62 results on '"Zhang EE"'
Search Results
2. In-plane crushing behavior and energy absorption of a novel graded honeycomb from hierarchical architecture
- Author
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Liu, Hu, Zhang, Ee Teng, Wang, Guangjian, and Ng, Bing Feng
- Published
- 2022
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3. Novel arc-shaped ligaments to enhance energy absorption capabilities of re-entrant anti-trichiral structures
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Zhang, Ee Teng, Liu, Hu, and Ng, Bing Feng
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- 2021
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4. In-plane dynamic crushing of a novel honeycomb with functionally graded fractal self-similarity
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Liu, Hu, Zhang, Ee Teng, and Ng, Bing Feng
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- 2021
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5. Mechanics of Re-Entrant Anti-Trichiral Honeycombs with Nature-
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Zhang, Ee Teng, primary, Liu, Hu, additional, and Ng, Bing Feng, additional
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- 2023
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6. A nature-inspired HIF stabilizer derived from a highland-adaptation insertion of plateau pika Epas1 protein.
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Yu Z, Ran G, Chai J, and Zhang EE
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- Animals, Humans, Mice, Ubiquitination, Protein Stability, Adaptation, Physiological, HEK293 Cells, Basic Helix-Loop-Helix Transcription Factors metabolism, Basic Helix-Loop-Helix Transcription Factors genetics, Von Hippel-Lindau Tumor Suppressor Protein metabolism, Von Hippel-Lindau Tumor Suppressor Protein genetics, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Hypoxia-Inducible Factor 1, alpha Subunit genetics
- Abstract
Hypoxia-inducible factors (HIFs) play pivotal roles in numerous diseases and high-altitude adaptation, and HIF stabilizers have emerged as valuable therapeutic tools. In our prior investigation, we identified a highland-adaptation 24-amino-acid insertion within the Epas1 protein. This insertion enhances the protein stability of Epas1, and mice engineered with this insertion display enhanced resilience to hypoxic conditions. In the current study, we delved into the biochemical mechanisms underlying the protein-stabilizing effects of this insertion. Our findings unveiled that the last 11 amino acids within this insertion adopt a helical conformation and interact with the α-domain of the von Hippel-Lindau tumor suppressor protein (pVHL), thereby disrupting the Eloc-pVHL interaction and impeding the ubiquitination of Epas1. Utilizing a synthesized peptide, E14-24, we demonstrated its favorable membrane permeability and ability to stabilize endogenous HIF-α proteins, inducing the expression of hypoxia-responsive element (HRE) genes. Furthermore, the administration of E14-24 to mice subjected to hypoxic conditions mitigated body weight loss, suggesting its potential to enhance hypoxia adaptation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2024
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7. Deformable anthropomorphic pelvis phantom for dose accumulation verification.
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Wong YM, Koh CWY, Lew KS, Chua CGA, Yeap PL, Zhang ET, Ong ALK, Tuan JKL, Ng BF, Lew WS, Lee JCL, and Tan HQ
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- Humans, Radiation Dosage, Image Processing, Computer-Assisted methods, Tomography, X-Ray Computed, Male, Printing, Three-Dimensional, Phantoms, Imaging, Pelvis diagnostic imaging
- Abstract
Objective. The validation of deformable image registration (DIR) for contour propagation is often done using contour-based metrics. Meanwhile, dose accumulation requires evaluation of voxel mapping accuracy, which might not be accurately represented by contour-based metrics. By fabricating a deformable anthropomorphic pelvis phantom, we aim to (1) quantify the voxel mapping accuracy for various deformation scenarios, in high- and low-contrast regions, and (2) identify any correlation between dice similarity coefficient (DSC), a commonly used contour-based metric, and the voxel mapping accuracy for each organ. Approach . Four organs, i.e. pelvic bone, prostate, bladder and rectum (PBR), were 3D printed using PLA and a Polyjet digital material, and assembled. The latter three were implanted with glass bead and CT markers within or on their surfaces. Four deformation scenarios were simulated by varying the bladder and rectum volumes. For each scenario, nine DIRs with different parameters were performed on RayStation v10B. The voxel mapping accuracy was quantified by finding the discrepancy between true and mapped marker positions, termed the target registration error (TRE). Pearson correlation test was done between the DSC and mean TRE for each organ. Main results . For the first time, we fabricated a deformable phantom purely from 3D printing, which successfully reproduced realistic anatomical deformations. Overall, the voxel mapping accuracy dropped with increasing deformation magnitude, but improved when more organs were used to guide the DIR or limit the registration region. DSC was found to be a good indicator of voxel mapping accuracy for prostate and rectum, but a comparatively poorer one for bladder. DSC > 0.85/0.90 was established as the threshold of mean TRE ⩽ 0.3 cm for rectum/prostate. For bladder, extra metrics in addition to DSC should be considered. Significance . This work presented a 3D printed phantom, which enabled quantification of voxel mapping accuracy and evaluation of correlation between DSC and voxel mapping accuracy., (© 2024 Institute of Physics and Engineering in Medicine.)
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- 2024
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8. TDP-43 deficiency in suprachiasmatic nucleus perturbs rhythmicity of neuroactivity in prefrontal cortex.
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Zhang H, Chen C, Zhang EE, and Huang X
- Abstract
Individuals within the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD) often experience disruptive mental behaviors and sleep-wake disturbances. The hallmark of ALS/FTD is the pathological involvement of TAR DNA-binding protein 43 (TDP-43). Understanding the role of TDP-43 in the circadian clock holds promise for addressing these behavioral abnormalities. In this study, we unveil TDP-43 as a pivotal regulator of the circadian clock. TDP-43 knockdown induces intracellular arrhythmicity, disrupts transcriptional activation regulation, and diminishes clock genes expression. Moreover, our experiments in adult mouse reveal that TDP-43 knockdown, specifically within the suprachiasmatic nucleus (SCN), induces locomotor arrhythmia, arrhythmic c-Fos expression, and depression-like behavior. This observation offers valuable insights into the substantial impact of TDP-43 on the behavioral aberrations associated with ALS/FTD. In summary, our study illuminates the significance of TDP-43 in circadian regulation, shedding light on the circadian regulatory mechanisms that may elucidate the pathological underpinnings of ALS/FTD., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)
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- 2024
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9. Factors influencing willingness to participate in ophthalmic clinical trials and strategies for effective recruitment.
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Liang JE, Liang MY, Zhang EE, Peng YY, Chen LL, Deng JY, Lin T, Fu J, Zhang JN, Li SL, Li F, Xiao HM, Huang WM, and Liu YH
- Abstract
Aim: To explore the factors influencing individuals' willingness to participate in ophthalmic clinical trials., Methods: A questionnaire survey was conducted from January to April 2021 among patients and their family members at Zhongshan Ophthalmic Center, Sun Yat-sen University, in Guangzhou, China. The survey gathered data on respondents' willingness, demographic and socioeconomic profiles, as well as their reasons and concerns regarding engagement in clinical trials., Results: Of the 1078 residents surveyed (mean age 31.2±13.1y; 65.8% females) in Guangzhou, 749 (69.5%) expressed a willingness to participate in future ophthalmic clinical trials. Specific characteristics associated with greater willingness included a younger age, lower annual income, higher education, prior participation experience, previous ophthalmic treatment, and a better understanding of clinical trials. With the exception of age, these characteristics were significantly linked to a higher willingness. The primary barrier to participation, expressed by 64.8% of those willing and 54.4% of those unwilling, was "Uncertain efficacy". In terms of motivations, the willing group ranked "Better therapeutic benefits" (35.0%), "Professional monitoring" (34.3%), and "Trust in healthcare professionals" (33.1%) as their top three reasons, whereas the unwilling participants indicated "Full comprehension of the protocol" (46.2%) as the key facilitator., Conclusion: This study reveals a substantial willingness to participate in ophthalmic clinical trials and demonstrates the predictive role of demographic and socioeconomic factors. Variations in motivators and concerns between willing and unwilling participants highlight the significance of tailored recruitment strategies. Importantly, the need for and trust in healthcare professionals stand out as powerful motivations, underscoring the importance of enhancing physician-patient relationships, adopting patient-centered communication approaches, and addressing individualized needs to improve accrual rates., (International Journal of Ophthalmology Press.)
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- 2024
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10. Prolonged sleep deprivation induces a cytokine-storm-like syndrome in mammals.
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Sang D, Lin K, Yang Y, Ran G, Li B, Chen C, Li Q, Ma Y, Lu L, Cui XY, Liu Z, Lv SQ, Luo M, Liu Q, Li Y, and Zhang EE
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- Animals, Mice, Cytokines metabolism, Inflammation, Prostaglandin D2, Sleep physiology, Syndrome, Humans, Rats, Cell Line, Cyclonic Storms, Neutrophils metabolism, Sleep Deprivation genetics, Sleep Deprivation metabolism
- Abstract
Most animals require sleep, and sleep loss induces serious pathophysiological consequences, including death. Previous experimental approaches for investigating sleep impacts in mice have been unable to persistently deprive animals of both rapid eye movement sleep (REMS) and non-rapid eye movement sleep (NREMS). Here, we report a "curling prevention by water" paradigm wherein mice remain awake 96% of the time. After 4 days of exposure, mice exhibit severe inflammation, and approximately 80% die. Sleep deprivation increases levels of prostaglandin D
2 (PGD2 ) in the brain, and we found that elevated PGD2 efflux across the blood-brain-barrier-mediated by ATP-binding cassette subfamily C4 transporter-induces both accumulation of circulating neutrophils and a cytokine-storm-like syndrome. Experimental disruption of the PGD2 /DP1 axis dramatically reduced sleep-deprivation-induced inflammation. Thus, our study reveals that sleep-related changes in PGD2 in the central nervous system drive profound pathological consequences in the peripheral immune system., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)- Published
- 2023
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11. Disrupted circadian rhythms in the plateau pika.
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Yu Z and Zhang EE
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- Animals, Humans, Tibet, Lagomorpha genetics
- Abstract
The plateau pika (Ochotona curzoniae) is the most populous mammal on the 'third pole', the Qinghai-Tibet Plateau, and is presumed to have inhabited the region before the plateau rose up from sea level. Herein we discuss the disrupted circadian rhythm in the plateau pika and the gene polymorphism behind this phenotype, placing these findings in the broader context of circadian rhythms under extreme conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)
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- 2023
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12. A signalling pathway for transcriptional regulation of sleep amount in mice.
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Zhou R, Wang G, Li Q, Meng F, Liu C, Gan R, Ju D, Liao M, Xu J, Sang D, Gao X, Zhou S, Wu K, Sun Q, Guo Y, Wu C, Chen Z, Chen L, Shi B, Wang H, Wang X, Li H, Cai T, Li B, Wang F, Funato H, Yanagisawa M, Zhang EE, and Liu Q
- Subjects
- Animals, Mice, Gene Expression Regulation, Phosphorylation, Sleep, Slow-Wave genetics, Gene Expression Profiling, Signal Transduction physiology, Sleep Duration, Transcription, Genetic
- Abstract
In mice and humans, sleep quantity is governed by genetic factors and exhibits age-dependent variation
1-3 . However, the core molecular pathways and effector mechanisms that regulate sleep duration in mammals remain unclear. Here, we characterize a major signalling pathway for the transcriptional regulation of sleep in mice using adeno-associated virus-mediated somatic genetics analysis4 . Chimeric knockout of LKB1 kinase-an activator of AMPK-related protein kinase SIK35-7 -in adult mouse brain markedly reduces the amount and delta power-a measure of sleep depth-of non-rapid eye movement sleep (NREMS). Downstream of the LKB1-SIK3 pathway, gain or loss-of-function of the histone deacetylases HDAC4 and HDAC5 in adult brain neurons causes bidirectional changes of NREMS amount and delta power. Moreover, phosphorylation of HDAC4 and HDAC5 is associated with increased sleep need, and HDAC4 specifically regulates NREMS amount in posterior hypothalamus. Genetic and transcriptomic studies reveal that HDAC4 cooperates with CREB in both transcriptional and sleep regulation. These findings introduce the concept of signalling pathways targeting transcription modulators to regulate daily sleep amount and demonstrate the power of somatic genetics in mouse sleep research., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2022
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13. The pancreatic clock is a key determinant of pancreatic fibrosis progression and exocrine dysfunction.
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Jiang W, Jin L, Ju D, Lu Z, Wang C, Guo X, Zhao H, Shen S, Cheng Z, Shen J, Zong G, Chen J, Li K, Yang L, Zhang Z, Feng Y, Shen JZ, Zhang EE, and Wan R
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- ARNTL Transcription Factors, Animals, Aryl Hydrocarbon Receptor Nuclear Translocator, Fibrosis, Interleukin-11 therapeutic use, Mice, Nuclear Receptor Subfamily 1, Group D, Member 1, Pancreas, Receptors, Retinoic Acid genetics, Receptors, Retinoic Acid therapeutic use, Retinoids therapeutic use, Melatonin therapeutic use, Pancreatitis, Chronic drug therapy, Pancreatitis, Chronic pathology
- Abstract
Chronic pancreatitis (CP) is characterized by progressive fibrosis and exocrine dysregulation, which have long been considered irreversible. As a peripheral oscillator, the pancreas harbors autonomous and self-sustained timekeeping systems in both its endocrine and exocrine compartments, although the role of the latter remains poorly understood. By using different models of CP established in mice with dysfunctional pancreatic clocks, we found that the local clock played an important role in CP pathology, and genetic or external disruption of the pancreatic clock exacerbated fibrogenesis and exocrine insufficiency. Mechanistically, an impaired retinoic acid receptor-related orphan receptor A (Rora)/nuclear receptor subfamily 1, group D, member 1 (Nr1d1)/aryl hydrocarbon receptor nuclear translocator-like (Arntl or Bmal1) loop, called the circadian stabilizing loop, resulted in the deficiency of pancreatic Bmal1, which was responsible for controlling the fibrogenic properties of pancreatic stellate cells (PSCs) and for rewiring the function of acinar cells in a clock-TGF signaling-IL-11/IL-11RA axis-dependent manner. During PSC activation, the antagonistic interaction between Nr1d1 and Rora was unbalanced in response to the loss of cytoplasmic retinoid-containing lipid droplets. Patients with CP also exhibited reduced production of endogenous melatonin. Enhancing the clock through pharmacological restoration of the circadian stabilizing loop using a combination of melatonin and the Rora agonist SR1078 attenuated intrapancreatic pathological changes in mouse models of CP. Collectively, this study identified a protective role of the pancreatic clock against pancreatic fibrosis and exocrine dysfunction. Pancreatic clock-targeted therapy may represent a potential strategy to treat CP.
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- 2022
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14. A highland-adaptation mutation of the Epas1 protein increases its stability and disrupts the circadian clock in the plateau pika.
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Liu N, Tian H, Yu Z, Zhao H, Li W, Sang D, Lin K, Cui Y, Liao M, Xu Z, Chen C, Guo Y, Wang Y, Huang HW, Wang J, Zhang H, Wu W, Huang H, Lv S, Guo Z, Wang W, Zheng S, Wang F, Zhang Y, Cai T, and Zhang EE
- Subjects
- Acclimatization, Animals, Circadian Rhythm genetics, Hypoxia genetics, Hypoxia metabolism, Mice, Mutation genetics, Basic Helix-Loop-Helix Transcription Factors, Circadian Clocks genetics, Lagomorpha genetics, Lagomorpha metabolism
- Abstract
The Qinghai-Tibet Plateau (QTP) harbors hundreds of species well adapted to its extreme conditions, including its low-oxygen (hypoxic) atmosphere. Here, we show that the plateau pika-a keystone mammal of the QTP-lacks robust circadian rhythms. The major form of the plateau pika Epas1 protein includes a 24-residue insert caused by a point mutation at the 5' juncture site of Intron14 and is more stable than other mammalian orthologs. Biochemical studies reveal that an Epas1-Bmal1 complex with lower trans-activation activity occupies the E1/E2 motifs at the promoter of the core-clock gene Per2, thus explaining how an Epas1 mutation-selected in the hypoxic conditions of the QTP-disrupts the molecular clockwork. Importantly, experiments with hypoxic chambers show that mice expressing the plateau pika Epas1 ortholog in their suprachiasmatic nucleus have dysregulated central clocks, and pika Epas1 knockin mice reared in hypoxic conditions exhibit dramatically reduced heart damage compared with wild-type animals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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15. Deep-learning models for the detection and incidence prediction of chronic kidney disease and type 2 diabetes from retinal fundus images.
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Zhang K, Liu X, Xu J, Yuan J, Cai W, Chen T, Wang K, Gao Y, Nie S, Xu X, Qin X, Su Y, Xu W, Olvera A, Xue K, Li Z, Zhang M, Zeng X, Zhang CL, Li O, Zhang EE, Zhu J, Xu Y, Kermany D, Zhou K, Pan Y, Li S, Lai IF, Chi Y, Wang C, Pei M, Zang G, Zhang Q, Lau J, Lam D, Zou X, Wumaier A, Wang J, Shen Y, Hou FF, Zhang P, Xu T, Zhou Y, and Wang G
- Subjects
- Area Under Curve, Blood Glucose metabolism, Body Height, Body Mass Index, Body Weight, Diabetes Mellitus, Type 2 metabolism, Diabetes Mellitus, Type 2 pathology, Disease Progression, Female, Fundus Oculi, Glomerular Filtration Rate, Humans, Male, Metadata statistics & numerical data, Middle Aged, Neural Networks, Computer, Photography methods, Prospective Studies, ROC Curve, Renal Insufficiency, Chronic metabolism, Renal Insufficiency, Chronic pathology, Retina metabolism, Retina pathology, Deep Learning, Diabetes Mellitus, Type 2 diagnostic imaging, Image Interpretation, Computer-Assisted statistics & numerical data, Photography statistics & numerical data, Renal Insufficiency, Chronic diagnostic imaging, Retina diagnostic imaging
- Abstract
Regular screening for the early detection of common chronic diseases might benefit from the use of deep-learning approaches, particularly in resource-poor or remote settings. Here we show that deep-learning models can be used to identify chronic kidney disease and type 2 diabetes solely from fundus images or in combination with clinical metadata (age, sex, height, weight, body-mass index and blood pressure) with areas under the receiver operating characteristic curve of 0.85-0.93. The models were trained and validated with a total of 115,344 retinal fundus photographs from 57,672 patients and can also be used to predict estimated glomerulal filtration rates and blood-glucose levels, with mean absolute errors of 11.1-13.4 ml min
-1 per 1.73 m2 and 0.65-1.1 mmol l-1 , and to stratify patients according to disease-progression risk. We evaluated the generalizability of the models for the identification of chronic kidney disease and type 2 diabetes with population-based external validation cohorts and via a prospective study with fundus images captured with smartphones, and assessed the feasibility of predicting disease progression in a longitudinal cohort.- Published
- 2021
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16. EyeHealer: A large-scale anterior eye segment dataset with eye structure and lesion annotations.
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Cai W, Xu J, Wang K, Liu X, Xu W, Cai H, Gao Y, Su Y, Zhang M, Zhu J, Zhang CL, Zhang EE, Wang F, Yin Y, Lai IF, Wang G, Zhang K, and Zheng Y
- Abstract
Anterior segment eye diseases account for a significant proportion of presentations to eye clinics worldwide, including diseases associated with corneal pathologies, anterior chamber abnormalities (e.g. blood or inflammation), and lens diseases. The construction of an automatic tool for segmentation of anterior segment eye lesions would greatly improve the efficiency of clinical care. With research on artificial intelligence progressing in recent years, deep learning models have shown their superiority in image classification and segmentation. The training and evaluation of deep learning models should be based on a large amount of data annotated with expertise; however, such data are relatively scarce in the domain of medicine. Herein, the authors developed a new medical image annotation system, called EyeHealer. It is a large-scale anterior eye segment dataset with both eye structures and lesions annotated at the pixel level. Comprehensive experiments were conducted to verify its performance in disease classification and eye lesion segmentation. The results showed that semantic segmentation models outperformed medical segmentation models. This paper describes the establishment of the system for automated classification and segmentation tasks. The dataset will be made publicly available to encourage future research in this area., (© The Author(s) 2021. Published by Oxford University Press on behalf of the West China School of Medicine & West China Hospital of Sichuan University.)
- Published
- 2021
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17. Identification of PCBP1 as a Novel Modulator of Mammalian Circadian Clock.
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Wu Y, Zhao H, Zhang EE, and Liu N
- Abstract
The circadian clock governs our daily cycle of behavior and physiology. Previous studies have identified a handful of core clock components and hundreds of circadian modifiers. Here, we report the discovery that poly(C)-binding protein 1 (PCBP1), displaying a circadian expression pattern, was a novel circadian clock regulator. We found that knocking down PCBP1 resulted in period shortening in human U2OS cells, and that manipulations of PCBP1 expression altered the activity of CLOCK/BMAL1 in an E-box-based reporter assay. Further mechanistic study demonstrated that this clock function of PCBP1 appears to work by enhancing the association of Cryptochrome 1 (CRY1) with the CLOCK/BMAL1 complex, thereby negatively regulating the latter's activation. Co-immunoprecipitation of PCBP1 and core clock molecules confirmed the interactions between PCBP1 and CRY1, and a time-course qPCR assay revealed the rhythmic expression of PCBP1 in mouse hearts in vivo . Given that the RNA interference of mushroom-body expressed ( mub ), the poly(rC) binding protein (PCBP) homolog of Drosophila , in the clock neurons also led to a circadian phenotype in the locomotor assay, our study deemed PCBP1 a novel clock modifier whose circadian regulatory mechanism is conserved during evolution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wu, Zhao, Zhang and Liu.)
- Published
- 2021
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18. Chemical perturbations reveal that RUVBL2 regulates the circadian phase in mammals.
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Ju D, Zhang W, Yan J, Zhao H, Li W, Wang J, Liao M, Xu Z, Wang Z, Zhou G, Mei L, Hou N, Ying S, Cai T, Chen S, Xie X, Lai L, Tang C, Park N, Takahashi JS, Huang N, Qi X, and Zhang EE
- Subjects
- ATPases Associated with Diverse Cellular Activities, Animals, CLOCK Proteins genetics, CLOCK Proteins metabolism, Circadian Rhythm, DNA Helicases, Gene Expression Regulation, Humans, Mammals metabolism, Mice, ARNTL Transcription Factors genetics, ARNTL Transcription Factors metabolism, Circadian Clocks genetics
- Abstract
Transcriptional regulation lies at the core of the circadian clockwork, but how the clock-related transcription machinery controls the circadian phase is not understood. Here, we show both in human cells and in mice that RuvB-like ATPase 2 (RUVBL2) interacts with other known clock proteins on chromatin to regulate the circadian phase. Pharmacological perturbation of RUVBL2 with the adenosine analog compound cordycepin resulted in a rapid-onset 12-hour clock phase-shift phenotype at human cell, mouse tissue, and whole-animal live imaging levels. Using simple peripheral injection treatment, we found that cordycepin penetrated the blood-brain barrier and caused rapid entrainment of the circadian phase, facilitating reduced duration of recovery in a mouse jet-lag model. We solved a crystal structure for human RUVBL2 in complex with a physiological metabolite of cordycepin, and biochemical assays showed that cordycepin treatment caused disassembly of an interaction between RUVBL2 and the core clock component BMAL1. Moreover, we showed with spike-in ChIP-seq analysis and binding assays that cordycepin treatment caused disassembly of the circadian super-complex, which normally resides at E-box chromatin loci such as PER1 , PER2 , DBP, and NR1D1 Mathematical modeling supported that the observed type 0 phase shifts resulted from derepression of E-box clock gene transcription., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2020
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19. A microfluidic approach for experimentally modelling the intercellular coupling system of a mammalian circadian clock at single-cell level.
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Han K, Mei L, Zhong R, Pang Y, Zhang EE, and Huang Y
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- Animals, Circadian Rhythm, Mammals, Mice, Microfluidics, Suprachiasmatic Nucleus, Circadian Clocks
- Abstract
In mammals, it is believed that the intercellular coupling mechanism between neurons in the suprachiasmatic nucleus (SCN) confers robustness and distinguishes the central clock from peripheral circadian oscillators. Current in vitro culturing methods used in Petri dishes to study intercellular coupling by exogenous factors invariably cause perturbations, such as simple media changes. Here, we design a microfluidic device to quantitatively study the intercellular coupling mechanism of circadian clock at the single cell level, and demonstrate that vasoactive intestinal peptide (VIP) induced coupling in clock mutant Cry1-/- mouse adult fibroblasts engineered to express the VIP receptor, VPAC2, is sufficient to synchronize and maintain robust circadian oscillations. Our study provides a proof-of-concept platform to reconstitute the intercellular coupling system of the central clock using uncoupled, single fibroblast cells in vitro, to mimic SCN slice cultures ex vivo and mouse behavior in vivo phenotypically. Such a versatile microfluidic platform may greatly facilitate the studies of intercellular regulation networks, and provide new insights into the coupling mechanisms of the circadian clock.
- Published
- 2020
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20. Diurnal oscillations of endogenous H 2 O 2 sustained by p66 Shc regulate circadian clocks.
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Pei JF, Li XK, Li WQ, Gao Q, Zhang Y, Wang XM, Fu JQ, Cui SS, Qu JH, Zhao X, Hao DL, Ju D, Liu N, Carroll KS, Yang J, Zhang EE, Cao JM, Chen HZ, and Liu DP
- Subjects
- Animals, Female, Liver metabolism, Liver physiology, Male, Mammals metabolism, Mammals physiology, Mice, Mice, Knockout, Oxidation-Reduction, Period Circadian Proteins metabolism, Signal Transduction physiology, Suprachiasmatic Nucleus metabolism, Suprachiasmatic Nucleus physiology, Circadian Clocks physiology, Circadian Rhythm physiology, Hydrogen Peroxide metabolism, Src Homology 2 Domain-Containing, Transforming Protein 1 metabolism
- Abstract
Redox balance, an essential feature of healthy physiological steady states, is regulated by circadian clocks, but whether or how endogenous redox signalling conversely regulates clockworks in mammals remains unknown. Here, we report circadian rhythms in the levels of endogenous H
2 O2 in mammalian cells and mouse livers. Using an unbiased method to screen for H2 O2 -sensitive transcription factors, we discovered that rhythmic redox control of CLOCK directly by endogenous H2 O2 oscillations is required for proper intracellular clock function. Importantly, perturbations in the rhythm of H2 O2 levels induced by the loss of p66Shc , which oscillates rhythmically in the liver and suprachiasmatic nucleus (SCN) of mice, disturb the rhythmic redox control of CLOCK function, reprogram hepatic transcriptome oscillations, lengthen the circadian period in mice and modulate light-induced clock resetting. Our findings suggest that redox signalling rhythms are intrinsically coupled to the circadian system through reversible oxidative modification of CLOCK and constitute essential mechanistic timekeeping components in mammals.- Published
- 2019
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21. Identification of entacapone as a chemical inhibitor of FTO mediating metabolic regulation through FOXO1.
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Peng S, Xiao W, Ju D, Sun B, Hou N, Liu Q, Wang Y, Zhao H, Gao C, Zhang S, Cao R, Li P, Huang H, Ma Y, Wang Y, Lai W, Ma Z, Zhang W, Huang S, Wang H, Zhang Z, Zhao L, Cai T, Zhao YL, Wang F, Nie Y, Zhi G, Yang YG, Zhang EE, and Huang N
- Subjects
- Animals, Blood Glucose drug effects, Body Weight drug effects, Body Weight physiology, Catechol O-Methyltransferase genetics, Enzyme Inhibitors pharmacology, Forkhead Box Protein O1 genetics, Humans, Mice, RNA, Messenger genetics, Thermogenesis drug effects, Thermogenesis genetics, Thermogenesis physiology, Catechol O-Methyltransferase metabolism, Catechols pharmacology, Forkhead Box Protein O1 metabolism, Nitriles pharmacology, RNA, Messenger metabolism
- Abstract
Recent studies have established the involvement of the fat mass and obesity-associated gene ( FTO ) in metabolic disorders such as obesity and diabetes. However, the precise molecular mechanism by which FTO regulates metabolism remains unknown. Here, we used a structure-based virtual screening of U.S. Food and Drug Administration-approved drugs to identify entacapone as a potential FTO inhibitor. Using structural and biochemical studies, we showed that entacapone directly bound to FTO and inhibited FTO activity in vitro. Furthermore, entacapone administration reduced body weight and lowered fasting blood glucose concentrations in diet-induced obese mice. We identified the transcription factor forkhead box protein O1 ( FOXO1 ) mRNA as a direct substrate of FTO, and demonstrated that entacapone elicited its effects on gluconeogenesis in the liver and thermogenesis in adipose tissues in mice by acting on an FTO-FOXO1 regulatory axis., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2019
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22. The lived experiences of patients undergoing acellular porcine corneal stroma transplantation.
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Tian BS, Li SQ, Zhang EE, Xiao HM, Su LJ, Zhang JE, and Yuan J
- Abstract
To explore the lived experiences of patients undergoing acellular porcine corneal stroma (APCS) transplantation, a descriptive, qualitative design was performed. A purposive sample of 13 patients who underwent APCS transplantation to treat progressive infectious keratitis were enrolled in the semi-structured, open-ended interviews. The taped and transcribed interviews were analyzed using a thematic analysis approach. Alterations in the transparency of APCS grafts were accompanied by a gradual improved visual acuity (before surgery: 1.38± 0.91 logMAR; 3mo postoperatively: 0.40±0.24 logMAR, respectively). Accordingly, in terms of lived experiences, the patients generally reported "negative" experiences before the operation and during the early postoperative period, but this was greatly improved 3mo after surgery. Four main themes were derived: anxiety and fear, stigma, lifestyle change, and gratitude and insights. Conclusively, health care professionals should provide holistic care for patients, proactively promoting patients' physical and mental health.
- Published
- 2018
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23. Methylation-mediated miR-155-FAM133A axis contributes to the attenuated invasion and migration of IDH mutant gliomas.
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Huang GH, Du L, Li N, Zhang Y, Xiang Y, Tang JH, Xia S, Zhang EE, and Lv SQ
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- Antigens, Neoplasm genetics, Apoptosis, Biomarkers, Tumor genetics, Biomarkers, Tumor metabolism, Brain Neoplasms genetics, Brain Neoplasms metabolism, Brain Neoplasms pathology, Case-Control Studies, Cell Movement, Cell Proliferation, Cohort Studies, CpG Islands, Gene Expression Regulation, Neoplastic, Glioma genetics, Glioma metabolism, Humans, Neoplasm Invasiveness, Prognosis, Promoter Regions, Genetic, Tumor Cells, Cultured, Antigens, Neoplasm metabolism, DNA Methylation, Glioma pathology, Isocitrate Dehydrogenase genetics, MicroRNAs genetics, Mutation
- Abstract
Gliomas with isocitrate dehydrogenases gene mutations (IDH
MT ) were found to be less aggressive than their wildtype (IDHWT ) counterparts. However, the mechanism remains unclear. The current study aims to investigate the role of silenced oncogenic microRNAs in IDHMT gliomas, which were largely ignored and may contribute to the less aggressive behavior of IDHMT gliomas. Microarrays, bioinformatics analysis of the data from TCGA and qPCR analysis of samples from our experimental cohort (LGG: IDHWT = 10, IDHMT = 31; GBM: IDHWT = 34, IDHMT = 9) were performed. The results show that miR-155 was consistently down-regulated in IDHMT gliomas. Establishment of IDH1R132H overexpressing glioma cell line and bisulfite sequencing PCR suggested that miR-155 down-regulation was associated with IDH1R132H mutation induced promoter CpG islands methylation. The cancer testis antigen FAM133A is a direct downstream target of miR-155 and is a negative regulator of glioma invasion and migration possibly by regulating matrix metallopeptidase 14 (MMP14). Together, we found that methylation-regulated miR-155-FAM133A axis may contribute to the attenuated invasion and migration of IDHMT gliomas by targeting MMP14., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2018
- Full Text
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24. In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters.
- Author
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Mei L, Zhan C, and Zhang EE
- Subjects
- Animals, Mice, Circadian Clocks genetics, Circadian Rhythm genetics, Fluorescence, Genes, Reporter genetics, Suprachiasmatic Nucleus metabolism
- Abstract
This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 × 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.
- Published
- 2018
- Full Text
- View/download PDF
25. Long-term in vivo recording of circadian rhythms in brains of freely moving mice.
- Author
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Mei L, Fan Y, Lv X, Welsh DK, Zhan C, and Zhang EE
- Subjects
- Animals, Bacterial Proteins analysis, Bacterial Proteins genetics, CA1 Region, Hippocampal metabolism, CA2 Region, Hippocampal metabolism, Cells, Cultured, Circadian Rhythm genetics, Cryptochromes deficiency, Cryptochromes genetics, Dependovirus genetics, Fiber Optic Technology instrumentation, Fluorometry instrumentation, Genes, Reporter, Genetic Vectors administration & dosage, Genetic Vectors genetics, Hypothalamus, Anterior metabolism, Longitudinal Studies, Luminescent Proteins analysis, Luminescent Proteins genetics, Mice, Mice, Inbred C57BL, Movement, Neurons chemistry, Neurons classification, Optical Fibers, Organ Specificity, Period Circadian Proteins genetics, Photoperiod, Suprachiasmatic Nucleus cytology, Transcription, Genetic, Vasoactive Intestinal Peptide analysis, Circadian Rhythm physiology, Cryptochromes biosynthesis, Fiber Optic Technology methods, Fluorometry methods, Neurons metabolism, Period Circadian Proteins biosynthesis, Suprachiasmatic Nucleus metabolism
- Abstract
Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1
-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle., Competing Interests: The authors declare no conflict of interest.- Published
- 2018
- Full Text
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26. The MiR-135b-BMAL1-YY1 loop disturbs pancreatic clockwork to promote tumourigenesis and chemoresistance.
- Author
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Jiang W, Zhao S, Shen J, Guo L, Sun Y, Zhu Y, Ma Z, Zhang X, Hu Y, Xiao W, Li K, Li S, Zhou L, Huang L, Lu Z, Feng Y, Xiao J, Zhang EE, Yang L, and Wan R
- Subjects
- Animals, Carcinogenesis pathology, Cell Line, Tumor, Cell Movement, Cell Proliferation, Epithelial Cells metabolism, Epithelial Cells pathology, Feedback, Physiological, Gene Expression Regulation, Neoplastic, Humans, Male, Mice, Inbred BALB C, Mice, Nude, MicroRNAs genetics, Neoplasm Invasiveness, Pancreatic Ducts metabolism, Pancreatic Ducts pathology, Prognosis, ARNTL Transcription Factors metabolism, Biological Clocks, Carcinogenesis genetics, Drug Resistance, Neoplasm genetics, MicroRNAs metabolism, Pancreatic Neoplasms genetics, Pancreatic Neoplasms pathology, YY1 Transcription Factor metabolism
- Abstract
Circadian disruption has been implicated in tumour development, but the underlying mechanism remains unclear. Here, we show that the molecular clockwork within malignant human pancreatic epithelium is disrupted and that this disruption is mediated by miR-135b-induced BMAL1 repression. miR-135b directly targets the BMAL1 3'-UTR and thereby disturbs the pancreatic oscillator, and the downregulation of miR-135b is essential for the realignment of the cellular clock. Asynchrony between miR-135b and BMAL1 expression impairs the local circadian gating control of tumour suppression and significantly promotes tumourigenesis and resistance to gemcitabine in pancreatic cancer (PC) cells, as demonstrated by bioinformatics analyses of public PC data sets and in vitro and in vivo functional studies. Moreover, we found that YY1 transcriptionally activated miR-135b and formed a 'miR-135b-BMAL1-YY1' loop, which holds significant predictive and prognostic value for patients with PC. Thus, our work has identified a novel signalling loop that mediates pancreatic clock disruption as an important mechanism of PC progression and chemoresistance.
- Published
- 2018
- Full Text
- View/download PDF
27. Pyrrolidine dithiocarbamate sensitizes U251 brain glioma cells to temozolomide via downregulation of MGMT and BCL-XL .
- Author
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Tang JH, Huang GH, Mou KJ, Zhang EE, Li N, Du L, Zhu XP, Chen L, Yang H, Zhang KB, and Lv SQ
- Abstract
The current study investigated the effect of pyrrolidine dithiocarbamate (PDTC) on the proliferation, apoptosis, cell cycle and sensitivity to temozolomide (TMZ) of the U251 glioma cell line. Proliferation, apoptosis and cell cycle analysis of U251 cells following treatment with PDTC and TMZ was determined by an MTT assay and flow cytometry, respectively. The mRNA and protein expression levels of O-6-methylguanine-DNA methyltransferase ( MGMT ), B-cell lymphoma extra-large (BCL-XL) and survivin were further determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting analysis. The results revealed that treatment with TMZ, PDTC and TMZ + PDTC significantly inhibited cell proliferation, induced apoptosis and contributed to cell cycle arrest in U251 cells. A combination of PDTC and TMZ induced the highest rates of proliferation inhibition and apoptosis. PDTC treatment markedly reduced the expression levels of MGMT , BCL-XL and survi v in . The expression levels of MGMT and BCL-XL , were significantly upregulated by TMZ but not by combination treatment of TMZ and PDTC. The results of the present study suggest that treatment with PDTC inhibits cell proliferation, induces apoptosis and cell cycle arrest, and enhances sensitivity to TMZ in U251 cells, which is partly induced by downregulation of MGMT and BCL-XL .
- Published
- 2017
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28. Guidelines for Genome-Scale Analysis of Biological Rhythms.
- Author
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Hughes ME, Abruzzi KC, Allada R, Anafi R, Arpat AB, Asher G, Baldi P, de Bekker C, Bell-Pedersen D, Blau J, Brown S, Ceriani MF, Chen Z, Chiu JC, Cox J, Crowell AM, DeBruyne JP, Dijk DJ, DiTacchio L, Doyle FJ, Duffield GE, Dunlap JC, Eckel-Mahan K, Esser KA, FitzGerald GA, Forger DB, Francey LJ, Fu YH, Gachon F, Gatfield D, de Goede P, Golden SS, Green C, Harer J, Harmer S, Haspel J, Hastings MH, Herzel H, Herzog ED, Hoffmann C, Hong C, Hughey JJ, Hurley JM, de la Iglesia HO, Johnson C, Kay SA, Koike N, Kornacker K, Kramer A, Lamia K, Leise T, Lewis SA, Li J, Li X, Liu AC, Loros JJ, Martino TA, Menet JS, Merrow M, Millar AJ, Mockler T, Naef F, Nagoshi E, Nitabach MN, Olmedo M, Nusinow DA, Ptáček LJ, Rand D, Reddy AB, Robles MS, Roenneberg T, Rosbash M, Ruben MD, Rund SSC, Sancar A, Sassone-Corsi P, Sehgal A, Sherrill-Mix S, Skene DJ, Storch KF, Takahashi JS, Ueda HR, Wang H, Weitz C, Westermark PO, Wijnen H, Xu Y, Wu G, Yoo SH, Young M, Zhang EE, Zielinski T, and Hogenesch JB
- Subjects
- Biostatistics, Computational Biology methods, Humans, Metabolomics, Proteomics, Software, Systems Biology, Circadian Rhythm genetics, Genome, Genomics statistics & numerical data, Statistics as Topic methods
- Abstract
Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding "big data" that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.
- Published
- 2017
- Full Text
- View/download PDF
29. Reciprocal Regulation between the Circadian Clock and Hypoxia Signaling at the Genome Level in Mammals.
- Author
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Wu Y, Tang D, Liu N, Xiong W, Huang H, Li Y, Ma Z, Zhao H, Chen P, Qi X, and Zhang EE
- Subjects
- ARNTL Transcription Factors genetics, ARNTL Transcription Factors metabolism, Animals, Base Sequence, Chromatin Immunoprecipitation, Disease Models, Animal, E-Box Elements genetics, Gene Expression Regulation, HEK293 Cells, Humans, Hypoxia-Inducible Factor 1, alpha Subunit genetics, Mice, Period Circadian Proteins genetics, Period Circadian Proteins metabolism, Promoter Regions, Genetic genetics, Protein Binding genetics, Sequence Analysis, DNA, Circadian Clocks genetics, Genome, Hypoxia genetics, Mammals genetics, Signal Transduction genetics
- Abstract
Circadian regulation is critically important in maintaining metabolic and physiological homeostasis. However, little is known about the possible influence of the clock on physiological abnormalities occurring under pathological conditions. Here, we report the discovery that hypoxia, a condition that causes catastrophic bodily damage, is gated by the circadian clock in vivo. Hypoxia signals conversely regulate the clock by slowing the circadian cycle and dampening the amplitude of oscillations in a dose-dependent manner. ChIP-seq analyses of hypoxia-inducible factor HIF1A and the core clock component BMAL1 revealed crosstalk between hypoxia and the clock at the genome level. Further, severe consequences caused by acute hypoxia, such as those that occur with heart attacks, were correlated with defects in circadian rhythms. We propose that the clock plays functions in fine-tuning hypoxic responses under pathophysiological conditions. We argue that the clock can, and likely should, be exploited therapeutically to reduce the severity of fatal hypoxia-related diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
30. Orexin signaling regulates both the hippocampal clock and the circadian oscillation of Alzheimer's disease-risk genes.
- Author
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Ma Z, Jiang W, and Zhang EE
- Subjects
- ARNTL Transcription Factors chemistry, ARNTL Transcription Factors metabolism, Algorithms, Alzheimer Disease metabolism, Alzheimer Disease veterinary, Amyloid Precursor Protein Secretases antagonists & inhibitors, Amyloid Precursor Protein Secretases chemistry, Amyloid Precursor Protein Secretases metabolism, Amyloid beta-Peptides metabolism, Animals, Apolipoproteins E metabolism, Aspartic Acid Endopeptidases antagonists & inhibitors, Aspartic Acid Endopeptidases chemistry, Aspartic Acid Endopeptidases metabolism, Basic-Leucine Zipper Transcription Factors chemistry, Basic-Leucine Zipper Transcription Factors metabolism, CLOCK Proteins genetics, CLOCK Proteins metabolism, HEK293 Cells, Humans, Mice, Mice, Transgenic, Period Circadian Proteins genetics, Signal Transduction, Alzheimer Disease pathology, Circadian Clocks genetics, Circadian Rhythm genetics, Hippocampus metabolism, Orexins metabolism
- Abstract
Alzheimer's disease (AD) is a circadian clock-related disease. However, it is not very clear whether pre-symptomatic AD leads to circadian disruption or whether malfunction of circadian rhythms exerts influence on development of AD. Here, we report a functional clock that exists in the hippocampus. This oscillator both receives input signals and maintains the cycling of the hippocampal Per2 gene. One of the potential inputs to the oscillator is orexin signaling, which can shorten the hippocampal clock period and thereby regulate the expression of clock-controlled-genes (CCGs). A 24-h time course qPCR analysis followed by a JTK_CYCLE algorithm analysis indicated that a number of AD-risk genes are potential CCGs in the hippocampus. Specifically, we found that Bace1 and Bace2, which are related to the production of the amyloid-beta peptide, are CCGs. BACE1 is inhibited by E4BP4, a repressor of D-box genes, while BACE2 is activated by CLOCK:BMAL1. Finally, we observed alterations in the rhythmic expression patterns of Bace2 and ApoE in the hippocampus of aged APP/PS1dE9 mice. Our results therefore indicate that there is a circadian oscillator in the hippocampus whose oscillation could be regulated by orexins. Hence, orexin signaling regulates both the hippocampal clock and the circadian oscillation of AD-risk genes.
- Published
- 2016
- Full Text
- View/download PDF
31. Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period.
- Author
-
Liu N and Zhang EE
- Abstract
The core circadian oscillator in mammals is composed of transcription/translation feedback loop, in which cryptochrome (CRY) proteins play critical roles as repressors of their own gene expression. Although post-translational modifications, such as phosphorylation of CRY1, are crucial for circadian rhythm, little is known about how phosphorylated CRY1 contributes to the molecular clockwork. To address this, we created a series of CRY1 mutants with single amino acid substitutions at potential phosphorylation sites and performed a cell-based, phenotype-rescuing screen to identify mutants with aberrant rhythmicity in CRY-deficient cells. We report 10 mutants with an abnormal circadian period length, including long period (S280D and S588D), short period (S158D, S247D, T249D, Y266D, Y273D, and Y432D), and arrhythmicity (S71D and S404D). When expressing mutated CRY1 in HEK293 cells, we show that most of the mutants (S71D, S247D, T249D, Y266D, Y273D, and Y432D) exhibited reduction in repression activity compared with wild-type (WT) CRY1, whereas other mutants had no obvious change. Correspondingly, these mutants also showed differences in protein stability and cellular localization. We show that most of mutants are more stable than WT, except S158D, T249D, and S280D. Although the characteristics of the 10 mutants are various, they all impair the ratio balance of intracellular CRY1 protein. Thus, we conclude that the mutations caused distinct phenotypes most likely through the ratio of functional CRY1 protein in cells.
- Published
- 2016
- Full Text
- View/download PDF
32. Downregulation of HIF-1a sensitizes U251 glioma cells to the temozolomide (TMZ) treatment.
- Author
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Tang JH, Ma ZX, Huang GH, Xu QF, Xiang Y, Li N, Sidlauskas K, Zhang EE, and Lv SQ
- Subjects
- Apoptosis drug effects, Brain Neoplasms pathology, Cell Differentiation drug effects, Cell Line, Tumor, Cell Movement drug effects, Cell Proliferation drug effects, Dacarbazine pharmacology, Dacarbazine therapeutic use, Down-Regulation genetics, Gene Expression Regulation, Neoplastic drug effects, Glioma pathology, Humans, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Neoplasm Invasiveness, Neoplasm Proteins metabolism, RNA, Small Interfering metabolism, Signal Transduction drug effects, Temozolomide, Transfection, Brain Neoplasms drug therapy, Brain Neoplasms genetics, Dacarbazine analogs & derivatives, Down-Regulation drug effects, Glioma drug therapy, Glioma genetics, Hypoxia-Inducible Factor 1, alpha Subunit genetics
- Abstract
Purpose: The aim of this study was to investigate the effect of downregulation of HIF-1α gene on human U251 glioma cells and examine the consequent changes of TMZ induced effects and explore the molecular mechanisms., Methods: U251 cell line stably expressing HIF-1α shRNA was acquired via lentiviral vector transfection. The mRNA and protein expression alterations of genes involved in our study were determined respectively by qRT-PCR and Western blot. Cell proliferation was measured by MTT assay and colony formation assay, cell invasion/migration capacity was determined by transwell invasion assay/wound healing assay, and cell apoptosis was detected by flow cytometry., Results: We successfully established a U251 cell line with highly efficient HIF-1α knockdown. HIF-1a downregulation sensitized U251 cells to TMZ treatment and enhanced the proliferation-inhibiting, invasion/migration-suppressing, apoptosis-inducing and differentiation-promoting effects exerted by TMZ. The related molecular mechanisms demonstrated that expression of O(6)-methylguanine DNA methyltransferase gene (MGMT) and genes of Notch1 pathway were significantly upregulated by TMZ treatment. However, this upregulation was abrogated by HIF-1α knockdown. We further confirmed important regulatory roles of HIF-1α in the expression of MGMT and activation of Notch1 pathways., Conclusion: HIF-1α downregulation sensitizes U251 glioma cells to the temozolomide treatment via inhibiting MGMT expression and Notch1 pathway activation., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
33. The ratio of intracellular CRY proteins determines the clock period length.
- Author
-
Li Y, Xiong W, and Zhang EE
- Subjects
- Active Transport, Cell Nucleus physiology, Feedback, Physiological physiology, HEK293 Cells, Humans, Cell Nucleus physiology, Circadian Clocks physiology, Circadian Rhythm physiology, Cryptochromes metabolism, Fibroblasts physiology
- Abstract
Although a deficiency in CRY1 or CRY2 correlates with a shorter or longer circadian period, the regulation of CRY proteins in the circadian period has not been well studied. In this study, we found that both CRY1 and CRY2 were able to rescue oscillation in CRY null cells and that they displayed different periods. Furthermore, we demonstrated that protein nuclear import rates, not protein stability, regulate the period-length at the cellular level. Co-transfection of CRY1 and CRY2 in various ratios in the same cells gives rise to the predicted period length in a dose-dependent manner. Given the distinct characteristics of the C-terminal tails of the CRY1 and CRY2 proteins, our study addresses a long-standing hypothesis that the ratio of these two CRY molecules affects the clock period., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
34. Visualizing the Ensemble Structures of Protein Complexes Using Chemical Cross-Linking Coupled with Mass Spectrometry.
- Author
-
Gong Z, Ding YH, Dong X, Liu N, Zhang EE, Dong MQ, and Tang C
- Abstract
Abstract: Chemical cross-linking coupled with mass spectrometry (CXMS) identifies protein residues that are close in space, and has been increasingly used for modeling the structures of protein complexes. Here we show that a single structure is usually sufficient to account for the intermolecular cross-links identified for a stable complex with sub-µmol/L binding affinity. In contrast, we show that the distance between two cross-linked residues in the different subunits of a transient or fleeting complex may exceed the maximum length of the cross-linker used, and the cross-links cannot be fully accounted for with a unique complex structure. We further show that the seemingly incompatible cross-links identified with high confidence arise from alternative modes of protein-protein interactions. By converting the intermolecular cross-links to ambiguous distance restraints, we established a rigid-body simulated annealing refinement protocol to seek the minimum set of conformers collectively satisfying the CXMS data. Hence we demonstrate that CXMS allows the depiction of the ensemble structures of protein complexes and elucidates the interaction dynamics for transient and fleeting complexes.
- Published
- 2015
- Full Text
- View/download PDF
35. [Emergency use of extracorporeal membrane oxygenation in pediatric critically ill patients].
- Author
-
Lin R, Zhang CM, Tan LH, Shi LP, Xiong QX, Zhang EW, Shu Q, and Du LZ
- Subjects
- Cardiac Output, Low etiology, Cause of Death, Child, Child, Preschool, Critical Illness mortality, Critical Illness therapy, Female, Heart Failure etiology, Heart Failure mortality, Hemorrhage epidemiology, Humans, Infant, Infant, Newborn, Male, Postoperative Complications mortality, Postoperative Complications therapy, Respiratory Insufficiency etiology, Respiratory Insufficiency mortality, Retrospective Studies, Survival Analysis, Thrombosis epidemiology, Thrombosis etiology, Treatment Outcome, Cardiac Output, Low therapy, Extracorporeal Membrane Oxygenation adverse effects, Heart Failure therapy, Hemorrhage etiology, Respiratory Insufficiency therapy
- Abstract
Objective: The history of clinical application of extracorporeal membrane oxygenation (ECMO) has been more than 30 years. But in China, there were only a few ECMO centers with limited successful cases reported by the end of twentieth century. The high morbidities and mortalities in current pediatric ECMO practice are noted in China. Therefore, it is necessary to review the experience on rescue use of ECMO in critically ill pediatric patients., Method: A retrospective analysis was done for patients who had been receiving ECMO treatment to rescue refractory cardiorespiratory failure from different causes in a hospital between July 2007 and May 2011., Result: A total of 12 patients were treated with ECMO; 7 of them were male and 5 female, they aged 6 days to 11 years, weighed 2.8 - 35 (17.21 ± 11.64) kg. The underlying causes of cardiorespiratory failure were as follows: two cases with acute respiratory distress syndrome (ARDS) leading to respiratory failure, 4 with failure of weaning from cardiopulmonary bypass, 3 with fulminant myocarditis, 1 with right ventricular cardiomyopathy leading to repeated cardiac arrest, 1 with preoperative severe hypoxemia, and 1 with anaphylactic shock complicated with massive pulmonary hemorrhage and severe hypoxemia. Of the 12 cases, 3 were established ECMO (E-CPR) while underwent chest compression cardiopulmonary resuscitation (CPR). The mean ECMO support time was 151.75 (15 - 572) h. Seven patients (58.33%) were weaned from ECMO, 6 patients (50.00%) were successfully discharged. Six cases had bleeding from sutures, 2 cases with severe bleeding underwent thoracotomy hemostasis, 2 presented with acute renal failure. Infection was documented in 3 cases, hyperbilirubinemia in 2 cases, lower limb ischemia in 1 case, hyperglycemia in 3 cases, disseminated intravascular coagulation in 1 case, membrane lung leakage in 2 cases, systemic hemolysis in 3 cases, oxygenator failure in 2 cases and oxygenator thrombosis in one case. During the follow-up between 6 months and 4.5 years, 5 patients survived with good quality of life, without any documented central nervous system disorders. One case survived with the right lower extremity disorder from ischemic damage. His motor function has been improved following orthopedic operation at one year after discharge., Conclusion: ECMO is a justifiable alternative treatment for reversible severe cardiopulmonary failure in critically ill children.
- Published
- 2012
36. High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase.
- Author
-
Hirota T, Lee JW, Lewis WG, Zhang EE, Breton G, Liu X, Garcia M, Peters EC, Etchegaray JP, Traver D, Schultz PG, and Kay SA
- Subjects
- Adenine metabolism, Animals, Biological Clocks genetics, Casein Kinase I metabolism, Cell Line, Tumor, Circadian Rhythm genetics, Cyclin-Dependent Kinases metabolism, Gene Expression Regulation drug effects, Gene Expression Regulation genetics, Gene Knockdown Techniques, Genome-Wide Association Study, Histones metabolism, Humans, Mice, Mice, Inbred Strains, Mitogen-Activated Protein Kinase 1 metabolism, Period Circadian Proteins metabolism, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases metabolism, RNA Interference, Transcription Factors genetics, Transcription Factors physiology, Zebrafish genetics, Zebrafish physiology, Adenine analogs & derivatives, Biological Clocks drug effects, CLOCK Proteins genetics, CLOCK Proteins metabolism, Circadian Rhythm drug effects, Circadian Rhythm physiology
- Abstract
The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ∼120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKIδ, CKIα, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKIδ, CKIα, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIα in the clock mechanism and found that CKIα-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIδ. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIα as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species., Competing Interests: SAK is a SAB member of Reset Therapeutics.
- Published
- 2010
- Full Text
- View/download PDF
37. Clocks not winding down: unravelling circadian networks.
- Author
-
Zhang EE and Kay SA
- Subjects
- Animals, CLOCK Proteins genetics, Gene Expression Profiling, Humans, Models, Genetic, Biological Clocks genetics, Circadian Rhythm genetics, Energy Metabolism genetics, Gene Regulatory Networks
- Abstract
An intrinsic clock enables an organism to anticipate environmental changes and use energy sources more efficiently, thereby conferring an adaptive advantage. Having an intrinsic clock to orchestrate rhythms is also important for human health. The use of systems biology approaches has advanced our understanding of mechanistic features of circadian oscillators over the past decade. The field is now in a position to develop a multiscale view of circadian systems, from the molecular level to the intact organism, and to apply this information for the development of new therapeutic strategies or for enhancing agricultural productivity in crops.
- Published
- 2010
- Full Text
- View/download PDF
38. Emergence of noise-induced oscillations in the central circadian pacemaker.
- Author
-
Ko CH, Yamada YR, Welsh DK, Buhr ED, Liu AC, Zhang EE, Ralph MR, Kay SA, Forger DB, and Takahashi JS
- Subjects
- ARNTL Transcription Factors genetics, ARNTL Transcription Factors metabolism, Animals, Cell Communication physiology, Cyclic AMP metabolism, Mice, Mice, Knockout, Neurons metabolism, Period Circadian Proteins genetics, Period Circadian Proteins metabolism, Stochastic Processes, Suprachiasmatic Nucleus cytology, Tissue Culture Techniques, Circadian Clocks physiology, Circadian Rhythm physiology, Suprachiasmatic Nucleus physiology
- Abstract
Bmal1 is an essential transcriptional activator within the mammalian circadian clock. We report here that the suprachiasmatic nucleus (SCN) of Bmal1-null mutant mice, unexpectedly, generates stochastic oscillations with periods that overlap the circadian range. Dissociated SCN neurons expressed fluctuating levels of PER2 detected by bioluminescence imaging but could not generate circadian oscillations intrinsically. Inhibition of intercellular communication or cyclic-AMP signaling in SCN slices, which provide a positive feed-forward signal to drive the intracellular negative feedback loop, abolished the stochastic oscillations. Propagation of this feed-forward signal between SCN neurons then promotes quasi-circadian oscillations that arise as an emergent property of the SCN network. Experimental analysis and mathematical modeling argue that both intercellular coupling and molecular noise are required for the stochastic rhythms, providing a novel biological example of noise-induced oscillations. The emergence of stochastic circadian oscillations from the SCN network in the absence of cell-autonomous circadian oscillatory function highlights a previously unrecognized level of circadian organization., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2010
- Full Text
- View/download PDF
39. Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis.
- Author
-
Zhang EE, Liu Y, Dentin R, Pongsawakul PY, Liu AC, Hirota T, Nusinow DA, Sun X, Landais S, Kodama Y, Brenner DA, Montminy M, and Kay SA
- Subjects
- Animals, Cells, Cultured, Cyclic AMP Response Element-Binding Protein physiology, Male, Mice, Mice, Inbred C57BL, Receptors, G-Protein-Coupled physiology, Circadian Rhythm physiology, Cryptochromes physiology, Cyclic AMP physiology, Gluconeogenesis, Liver metabolism, Signal Transduction physiology
- Abstract
During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element-binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)--two key transcriptional regulators of this process. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein-coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with G(s)α. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.
- Published
- 2010
- Full Text
- View/download PDF
40. A genome-wide RNAi screen for modifiers of the circadian clock in human cells.
- Author
-
Zhang EE, Liu AC, Hirota T, Miraglia LJ, Welch G, Pongsawakul PY, Liu X, Atwood A, Huss JW 3rd, Janes J, Su AI, Hogenesch JB, and Kay SA
- Subjects
- Cell Line, Gene Knockdown Techniques, Humans, RNA Interference, RNA, Small Interfering metabolism, Biological Clocks, Circadian Rhythm, Genome-Wide Association Study
- Abstract
Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.
- Published
- 2009
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41. Shp2 acts downstream of SDF-1alpha/CXCR4 in guiding granule cell migration during cerebellar development.
- Author
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Hagihara K, Zhang EE, Ke YH, Liu G, Liu JJ, Rao Y, and Feng GS
- Subjects
- Animals, Brain metabolism, Cell Differentiation, Chemokine CXCL12 genetics, Mice, Mice, Transgenic, Phosphorylation, Protein Tyrosine Phosphatase, Non-Receptor Type 11 genetics, Receptors, CXCR4 genetics, Signal Transduction physiology, Cell Movement physiology, Cerebellum growth & development, Chemokine CXCL12 metabolism, Protein Tyrosine Phosphatase, Non-Receptor Type 11 metabolism, Receptors, CXCR4 metabolism
- Abstract
Shp2 is a non-receptor protein tyrosine phosphatase containing two Src homology 2 (SH2) domains that is implicated in intracellular signaling events controlling cell proliferation, differentiation and migration. To examine the role of Shp2 in brain development, we created mice with Shp2 selectively deleted in neural stem/progenitor cells. Homozygous mutant mice exhibited early postnatal lethality with defects in neural stem cell self-renewal and neuronal/glial cell fate specification. Here we report a critical role of Shp2 in guiding neuronal cell migration in the cerebellum. In homozygous mutants, we observed reduced and less foliated cerebellum, ectopic presence of external granule cells and mispositioned Purkinje cells, a phenotype very similar to that of mutant mice lacking either SDF-1alpha or CXCR4. Consistently, Shp2-deficient granule cells failed to migrate toward SDF-1alpha in an in vitro cell migration assay, and SDF-1alpha treatment triggered a robust induction of tyrosyl phosphorylation on Shp2. Together, these results suggest that although Shp2 is involved in multiple signaling events during brain development, a prominent role of the phosphatase is to mediate SDF-1alpha/CXCR4 signal in guiding cerebellar granule cell migration.
- Published
- 2009
- Full Text
- View/download PDF
42. Antitumor efficiency of the cytosine deaminase/5-fluorocytosine suicide gene therapy system on malignant gliomas: an in vivo study.
- Author
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Lv SQ, Zhang KB, Zhang EE, Gao FY, Yin CL, Huang CJ, He JQ, and Yang H
- Subjects
- Animals, Cell Line, Tumor, Chromatography, High Pressure Liquid, Cytosine Deaminase metabolism, DNA Primers genetics, Escherichia coli, Fluorescent Antibody Technique, Fluorouracil metabolism, Glioma drug therapy, Immunoblotting, Mice, Mice, Nude, Reverse Transcriptase Polymerase Chain Reaction, Tetrazolium Salts, Thiazoles, Cytosine Deaminase genetics, Flucytosine metabolism, Fluorouracil therapeutic use, Genetic Therapy methods, Glioma genetics, Glioma therapy
- Abstract
Background: Suicide gene therapy, particularly that utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, represents a novel and attractive methodology of cancer research. Mechanistically, the CD enzyme can convert the antifungal agent 5-FC into the antimetabolite agent 5-fluorouracil (5-FU), thereby killing tumor cells. The purpose of this study was to investigate the antitumor efficiency of the CD/5-FC system in malignant gliomas using a nude mouse model., Material/methods: The eukaryotic expression plasmid pCMV-CD was transfected into U251 malignant glioma cells. Resistant clones (labeled U251/CD cells) were subsequently isolated and further confirmed by reverse transcription polymerase chain reaction (RT-PCR), immunofluoroscence, and immunoblot. Then U251/CD cells were incubated with 5-FC at various concentrations to measure viability ratios as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. 5-FU concentrations in the media were measured by high-performance liquid chromatography (HPLC). Finally, the volumes and weights of tumors from glioma-bearing nude mice after 5-FC intervention were evaluated., Results: The results revealed that the untreated U251 cells were insensitive to 5-FC whereas the U251/CD cells were highly sensitive. Apoptosis and cell death were observed on the U251/CD cells after 5-FC administration. HPLC analysis showed that 5-FU was detected in the U251/CD cell media. These in vivo animal data showed that the volumes and weights of the implanted tumors were dramatically decreased due to cell apoptosis and tumor necrosis., Conclusions: The in vivo results encourage a further investigation in a controlled trial on the treatment of malignant gliomas via the CD/5-FC gene therapy system.
- Published
- 2009
43. Development of diabesity in mice with neuronal deletion of Shp2 tyrosine phosphatase.
- Author
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Krajewska M, Banares S, Zhang EE, Huang X, Scadeng M, Jhala US, Feng GS, and Krajewski S
- Subjects
- Animals, Calcium-Calmodulin-Dependent Protein Kinase Type 2 genetics, Crosses, Genetic, Diabetes Complications pathology, Diabetes Complications physiopathology, Diabetes Mellitus pathology, Diabetes Mellitus physiopathology, Eating, Female, Hyperglycemia metabolism, Hyperglycemia physiopathology, Hyperinsulinism metabolism, Hyperinsulinism physiopathology, Insulin Resistance, Leptin pharmacology, Leptin physiology, Male, Mice, Mice, Mutant Strains, Mice, Transgenic, Obesity pathology, Obesity physiopathology, Protein Tyrosine Phosphatase, Non-Receptor Type 11 genetics, Signal Transduction, Diabetes Complications metabolism, Diabetes Mellitus metabolism, Neurons metabolism, Obesity metabolism, Protein Tyrosine Phosphatase, Non-Receptor Type 11 physiology
- Abstract
Obesity and diabetes, termed "diabesity," are serious health problems that are increasing in frequency. However, the molecular mechanisms and neuronal regulation of these metabolic disorders are not fully understood. We show here that Shp2, a widely expressed Src homology 2-containing Tyr phosphatase, plays a critical role in the adult brain to control food intake, energy balance, and metabolism. Mice with a neuron-specific, conditional Shp2 deletion were generated by crossing a pan-neuronal Cre-line (CRE3) with Shp2(flox/flox) mice. These congenic mice, CRE3/Shp2-KO, developed obesity and diabetes and the associated pathophysiological complications that resemble those encountered in humans, including hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin and leptin resistance, vasculitis, diabetic nephropathy, urinary bladder infections, prostatitis, gastric paresis, and impaired spermatogenesis. This mouse model may help to elucidate the molecular mechanisms that lead to the development of diabesity in humans and provide a tool to study the in vivo complications of uncontrolled diabetes.
- Published
- 2008
- Full Text
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44. Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms.
- Author
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Liu AC, Tran HG, Zhang EE, Priest AA, Welsh DK, and Kay SA
- Subjects
- ARNTL Transcription Factors, Animals, Cryptochromes, Feedback, Fibroblasts metabolism, Flavoproteins genetics, Flavoproteins metabolism, Liver metabolism, Mice, Mice, Knockout, Mice, Transgenic, Models, Biological, Nuclear Receptor Subfamily 1, Group D, Member 1, RNA, Messenger genetics, RNA, Messenger metabolism, Signal Transduction, Tissue Distribution, Transcription, Genetic, Basic Helix-Loop-Helix Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors metabolism, Circadian Rhythm genetics, Circadian Rhythm physiology, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Receptors, Cytoplasmic and Nuclear genetics, Receptors, Cytoplasmic and Nuclear metabolism, Repressor Proteins genetics, Repressor Proteins metabolism
- Abstract
The mammalian circadian clockwork is composed of a core PER/CRY feedback loop and additional interlocking loops. In particular, the ROR/REV/Bmal1 loop, consisting of ROR activators and REV-ERB repressors that regulate Bmal1 expression, is thought to "stabilize" core clock function. However, due to functional redundancy and pleiotropic effects of gene deletions, the role of the ROR/REV/Bmal1 loop has not been accurately defined. In this study, we examined cell-autonomous circadian oscillations using combined gene knockout and RNA interference and demonstrated that REV-ERBalpha and beta are functionally redundant and are required for rhythmic Bmal1 expression. In contrast, the RORs contribute to Bmal1 amplitude but are dispensable for Bmal1 rhythm. We provide direct in vivo genetic evidence that the REV-ERBs also participate in combinatorial regulation of Cry1 and Rorc expression, leading to their phase-delay relative to Rev-erbalpha. Thus, the REV-ERBs play a more prominent role than the RORs in the basic clock mechanism. The cellular genetic approach permitted testing of the robustness of the intracellular core clock function. We showed that cells deficient in both REV-ERBalpha and beta function, or those expressing constitutive BMAL1, were still able to generate and maintain normal Per2 rhythmicity. Our findings thus underscore the resilience of the intracellular clock mechanism and provide important insights into the transcriptional topologies underlying the circadian clock. Since REV-ERB function and Bmal1 mRNA/protein cycling are not necessary for basic clock function, we propose that the major role of the ROR/REV/Bmal1 loop and its constituents is to control rhythmic transcription of clock output genes., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2008
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45. Bud specific N-sulfation of heparan sulfate regulates Shp2-dependent FGF signaling during lacrimal gland induction.
- Author
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Pan Y, Carbe C, Powers A, Zhang EE, Esko JD, Grobe K, Feng GS, and Zhang X
- Subjects
- Animals, Epithelium embryology, Epithelium enzymology, Extracellular Signal-Regulated MAP Kinases metabolism, Fibroblast Growth Factor 10 genetics, Fibroblast Growth Factor 10 metabolism, Gene Expression Regulation, Developmental, Genes, Reporter, Green Fluorescent Proteins metabolism, Integrases metabolism, Lacrimal Apparatus cytology, Lacrimal Apparatus enzymology, Mice, Mutation genetics, Organ Specificity, Protein Tyrosine Phosphatase, Non-Receptor Type 11 genetics, Receptor, Fibroblast Growth Factor, Type 2 genetics, Receptor, Fibroblast Growth Factor, Type 2 metabolism, Sulfotransferases metabolism, Embryonic Induction, Fibroblast Growth Factors metabolism, Heparitin Sulfate metabolism, Lacrimal Apparatus embryology, Protein Tyrosine Phosphatase, Non-Receptor Type 11 metabolism, Signal Transduction, Sulfur metabolism
- Abstract
Preferential outgrowth of the bud cells forms the basis of branching morphogenesis. Here, we show that lacrimal gland development requires specific modification of heparan sulfates by Ndst genes at the tip of the lacrimal gland bud. Systemic and conditional knockout experiments demonstrate the tissue specific requirement of Ndst1 and Ndst2 in the lacrimal gland epithelial, but not mesenchymal, cells, and the functional importance of Ndst1 in Fgf10-Fgfr2b, but not of Fgf1-Fgfr2b, complex formation. Consistent with this, Fgf10-induced ectopic lacrimal gland budding in explant cultures is dependent upon Ndst gene dose, and epithelial deletion of Fgfr2 abolishes lacrimal gland budding, its specific modification of heparan sulfate and its phosphorylation of Shp2 (Ptpn11 - Mouse Genome Informatics). Finally, we show that genetic ablation of Ndst1, Fgfr2 or Shp2 disrupts ERK signaling in lacrimal gland budding. Given the evolutionarily conserved roles of these genes, the localized activation of the Ndst-Fgfr-Shp2 genetic cascade is probably a general regulatory mechanism of FGF signaling in branching morphogenesis.
- Published
- 2008
- Full Text
- View/download PDF
46. Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality.
- Author
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Ke Y, Zhang EE, Hagihara K, Wu D, Pang Y, Klein R, Curran T, Ranscht B, and Feng GS
- Subjects
- Animals, Astrocytes cytology, Astrocytes physiology, Female, In Situ Hybridization, Male, Mice, Mice, Transgenic, Neurons cytology, Nuclear Proteins genetics, Polycomb Repressive Complex 1, Protein Tyrosine Phosphatase, Non-Receptor Type 11 genetics, Proto-Oncogene Proteins genetics, Repressor Proteins genetics, Stem Cells cytology, Brain cytology, Brain embryology, Brain enzymology, Brain growth & development, Cell Differentiation, Cell Proliferation, Neurons physiology, Nuclear Proteins metabolism, Protein Tyrosine Phosphatase, Non-Receptor Type 11 metabolism, Proto-Oncogene Proteins metabolism, Repressor Proteins metabolism, Signal Transduction physiology, Stem Cells physiology
- Abstract
The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.
- Published
- 2007
- Full Text
- View/download PDF
47. Intercellular coupling confers robustness against mutations in the SCN circadian clock network.
- Author
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Liu AC, Welsh DK, Ko CH, Tran HG, Zhang EE, Priest AA, Buhr ED, Singer O, Meeker K, Verma IM, Doyle FJ 3rd, Takahashi JS, and Kay SA
- Subjects
- Animals, Cell Cycle Proteins genetics, Cells, Cultured, Cryptochromes, Fibroblasts, Flavoproteins genetics, Mice, Motor Activity, Mutation, Neurons metabolism, Nuclear Proteins genetics, Period Circadian Proteins, Suprachiasmatic Nucleus cytology, Transcription Factors genetics, Biological Clocks physiology, Cell Cycle Proteins physiology, Circadian Rhythm physiology, Flavoproteins physiology, Nuclear Proteins physiology, Suprachiasmatic Nucleus physiology, Transcription Factors physiology
- Abstract
Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.
- Published
- 2007
- Full Text
- View/download PDF
48. Conditional deletion of Shp2 in the mammary gland leads to impaired lobulo-alveolar outgrowth and attenuated Stat5 activation.
- Author
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Ke Y, Lesperance J, Zhang EE, Bard-Chapeau EA, Oshima RG, Muller WJ, and Feng GS
- Subjects
- Animals, Apoptosis, Down-Regulation, Epithelial Cells metabolism, Epithelial Cells pathology, Female, Gene Expression Regulation, Genotype, Immunoenzyme Techniques, Intracellular Signaling Peptides and Proteins genetics, Lactation, Mammary Glands, Animal cytology, Mammary Glands, Animal metabolism, Mice, Mice, Knockout, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Polymerase Chain Reaction, Pregnancy, Prolactin metabolism, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Protein Tyrosine Phosphatases genetics, STAT3 Transcription Factor genetics, STAT3 Transcription Factor metabolism, STAT5 Transcription Factor genetics, Sequence Deletion, Transgenes, Intracellular Signaling Peptides and Proteins physiology, Mammary Glands, Animal growth & development, Protein Tyrosine Phosphatases physiology, STAT5 Transcription Factor metabolism
- Abstract
Stat5 and Stat3, two members of the Stat (signal transducer and activator of transcription) family, are known to play critical roles in mammopoiesis/lactogenesis and involution, respectively, in the mammary gland. Phosphotyrosine phosphatase Shp2 has been shown to dephosphorylate and thus inactivate both Stat5 and Stat3 in vitro. Paradoxically, cell culture studies also suggest a positive role of Shp2 in promoting prolactin-stimulated Stat5 activation. We have shown here that selective deletion of Shp2 in mouse mammary glands suppresses Stat5 activity during pregnancy and lactation, resulting in significant impairment of lobulo-alveolar outgrowth and lactation. In contrast, Stat3 activity was slightly up-regulated shortly before/at involution, leading to normal epithelial cell apoptosis/involution in Shp2-deficient mammary gland. Thus, Shp2 acts to promote Stat5 activation by the JAK2.prolactin receptor complex, while negatively modulating Stat3 activity before the onset of involution. This is the first demonstration that Shp2 manipulates Stat5 and Stat3 activities reciprocally in mammary epithelial cells, providing novel insight into the complex mechanisms for regulation of various Stat family members by a cytoplasmic tyrosine phosphatase.
- Published
- 2006
- Full Text
- View/download PDF
49. Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals.
- Author
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Nguyen TV, Ke Y, Zhang EE, and Feng GS
- Subjects
- Animals, Cell Proliferation, Enzyme Activation, Extracellular Signal-Regulated MAP Kinases metabolism, Gene Deletion, Intracellular Signaling Peptides and Proteins genetics, Lymphocyte Count, Mice, Mice, Knockout, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Protein Tyrosine Phosphatases genetics, Receptors, Antigen, T-Cell genetics, Signal Transduction, Spleen cytology, Spleen immunology, T-Lymphocytes enzymology, Thymus Gland cytology, Thymus Gland enzymology, Cell Differentiation genetics, Intracellular Signaling Peptides and Proteins physiology, Protein Tyrosine Phosphatases physiology, Receptors, Antigen, T-Cell metabolism, T-Lymphocytes immunology, Thymus Gland immunology
- Abstract
It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.
- Published
- 2006
- Full Text
- View/download PDF
50. Concerted functions of Gab1 and Shp2 in liver regeneration and hepatoprotection.
- Author
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Bard-Chapeau EA, Yuan J, Droin N, Long S, Zhang EE, Nguyen TV, and Feng GS
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Base Sequence, Cell Proliferation, Cytokines genetics, DNA genetics, Down-Regulation, Genes, Immediate-Early, Growth Substances genetics, Hepatectomy, Hepatocytes cytology, Hepatocytes metabolism, Interleukin-6 metabolism, Intracellular Signaling Peptides and Proteins deficiency, Intracellular Signaling Peptides and Proteins genetics, Liver injuries, Liver metabolism, Liver Regeneration genetics, MAP Kinase Signaling System, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Mutant Strains, Phosphoproteins deficiency, Phosphoproteins genetics, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Protein Tyrosine Phosphatases deficiency, Protein Tyrosine Phosphatases genetics, Proto-Oncogene Proteins c-akt metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, STAT3 Transcription Factor metabolism, Intracellular Signaling Peptides and Proteins physiology, Liver Regeneration physiology, Phosphoproteins physiology, Protein Tyrosine Phosphatases physiology
- Abstract
Liver regeneration is a rapid and concerted response to injury, in which growth factor-generated intracellular signals result in activation of transcription factors, DNA synthesis, and hepatocyte proliferation. However, the link between cytoplasmic signals resulting in proliferative response to liver injury remains to be elucidated. We show here that association of Gab1 adaptor protein and Shp2 tyrosine phosphatase is a critical event at the early phase of liver regeneration. Partial hepatectomy (PH) rapidly and transiently induced assembly of a complex comprising Shp2 and tyrosine-phosphorylated Gab1 in wild-type hepatocytes. Consistently, liver-specific Shp2 knockout (LSKO) and liver-specific Gab1 knockout (LGKO) mice displayed very similar phenotypes of defective liver regeneration triggered by PH, including blunted extracellular signal-regulated kinase 1/2 (Erk1/2) activation, decreased expression of immediate-early genes, and reduced levels of cyclins A, E, and B1, as well as suppression of hepatocyte proliferation. In contrast, the Akt and interleukin-6/Stat3 pathways were up-regulated posthepatectomy in LSKO and LGKO mice, accompanied by improved hepatoprotection. Collectively, this study establishes the physiological significance of the Gab1/Shp2 link in promoting mitogenic signaling through the Erk pathway in mammalian liver regeneration.
- Published
- 2006
- Full Text
- View/download PDF
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