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1.
Sci Rep ; 13(1): 14907, 2023 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-37689790

RESUMO

All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA's anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA's anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice-a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.


Assuntos
Linfócitos T CD8-Positivos , Neoplasias Pulmonares , Animais , Camundongos , Interleucina-13 , Interleucina-5 , Microambiente Tumoral , Receptores do Ácido Retinoico , Neoplasias Pulmonares/tratamento farmacológico , Tretinoína , Carcinogênese
2.
Anal Methods ; 13(46): 5564-5572, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34780584

RESUMO

Level 3 details play essential roles in practical latent fingerprint (LFP) identification. To reliably extract reproducible and identifiable level 3 features, high-resolution images of fingerprints with adequate quality are required. Conventional methods for acquiring level 3 details often involve specific pretreatment, intricate peripheral, leading to time-consuming analysis. Herein, we simply used water to develop the sebaceous LFPs deposited on nitrocellulose (NC) membranes with only one step, and then the high-resolution (2048 pixels per inch) optical micrographs were captured to reflect the live fingertip with high fidelity. From the pictures, level 3 features, including all dimensional attributes of the ridges and pores such as number, size, location, shape, and edge contour can be extracted accurately and reproducibly. Among them, qualitative features (the structures of ridge edges) and several quantitative characteristics (the number and the relative location of sweat pores) exhibit good reproducibility. Remarkably, we proposed a new parameter termed "frequency distribution of the distance between adjacent sweat pores", short form "FDDasp", which was further proved highly identifiable in different individuals, enabling the successful distinguishment between two fragmentary fingerprints with similar level 2 structures. We believe that this methodology provides a fast and quantitative analytical paradigm for latent fingerprint identification at level 3 details.


Assuntos
Dermatoglifia , Água , Humanos , Reprodutibilidade dos Testes
3.
Anal Chem ; 93(36): 12417-12425, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34464090

RESUMO

The cellular heterogeneity and plasticity are often overlooked due to the averaged bulk assay in conventional methods. Optical imaging-based single-cell analysis usually requires specific labeling of target molecules inside or on the surface of the cell membrane, interfering with the physiological homeostasis of the cell. Scanning electrochemical microscopy (SECM), as an alternative approach, enables label-free imaging of single cells, which still confronts the challenge that the long-time scanning process is not feasible for large-scale analysis at the single-cell level. Herein, we developed a methodology combining a programmable SECM (P-SECM) with an addressable microwell array, which dramatically shortened the time consumption for the topography detection of the micropits array occupied by the polystyrene beads as well as the evaluation of alkaline phosphatase (ALP) activity of the 82 single cells compared with the traditional SECM imaging. This new arithmetic was based on the line scanning approach, enabling analysis of over 900 microwells within 1.2 h, which is 10 times faster than conventional SECM imaging. By implementing this configuration with the dual-mediator-based voltage-switching (VSM) mode, we investigated the activity of ALP, a promising marker for cancer stem cells, in hundreds of tumor and stromal cells on a single microwell device. The results discovered that not only a higher ALP activity is presented in cancer cells but also the heterogeneous distribution of kinetic constant (kf value) of ALP activity can be obtained at the single-cell level. By directly relating large numbers of addressed cells on the scalable microfluidic device to the deterministic routing of the above SECM tip, our platform holds potential as a high-content screening tool for label-free single-cell analysis.


Assuntos
Dispositivos Lab-On-A-Chip , Análise de Célula Única , Microscopia Eletroquímica de Varredura , Imagem Óptica
4.
Elife ; 102021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34085929

RESUMO

Sleep is a conserved and essential behavior, but its mechanistic and functional underpinnings remain poorly defined. Through unbiased genetic screening in Drosophila, we discovered a novel short-sleep mutant we named argus. Positional cloning and subsequent complementation, CRISPR/Cas9 knock-out, and RNAi studies identified Argus as a transmembrane protein that acts in adult peptidergic neurons to regulate sleep. argus mutants accumulate undigested Atg8a(+) autophagosomes, and genetic manipulations impeding autophagosome formation suppress argus sleep phenotypes, indicating that autophagosome accumulation drives argus short-sleep. Conversely, a blue cheese neurodegenerative mutant that impairs autophagosome formation was identified independently as a gain-of-sleep mutant, and targeted RNAi screens identified additional genes involved in autophagosome formation whose knockdown increases sleep. Finally, autophagosomes normally accumulate during the daytime and nighttime sleep deprivation extends this accumulation into the following morning, while daytime gaboxadol feeding promotes sleep and reduces autophagosome accumulation at nightfall. In sum, our results paradoxically demonstrate that wakefulness increases and sleep decreases autophagosome levels under unperturbed conditions, yet strong and sustained upregulation of autophagosomes decreases sleep, whereas strong and sustained downregulation of autophagosomes increases sleep. The complex relationship between sleep and autophagy suggested by our findings may have implications for pathological states including chronic sleep disorders and neurodegeneration, as well as for integration of sleep need with other homeostats, such as under conditions of starvation.


Assuntos
Autofagossomos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mutação com Ganho de Função , Macroautofagia/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurônios , Sono/genética , Animais , Animais Geneticamente Modificados , Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Genótipo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fenótipo , Fatores de Tempo , Vigília
5.
Insects ; 12(2)2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33671950

RESUMO

Drosophila suzukii (Diptera: Drosophilidae) infests a variety of commercial fruits, including cherries and other soft-skinned fruits. After the cropping season of most cultivated crop hosts, it heavily infests the fruit of a wild host-plant, Osyris wightiana in southwest China. Here, we employ gas chromatography-electroantennographic detection (GC-EAD) together with behavioral bioassays and a trapping experiment to identify volatile semiochemicals emitted by O. wightiana that are involved in D. suzukii attraction. GC-EAD recordings of D. suzukii antenna showed responses to 13 compounds, including α-pinene, 3-methylbutyl acetate, 2-hexanol, E-ß-ocimene, Z-3-hexenol, ß-caryophyllene, α-humulene, and six unidentified compounds. The flies were attracted by seven individual EAD-active compounds at low doses (0.01 and 0.1 µg), but were repelled at high doses (10 and 100 µg). In a similar manner, a blend of seven EAD-active compounds at low doses (0.1 and 1 µg) was attractive to female flies, but became repulsive at high doses (10 µg). The low dose of the blend was as attractive as the fruit volatiles, although both were less attractive than the fruits. The blend attracted both female and male D. suzukii and other Drosophila flies. The percentage of D. suzukii out of all flies captured by the blend was significantly greater than that captured by the control. These results indicate that the EAD-active volatile compounds emitted by fruits of O. wightiana play an important role in D. suzukii attraction, and have the potential to be used for management of D. suzukii.

6.
Mol Cancer Res ; 19(4): 667-677, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33380466

RESUMO

Ubiquitin specific peptidase 18 (USP18), previously known as UBP43, is the IFN-stimulated gene 15 (ISG15) deconjugase. USP18 removes ISG15 from substrate proteins. This study reports that USP18-null mice (vs. wild-type mice) exhibited lower lipolysis rates, altered fat to body weight ratios, and cold sensitivity. USP18 is a regulator of lipid and fatty acid metabolism. Prior work established that USP18 promotes lung tumorigenesis. We sought to learn whether this occurs through altered lipid and fatty acid metabolism. Loss of USP18 repressed adipose triglyceride lipase (ATGL) expression; gain of USP18 expression upregulated ATGL in lung cancer cells. The E1-like ubiquitin activating enzyme promoted ISG15 conjugation of ATGL and destabilization. Immunoprecipitation assays confirmed that ISG15 covalently conjugates to ATGL. Protein expression of thermogenic regulators was examined in brown fat of USP18-null versus wild-type mice. Uncoupling protein 1 (UCP1) was repressed in USP18-null fat. Gain of USP18 expression augmented UCP1 protein via reduced ubiquitination. Gain of UCP1 expression in lung cancer cell lines enhanced cellular proliferation. UCP1 knockdown inhibited proliferation. Beta-hydroxybutyrate colorimetric assays performed after gain of UCP1 expression revealed increased cellular fatty acid beta-oxidation, augmenting fatty acid beta-oxidation in Seahorse assays. Combined USP18, ATGL, and UCP1 profiles were interrogated in The Cancer Genome Atlas. Intriguingly, lung cancers with increased USP18, ATGL, and UCP1 expression had an unfavorable survival. These findings reveal that USP18 is a pharmacologic target that controls fatty acid metabolism. IMPLICATIONS: USP18 is an antineoplastic target that affects lung cancer fatty acid metabolism.


Assuntos
Ácidos Graxos/metabolismo , Neoplasias Pulmonares/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Feminino , Humanos , Lipólise , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Knockout , Oxirredução , Smegmamorpha , Ubiquitinação
7.
Analyst ; 146(1): 33-47, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33140753

RESUMO

Fingermarks have long been recognized as one of the most reliable and valuable evidence for personal identification. In practice, fingerprint analysis primarily concentrates on latent fingerprint visualization. However, fingerprint visualization techniques do not always enable individualization when fingermarks collected in crime scenes are fragmentary, ambiguous, or deformed. Age determination techniques based on physical and chemical composition changes in fingerprints over time have attracted researchers' attention in recent years. Nevertheless, the components of fingerprints are liable to factors including donor features, deposition conditions, substrate properties, environmental conditions and revealing methods. All the influences mainly contribute to unreliable outcomes of age estimation. Recent developments in fingermark age determination have moved forward to more precise approaches. The advanced methods can be classified into two categories including techniques based on the modifications of physical characteristics and chemical composition characteristics. Herein, the review includes the five types of variables that influence the aging process. The methodologies are subsequently highlighted along with their advantages and disadvantages. Furthermore, photography, optical, microscopy and electrochemical methods, and vibrational spectroscopy and mass spectrometry (MS) techniques are summarized in detail, with an emphasis on their utilization.


Assuntos
Dermatoglifia , Técnicas Eletroquímicas , Espectrometria de Massas
8.
Sci Rep ; 9(1): 16838, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727959

RESUMO

Hemophilia A is a monogenic disease with a blood clotting factor VIII (FVIII) deficiency caused by mutation in the factor VIII (F8) gene. Current and emerging treatments such as FVIII protein injection and gene therapies via AAV-delivered F8 transgene in an episome are costly and nonpermanent. Here, we describe a CRISPR/Cas9-based in vivo genome editing method, combined with non-homologous end joining, enabling permanent chromosomal integration of a modified human B domain deleted-F8 (BDD-F8) at the albumin (Alb) locus in liver cells. To test the approach in mice, C57BL/6 mice received tail vein injections of two vectors, AAV8-SaCas9-gRNA, targeting Alb intron 13, and AAV8-BDD-F8. This resulted in BDD-F8 insertion at the Alb locus and FVIII protein expression in the liver of vector-, but not vehicle-, treated mice. Using this approach in hemophilic mice, BDD-F8 was expressed in liver cells as functional human FVIII, leading to increased plasma levels of FVIII and restoration of blood clotting properties in a dose-dependent manor for at least 7 months, with no detectable liver toxicity or meaningful off-target effects. Based on these findings, our BDD-F8 genome editing approach may offer an efficacious, long-term and safe treatment for patients with hemophilia A.


Assuntos
Dependovirus/genética , Fator VIII/genética , Edição de Genes/métodos , Hemofilia A/terapia , Albuminas/genética , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Fator VIII/química , Terapia Genética , Vetores Genéticos/administração & dosagem , Hemofilia A/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Resultado do Tratamento
9.
Anal Chem ; 91(7): 4307-4311, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30869520

RESUMO

The 3D cell spheroid is an emerging tool that allows better recapitulating of in vivo scenarios with multiple factors such as tissue-like morphology and membrane protein expression that intimately coordinates with enzyme activity, thus providing a psychological environment for tumorigenesis study. For analyzing different spheroids, conventional optical imaging may be hampered by the need for fluorescent labeling, which could cause toxicity side effects. As an alternative approach, scanning electrochemical microscopy (SECM) enables label-free imaging. However, SECM for cell spheroid imaging is currently suffering from incapability of systematically analyzing the cell aggregates from spheroid generation, electrochemical signal gaining, and the gene expression on different individual cell spheroids. Herein, we developed a top-removable microfluidic device for cell aggregate yielding and SECM imaging methodology to analyze heterotypic 3D cell spheroids on a single device. This technique allows not only on-chip culturing of cell aggregates but also SECM imaging of the spheroids after opening the chip and subsequent qPCR assay of corresponding clusters. Through employment of the micropit arrays (85 × 4) with a top withdrawable microfluidic layer, uniformly sized breast tumor cell and fibroblast spheroids can be simultaneously produced on a single device. By leveraging voltage-switching mode SECM at different potentials of dual mediators, we evaluated alkaline phosphatase without disturbance of substrate morphology for distinguishing the tumor aggregates from stroma. Moreover, this method also enables gene expression profiling on individual tumor or stromal spheroids. Therefore, this new strategy can seamlessly bridge SECM measurements and molecular biological analysis.


Assuntos
Fosfatase Alcalina/análise , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Microscopia Eletroquímica de Varredura/métodos , Esferoides Celulares/química , Fosfatase Alcalina/genética , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Fibroblastos/química , Perfilação da Expressão Gênica , Humanos , Células MCF-7 , Técnicas Analíticas Microfluídicas/instrumentação , Estudo de Prova de Conceito , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real
10.
Artigo em Inglês | MEDLINE | ID: mdl-30936393

RESUMO

Sleep is important for cognitive ability, and perturbations of sleep are associated with a myriad of brain disorders. However, how sleep promotes health and function during wake is poorly understood. To address the cellular and molecular mechanisms underlying sleep, we use the fruit fly Drosophila melanogaster as a genetic model. Forward genetic approaches in flies were critical for deciphering molecular mechanisms of the circadian clock. Using similar approaches, we and others are gaining insights into the pathways that control sleep amount.

11.
Sensors (Basel) ; 17(7)2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28696363

RESUMO

We demonstrate a facile method termed candle soot coating (CSC) for fast developing latent fingermarks (LFMs) on various kinds of surfaces (glass, ceramic, metal, paper and adhesive tape). The CSC method can be considered as simple, fast, and low-cost as well as providing high contrast for LFM visualization in potential forensic applications.

12.
J Environ Radioact ; 154: 60-7, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26854555

RESUMO

In order to study the bioreduction of U(VI) and stability of immobilized uranium under suboxic conditions, microcosm were amended with ethanol, lactate and glucose, and incubated under suboxic conditions. During the incubation, total dissolved U in amended microcosms decreased from 0.95 mg/L to 0.03 mg/L. Pyrosequencing results showed that, the proportion of anaerobic microorganisms capable of reducing U(VI) under suboxic conditions was small compared with that under anoxic conditions; the proportion of aerobic and facultative anaerobic microorganisms capable of consuming the dissolved oxygen was large; and some of the facultative anaerobic microorganisms could reduce U(VI). These results indicated that different microbial communities were responsible for the bioreduction of U(VI) under suboxic and anoxic conditions. After the electron donors were exhausted, total dissolved U in the amended microcosms remained unchanged, while the U(VI)/U(IV) ratio in the solid phase of sediments increased obviously. This implied that the performance of bioreduction of the U(VI) can be maintained under suboxic condition.


Assuntos
Bactérias/metabolismo , Urânio/metabolismo , Poluentes Radioativos da Água/metabolismo , Anaerobiose , Bactérias/genética , Biodegradação Ambiental , DNA Bacteriano/genética , Sedimentos Geológicos/análise , Oxirredução , RNA Ribossômico 16S/genética
13.
PLoS Genet ; 11(11): e1005655, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26544867

RESUMO

Individuals with Cornelia de Lange Syndrome (CdLS) display diverse developmental deficits, including slow growth, multiple limb and organ abnormalities, and intellectual disabilities. Severely-affected individuals most often have dominant loss-of-function mutations in the Nipped-B-Like (NIPBL) gene, and milder cases often have missense or in-frame deletion mutations in genes encoding subunits of the cohesin complex. Cohesin mediates sister chromatid cohesion to facilitate accurate chromosome segregation, and NIPBL is required for cohesin to bind to chromosomes. Individuals with CdLS, however, do not display overt cohesion or segregation defects. Rather, studies in human cells and model organisms indicate that modest decreases in NIPBL and cohesin activity alter the transcription of many genes that regulate growth and development. Sister chromatid cohesion factors, including the Nipped-B ortholog of NIPBL, are also critical for gene expression and development in Drosophila melanogaster. Here we describe how a modest reduction in Nipped-B activity alters growth and neurological function in Drosophila. These studies reveal that Nipped-B heterozygous mutant Drosophila show reduced growth, learning, and memory, and altered circadian rhythms. Importantly, the growth deficits are not caused by changes in systemic growth controls, but reductions in cell number and size attributable in part to reduced expression of myc (diminutive) and other growth control genes. The learning, memory and circadian deficits are accompanied by morphological abnormalities in brain structure. These studies confirm that Drosophila Nipped-B mutants provide a useful model for understanding CdLS, and provide new insights into the origins of birth defects.


Assuntos
Proteínas de Ligação a DNA/genética , Síndrome de Cornélia de Lange/genética , Proteínas de Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/fisiologia , Modelos Biológicos , Mutação , Animais , Drosophila/genética , Heterozigoto
14.
Bioprocess Biosyst Eng ; 38(4): 661-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25341364

RESUMO

To determine whether the U(VI) in groundwater under anoxic conditions at a decommissioned in situ leaching (ISL) uranium mine could be bioreduced, groundwater samples containing suspended sediments were taken from the mine, experimental setup was fabricated, and the jar containing the groundwater in the setup was amended with ethanol and incubated under anoxic conditions. The variations of pH, chemical oxygen demand, nitrate, sulfate, U(VI), and dissolved oxygen (DO) concentrations were monitored during the incubation. U(VI) concentration dropped to 0.043 mg/L when the stimulated microorganisms were active, and it then increased to 0.835 mg/L within 10 days after the metabolism of the stimulated microorganisms was inhibited. The DO variation was observed in the amended jar during the incubation, and the metabolism of the stimulated microorganisms was found to affect the DO concentration. Firmicutes were found to be dominant in the sediments in the amended jar through the 16S rRNA pyrosequencing. The results indicate that it is possible to bioreduce U(VI) in the groundwater under anoxic conditions at the decommissioned ISL uranium mine by adding carbon source into it without removing the oxygen from it.


Assuntos
Água Subterrânea/química , Mineração , Oxigênio/química , Urânio/química , Anaerobiose , Biodegradação Ambiental , Análise da Demanda Biológica de Oxigênio , Carbono/química , Firmicutes , Sedimentos Geológicos , Concentração de Íons de Hidrogênio , Nitratos/química , RNA Ribossômico 16S/química , Análise de Sequência de RNA , Sulfatos/química
15.
Elife ; 3: e01473, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24497543

RESUMO

In this study, we report a new protein involved in the homeostatic regulation of sleep in Drosophila. We conducted a forward genetic screen of chemically mutagenized flies to identify short-sleeping mutants and found one, redeye (rye) that shows a severe reduction of sleep length. Cloning of rye reveals that it encodes a nicotinic acetylcholine receptor α subunit required for Drosophila sleep. Levels of RYE oscillate in light-dark cycles and peak at times of daily sleep. Cycling of RYE is independent of a functional circadian clock, but rather depends upon the sleep homeostat, as protein levels are up-regulated in short-sleeping mutants and also in wild type animals following sleep deprivation. We propose that the homeostatic drive to sleep increases levels of RYE, which responds to this drive by promoting sleep. DOI: http://dx.doi.org/10.7554/eLife.01473.001.


Assuntos
Proteínas de Drosophila/metabolismo , Receptores Nicotínicos/metabolismo , Sono , Animais , Ritmo Circadiano , Proteínas de Drosophila/genética , Drosophila melanogaster , Regulação da Expressão Gênica , Genótipo , Homeostase , Mutação , Fenótipo , Fotoperíodo , Receptores Nicotínicos/genética , Transdução de Sinais , Sono/genética , Fatores de Tempo
16.
Acta Biochim Biophys Sin (Shanghai) ; 45(1): 61-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23257295

RESUMO

An endogenous circadian (∼24 h) clock regulates rhythmic processes of physiology, metabolism and behavior in most living organisms. While able to free-run under constant conditions, the circadian clock is coupled to day : night cycles to increase its amplitude and align the phase of circadian rhythms to the right time of the day. Disruptions of the circadian clock are correlated with brain dysfunctions, cardiovascular diseases and metabolic disorders. In this review, we focus on the interactions between the circadian clock and metabolism. We discuss recent findings on circadian clock regulation of feeding behavior and rhythmic expression of metabolic genes, and present evidence of metabolic input to the circadian clock. We emphasize how misalignment of circadian clocks within the body and with environmental cycles or daily schedules leads to the increasing prevalence of metabolic syndromes in modern society.


Assuntos
Relógios Circadianos , Metabolismo , Animais , Humanos
17.
Genetics ; 184(2): 351-61, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19948888

RESUMO

The Neurospora circadian oscillator comprises FREQUENCY (FRQ) and its transcription activator, the White Collar Complex (WCC). Repression of WCC's transcriptional activity by FRQ via negative feedback is indispensable for clock function. An unbiased genetic screen that targeted mutants with defects in negative feedback regulation yielded a fully viable arrhythmic strain bearing a novel allele of FRQ-interacting RNA helicase (frh), an essential gene that encodes a putative exosome component protein. In the allele, frh(R806H), clock function is completely disturbed, while roles of FRQ-interacting RNA helicase (FRH) essential for viability are left intact. FRH(R806H) still interacts with FRQ, but interaction between the FRQ-FRH(R806H) complex (FFC) and WCC is severely affected. Phosphorylation of WC-1 is reduced in the mutant leading to constantly elevated WCC activity, which breaks the negative feedback loop. WCC levels are considerably reduced in the mutant, especially those of WC-1, consistent both with loss of positive feedback (FRQ-dependent WC-1 stabilization) and with a reduced level of the FRQ-mediated WCC phosphorylation that leads to high WCC activity accompanied by rapid transcription-associated turnover. FRH overexpression promotes WC-1 accumulation, confirming that FRH together with FRQ plays a role in WC-1 stabilization. Identification of a viable allele of frh, displaying virtually complete loss of both negative and positive circadian feedback, positions FRH as a core component of the central oscillator that is permissive for rhythmicity but appears not to modulate periodicity. Moreover, the results suggest that there are clock-specific roles for FRH that are distinct from the predicted essential exosome-associated functions for the protein.


Assuntos
Ritmo Circadiano/fisiologia , Retroalimentação Fisiológica , Proteínas Fúngicas/metabolismo , Neurospora crassa/genética , Neurospora crassa/fisiologia , RNA Helicases/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Mutação de Sentido Incorreto/genética , Neurospora crassa/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Biossíntese de Proteínas , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Translocação Genética/genética
18.
Cell ; 137(4): 749-60, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19450520

RESUMO

Temperature compensation of circadian clocks is an unsolved problem with relevance to the general phenomenon of biological compensation. We identify casein kinase 2 (CK2) as a key regulator of temperature compensation of the Neurospora clock by determining that two long-standing clock mutants, chrono and period-3, displaying distinctive alterations in compensation encode the beta1 and alpha subunits of CK2, respectively. Reducing the dose of these subunits, particularly beta1, significantly alters temperature compensation without altering the enzyme's Q(10). By contrast, other kinases and phosphatases implicated in clock function do not play appreciable roles in temperature compensation. CK2 exerts its effects on the clock by directly phosphorylating FREQUENCY (FRQ), and this phosphorylation is compromised in CK2 hypomorphs. Finally, mutation of certain putative CK2 phosphosites on FRQ, shown to be phosphorylated in vivo, predictably alters temperature compensation profiles effectively phenocopying CK2 mutants.


Assuntos
Caseína Quinase II/fisiologia , Ritmo Circadiano , Neurospora crassa/enzimologia , Neurospora crassa/fisiologia , Caseína Quinase II/química , Caseína Quinase II/genética , Dosagem de Genes , Mutação , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Temperatura
19.
Genetics ; 181(2): 767-81, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19015548

RESUMO

We report the discovery and validation of a set of single nucleotide polymorphisms (SNPs) between the reference Neurospora crassa strain Oak Ridge and the Mauriceville strain (FGSC 2555), of sufficient density to allow fine mapping of most loci. Sequencing of Mauriceville cDNAs and alignment to the completed genomic sequence of the Oak Ridge strain identified 19,087 putative SNPs. Of these, a subset was validated by cleaved amplified polymorphic sequence (CAPS), a simple and robust PCR-based assay that reliably distinguishes between SNP alleles. Experimental confirmation resulted in the development of 250 CAPS markers distributed evenly over the genome. To demonstrate the applicability of this map, we used bulked segregant analysis followed by interval mapping to locate the csp-1 mutation to a narrow region on LGI. Subsequently, we refined mapping resolution to 74 kbp by developing additional markers, resequenced the candidate gene, NCU02713.3, in the mutant background, and phenocopied the mutation by gene replacement in the WT strain. Together, these techniques demonstrate a generally applicable and straightforward approach for the isolation of novel genes from existing mutants. Data on both putative and validated SNPs are deposited in a customized public database at the Broad Institute, which encourages augmentation by community users.


Assuntos
Neurospora crassa/genética , Polimorfismo de Nucleotídeo Único , Mapeamento Cromossômico , DNA Fúngico/genética , Bases de Dados de Ácidos Nucleicos , Etiquetas de Sequências Expressas , Genes Fúngicos , Marcadores Genéticos , Mutação , Neurospora crassa/classificação , Reação em Cadeia da Polimerase , Recombinação Genética , Especificidade da Espécie
20.
Proc Natl Acad Sci U S A ; 104(50): 20102-7, 2007 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-18056807

RESUMO

In Neurospora, metabolic oscillators coexist with the circadian transcriptional/translational feedback loop governed by the FRQ (Frequency) and WC (White Collar) proteins. One of these, a choline deficiency oscillator (CDO) observed in chol-1 mutants grown under choline starvation, drives an uncompensated long-period developmental cycle ( approximately 60-120 h). To assess possible contributions of this metabolic oscillator to the circadian system, molecular and physiological rhythms were followed in liquid culture under choline starvation, but these only confirmed that an oscillator with a normal circadian period length can run under choline starvation. This finding suggested that long-period developmental cycles elicited by nutritional stress could be masking output from the circadian system, although a caveat was that the CDO sometimes requires several days to become consolidated. To circumvent this and observe both oscillators simultaneously, we used an assay using a codon-optimized luciferase to follow the circadian oscillator. Under conditions where the long-period, uncompensated, CDO-driven developmental rhythm was expressed for weeks in growth tubes, the luciferase rhythm in the same cultures continued in a typical compensated manner with a circadian period length dependent on the allelic state of frq. Periodograms revealed no influence of the CDO on the circadian oscillator. Instead, the CDO appears as a cryptic metabolic oscillator that can, under appropriate conditions, assume control of growth and development, thereby masking output from the circadian system. frq-driven luciferase as a reporter of the circadian oscillator may in this way provide a means for assessing prospective role(s) of metabolic and/or ancillary oscillators within cellular circadian systems.


Assuntos
Colina/metabolismo , Ritmo Circadiano/fisiologia , Neurospora/crescimento & desenvolvimento , Neurospora/metabolismo , Alelos , Colina/farmacologia , Genes Reporter/genética , Neurospora/efeitos dos fármacos , Neurospora/genética , Temperatura
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