyrolysis was optimized using a combination of experimental and simulant methods. Combined with the traditional model-free and model-fitting approaches, the experimental kinetic model for AN pyrolysis was optimized and then reconstructed. Through the pyrolysis reaction of AN, a reliable methodology for processing TGA data of hazardous material is proposed in the paper, and the kinetic parameters can be accurately obtained by using such a kinetics method.

15.
PLoS Genet ; 14(12): e1007839, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30566447

RESUMO

MYB transcription factors are involved in many biological processes, including metabolism, development and responses to biotic and abiotic stresses. RADIALIS-LIKE SANT/MYB 1 (RSM1) belongs to a MYB-related subfamily, and previous transcriptome analysis suggests that RSM1 may play roles in plant development, stress responses and plant hormone signaling. However, the molecular mechanisms of RSM1 action in response to abiotic stresses remain obscure. We show that down-regulation or up-regulation of RSM1 expression alters the sensitivity of seed germination and cotyledon greening to abscisic acid (ABA), NaCl and mannitol in Arabidopsis. The expression of RSM1 is dynamically regulated by ABA and NaCl. Transcription factors ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG (HYH) regulate RSM1 expression via binding to the RSM1 promoter. Genetic analyses reveal that RSM1 mediates multiple functions of HY5 in responses of seed germination, post-germination development to ABA and abiotic stresses, and seedling tolerance to salinity. Pull-down and BiFC assays show that RSM1 interacts with HY5/HYH in vitro and in vivo. RSM1 and HY5/HYH may function as a regulatory module in responses to ABA and abiotic stresses. RSM1 binds to the promoter of ABA INSENSITIVE 5 (ABI5), thereby regulating its expression, while RSM1 interaction also stimulates HY5 binding to the ABI5 promoter. However, no evidence was found in the dual-luciferase transient expression assay to support that RSM enhances the activation of ABI5 expression by HY. In summary, HY5/HYH and RSM1 may converge on the ABI5 promoter and independently or somehow dependently regulate ABI5 expression and ABI5-downstream ABA and abiotic stress-responsive genes, thereby improving the adaption of plants to the environment.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Germinação/efeitos dos fármacos , Germinação/genética , Germinação/fisiologia , Modelos Biológicos , Proteínas Nucleares/genética , Pressão Osmótica , Reguladores de Crescimento de Planta/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Salinidade , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
16.
Proc Natl Acad Sci U S A ; 115(50): E11864-E11873, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30478060

RESUMO

Phytochrome A (phyA) is the only plant photoreceptor that perceives far-red light and then mediates various responses to this signal. Phosphorylation and dephosphorylation of oat phyA have been extensively studied, and it was shown that phosphorylation of a serine residue in the hinge region of oat phyA could regulate the interaction of phyA with its signal transducers. However, little is known about the role of the hinge region of Arabidopsis phyA. Here, we report that three sites in the hinge region of Arabidopsis phyA (i.e., S590, T593, and S602) are essential in regulating phyA function. Mutating all three of these sites to either alanines or aspartic acids impaired phyA function, changed the interactions of mutant phyA with FHY1 and FHL, and delayed the degradation of mutant phyA upon light exposure. Moreover, the in vivo formation of a phosphorylated phyA form was greatly affected by these mutations, while our data indicated that the abundance of this phosphorylated phyA form correlated well with the extent of phyA function, thus suggesting a pivotal role of the phosphorylated phyA in inducing the far-red light response. Taking these data together, our study reveals the important role of the hinge region of Arabidopsis phyA in regulating phyA phosphorylation and function, thus linking specific residues in the hinge region to the regulatory mechanisms of phyA phosphorylation.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fitocromo A/química , Fitocromo A/metabolismo , Transporte Ativo do Núcleo Celular , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Luz , Mutagênese Sítio-Dirigida , Fosforilação , Fitocromo/metabolismo , Fitocromo A/genética , Plantas Geneticamente Modificadas , Proteólise , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismo
17.
Adv Funct Mater ; 28(1)2018 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-30473658

RESUMO

The last decade has seen impressive progress in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that makes them ideal tools to repair injured hearts. To achieve an optimal outcome, advanced molecular imaging methods are essential to accurately track these transplanted cells in the heart. Herein, we demonstrate for the first time that a class of photoacoustic nanoparticles (PANPs) incorporating semiconducting polymers (SPs) as contrast agents can be used in the photoacoustic imaging (PAI) of transplanted hESC-CMs in living mouse hearts. This is achieved by virtue of two benefits of PANPs. First, strong PA signals and specific spectral features of SPs allow PAI to sensitively detect and distinguish a small number of PANP-labeled cells (2,000) from background tissues in vivo. Second, the PANPs show a high efficiency for hESC-CM labeling without adverse effects on cell structure, function, and gene expression. Assisted by ultrasound imaging, the delivery and engraftment of hESC-CMs in living mouse hearts can be assessed by PANP-based PAI with high spatial resolution (~100 µm). In summary, this study explores and validates a novel application of SPs as a PA contrast agent to track labeled cells with high sensitivity and accuracy in vivo, highlighting the advantages of integrating PAI and PANPs to advance cardiac regenerative therapies.

18.
Sci Rep ; 8(1): 12926, 2018 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150710

RESUMO

Diarrheagenic Escherichia coli (DEC) causes human diarrhea symptom in both healthy and immunocompromised individuals. An auto-microfluidic thin-film chip (AMTC) instrument integrating one-step multiplex PCR (mPCR) with reverse dot blot hybridization (RDBH) was developed for high-throughput detection of DEC. The novel mPCR method was developed by designing 14 specific primers and corresponding probes. 14 indexes including an endogenous gene (uidA) and 13 pathogenic genes (stx1, stx2, escV, ipaH, invE, estB, lt, pic, aggR, astA, bfpB, sth and stp) of DEC were detected. This one-step mPCR + RDBH approach is useful for simultaneous detection of numerous target genes in a single sample, whose specificity and availability have been confirmed on the positive control of 11 DEC strains. In addition, with 300 diarrheal stool samples being detected by this method, 21 were found to contain five major DEC strains. Compared with monoplex PCR and previous one-step mPCR approach, this method could detect ipaH and estB, and compared with current commercial kit, the relevance ratio of DEC detected by the AMTC method was increased by 1% in stool samples. Furthermore, the novel integration AMTC device could be a valuable detection tool for categorization of E. coli.


Assuntos
Escherichia coli/metabolismo , Microfluídica/métodos , Proteínas de Escherichia coli/metabolismo , Reação em Cadeia da Polimerase Multiplex/métodos
19.
J Am Coll Cardiol ; 72(1): 62-75, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29957233

RESUMO

BACKGROUND: The long QT syndrome (LQTS) is an arrhythmogenic disorder of QT interval prolongation that predisposes patients to life-threatening ventricular arrhythmias such as Torsades de pointes and sudden cardiac death. Clinical genetic testing has emerged as the standard of care to identify genetic variants in patients suspected of having LQTS. However, these results are often confounded by the discovery of variants of uncertain significance (VUS), for which there is insufficient evidence of pathogenicity. OBJECTIVES: The purpose of this study was to demonstrate that genome editing of patient-specific induced pluripotent stem cells (iPSCs) can be a valuable approach to delineate the pathogenicity of VUS in cardiac channelopathy. METHODS: Peripheral blood mononuclear cells were isolated from a carrier with a novel missense variant (T983I) in the KCNH2 (LQT2) gene and an unrelated healthy control subject. iPSCs were generated using an integration-free Sendai virus and differentiated to iPSC-derived cardiomyocytes (CMs). RESULTS: Whole-cell patch clamp recordings revealed significant prolongation of the action potential duration (APD) and reduced rapidly activating delayed rectifier K+ current (IKr) density in VUS iPSC-CMs compared with healthy control iPSC-CMs. ICA-105574, a potent IKr activator, enhanced IKr magnitude and restored normal action potential duration in VUS iPSC-CMs. Notably, VUS iPSC-CMs exhibited greater propensity to proarrhythmia than healthy control cells in response to high-risk torsadogenic drugs (dofetilide, ibutilide, and azimilide), suggesting a compromised repolarization reserve. Finally, the selective correction of the causal variant in iPSC-CMs using CRISPR/Cas9 gene editing (isogenic control) normalized the aberrant cellular phenotype, whereas the introduction of the homozygous variant in healthy control cells recapitulated hallmark features of the LQTS disorder. CONCLUSIONS: The results suggest that the KCNH2T983I VUS may be classified as potentially pathogenic.


Assuntos
Canalopatias/genética , Canal de Potássio ERG1/genética , Edição de Genes , Células-Tronco Pluripotentes Induzidas , Síndrome do QT Longo/genética , Adulto , Humanos , Masculino
20.
Circulation ; 138(23): 2666-2681, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29914921

RESUMO

BACKGROUND: The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. METHODS: To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a "healthy" asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be "likely pathogenic." Human-induced pluripotent stem cells (iPSCs) were derived from the heterozygous VUS MYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUS MYL3(170C>A); (3) heterozygous frameshift mutation MYL3(170C>A/fs); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUS MYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). RESULTS: The heterozygous VUS MYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUS MYL3(170C>A)- and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. CONCLUSIONS: Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrier-specific manner in a dish. As such, this platform represents a promising VUS risk-assessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.


Assuntos
Sistemas CRISPR-Cas/genética , Cardiomiopatias/patologia , Variação Genética , Sequência de Aminoácidos , Cálcio/metabolismo , Cardiomiopatias/genética , Diferenciação Celular , Mutação da Fase de Leitura , Edição de Genes/métodos , Expressão Gênica , Heterozigoto , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Alinhamento de Sequência
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