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1.
Sci Rep ; 9(1): 8646, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201348

RESUMO

Brain degeneration, including that caused by traumatic brain injury (TBI) often leads to severe bladder dysfunction, including incontinence and lower urinary tract symptoms; with the causes remaining unknown. Male C57BL/6J mice underwent repetitive moderate brain injury (rmdTBI) or sham injury, then mice received either cis P-tau monoclonal antibody (cis mAb), which prevents brain degeneration in TBI mice, or control (IgG). Void spot assays revealed age-dependent incontinence in IgG controls 8 months after injury, while cis mAb treated or sham mice showed no dysfunction. No obvious bladder pathology occurred in any group. Urodynamic cystometry in conscious mice revealed overactive bladder, reduced maximal voiding pressures and incontinence in IgG control, but not sham or cis mAb treated mice. Hyperphosphorylated tau deposition and neural tangle-like pathology occurred in cortical and hippocampal regions only of IgG control mice accompanied with post-traumatic neuroinflammation and was not seen in midbrain and hindbrain regions associated with bladder filling and voiding reflex arcs. In this model of brain degeneration bladder dysfunction results from rostral, and not hindbrain damage, indicating that rostral brain inputs are required for normal bladder functioning. Detailed analysis of the functioning of neural circuits controlling bladder function in TBI should lead to insights into how brain degeneration leads to bladder dysfunction, as well as novel strategies to treat these disorders.

2.
Chem Commun (Camb) ; 55(39): 5659-5662, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31032497

RESUMO

We report ionic self-assembly of two oppositely charged cobalt(iii) porphyrins (CoPs) on carbon coupled with subsequent mild pyrolysis at 350 °C, making CoPs lose some peripheral groups and become tightly adsorbed on the carbon with a high faradaic efficiency of 88 ± 1.5% and a current density of 8 mA cm-2 at a low overpotential of 430 mV toward electrochemical conversion of CO2 to CO.

3.
Methods ; 159-160: 115-123, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30797033

RESUMO

Dramatic increases in the scale of programmed synthesis of nucleic acid libraries coupled with deep sequencing have powered advances in understanding nucleic acid and protein biology. Biological systems centering on nucleic acids or encoded proteins greatly benefit from such high-throughput studies, given that large DNA variant pools can be synthesized and DNA, or RNA products of transcription, can be easily analyzed by deep sequencing. Here we review the scope of various high-throughput functional assays for studies of nucleic acids and proteins in general, followed by discussion of how these types of study have yielded insights into the RNA Polymerase II (Pol II) active site as an example. We discuss methodological considerations in the design and execution of these experiments that should be valuable to studies in any system.

4.
Nat Commun ; 9(1): 3069, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30093655

RESUMO

Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) combination safely cures fatal acute promyelocytic leukemia, but their mechanisms of action and efficacy are not fully understood. ATRA inhibits leukemia, breast, and liver cancer by targeting isomerase Pin1, a master regulator of oncogenic signaling networks. Here we show that ATO targets Pin1 and cooperates with ATRA to exert potent anticancer activity. ATO inhibits and degrades Pin1, and suppresses its oncogenic function by noncovalent binding to Pin1's active site. ATRA increases cellular ATO uptake through upregulating aquaporin-9. ATO and ATRA, at clinically safe doses, cooperatively ablate Pin1 to block numerous cancer-driving pathways and inhibit the growth of triple-negative breast cancer cells and tumor-initiating cells in cell and animal models including patient-derived orthotopic xenografts, like Pin1 knockout, which is substantiated by comprehensive protein and microRNA analyses. Thus, synergistic targeting of Pin1 by ATO and ATRA offers an attractive approach to combating breast and other cancers.

6.
J Hematol Oncol ; 11(1): 73, 2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-29848341

RESUMO

BACKGROUND: The increasing genomic complexity of acute myeloid leukemia (AML), the most common form of acute leukemia, poses a major challenge to its therapy. To identify potent therapeutic targets with the ability to block multiple cancer-driving pathways is thus imperative. The unique peptidyl-prolyl cis-trans isomerase Pin1 has been reported to promote tumorigenesis through upregulation of numerous cancer-driving pathways. Although Pin1 is a key drug target for treating acute promyelocytic leukemia (APL) caused by a fusion oncogene, much less is known about the role of Pin1 in other heterogeneous leukemia. METHODS: The mRNA and protein levels of Pin1 were detected in samples from de novo leukemia patients and healthy controls using real-time quantitative RT-PCR (qRT-PCR) and western blot. The establishment of the lentiviral stable-expressed short hairpin RNA (shRNA) system and the tetracycline-inducible shRNA system for targeting Pin1 were used to analyze the biological function of Pin1 in AML cells. The expression of cancer-related Pin1 downstream oncoproteins in shPin1 (Pin1 knockdown) and Pin1 inhibitor all-trans retinoic acid (ATRA) treated leukemia cells were examined by western blot, followed by evaluating the effects of genetic and chemical inhibition of Pin1 in leukemia cells on transformed phenotype, including cell proliferation and colony formation ability, using trypan blue, cell counting assay, and colony formation assay in vitro, as well as the tumorigenesis ability using in vivo xenograft mouse models. RESULTS: First, we found that the expression of Pin1 mRNA and protein was significantly increased in both de novo leukemia clinical samples and multiple leukemia cell lines, compared with healthy controls. Furthermore, genetic or chemical inhibition of Pin1 in human multiple leukemia cell lines potently inhibited multiple Pin1 substrate oncoproteins and effectively suppressed leukemia cell proliferation and colony formation ability in cell culture models in vitro. Moreover, tetracycline-inducible Pin1 knockdown and slow-releasing ATRA potently inhibited tumorigenicity of U937 and HL-60 leukemia cells in xenograft mouse models. CONCLUSIONS: We demonstrate that Pin1 is highly overexpressed in human AML and is a promising therapeutic target to block multiple cancer-driving pathways in AML.

8.
Front Microbiol ; 8: 1391, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28785254

RESUMO

Efficient and cost-effective bioethanol production from lignocellulosic materials requires co-fermentation of the main hydrolyzed sugars, including glucose, xylose, and L-arabinose. Saccharomyces cerevisiae is a glucose-fermenting yeast that is traditionally used for ethanol production. Fermentation of L-arabinose is also possible after metabolic engineering. Transport into the cell is the first and rate-limiting step for L-arabinose metabolism. The galactose permease, Gal2p, is a non-specific, endogenous monosaccharide transporter that has been shown to transport L-arabinose. However, Gal2p-mediated transport of L-arabinose occurs at a low efficiency. In this study, homologous modeling and L-arabinose docking were used to predict amino acids in Gal2p that are crucial for L-arabinose transport. Nine amino acid residues in Gal2p were identified and were the focus for site-directed mutagenesis. In the Gal2p transport-deficient chassis cells, the capacity for L-arabinose transport of the different Gal2p mutants was compared by testing growth rates using L-arabinose as the sole carbon source. Almost all the tested mutations affected L-arabinose transport capacity. Among them, F85 is a unique site. The F85S, F85G, F85C, and F85T point mutations significantly increased L-arabinose transport activities, while, the F85E and F85R mutations decreased L-arabinose transport activities compared to the Gal2p-expressing wild-type strain. These results verified F85 as a key residue in L-arabinose transport. The F85S mutation, having the most significant effect, elevated the exponential growth rate by 40%. The F85S mutation also improved xylose transport efficiency and weakened the glucose transport preference. Overall, enhancing the L-arabinose transport capacity further improved the L-arabinose metabolism of engineered S. cerevisiae.

9.
FEMS Yeast Res ; 17(4)2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28582494

RESUMO

The rapid co-fermentation of both glucose and xylose is important for the efficient conversion of lignocellulose biomass into fuels and chemicals. Saccharomyces cerevisiae is considered to be a potential cell factory and has been used to produce various fuels and chemicals, but it cannot metabolize xylose, which has greatly limited the utilization of lignocellulose materials. Therefore, numerous studies have attempted to develop xylose fermenting strains in past decades. The simple introduction of the xylose metabolic pathway does not enable yeast to rapidly utilize xylose, and several limitations still need to be addressed, including glucose repression and slow xylose transport, cofactor imbalance in the xylose reductase/xylitol dehydrogenase pathway, functional expression of a heterologous xylose isomerase, the low efficiency of downstream pathways and low ethanol production. In this review, we will discuss strategies to overcome these limitations and the recent progress in engineering xylose fermenting S. cerevisiae strains.


Assuntos
Glucose/metabolismo , Lignina/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Etanol/metabolismo , Fermentação , Regulação da Expressão Gênica , Microbiologia Industrial , Cinética , Saccharomyces cerevisiae/genética , Transgenes
10.
Biomed Res Int ; 2017: 5318232, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28459063

RESUMO

Efficient and cost-effective fuel ethanol production from lignocellulosic materials requires simultaneous cofermentation of all hydrolyzed sugars, mainly including D-glucose, D-xylose, and L-arabinose. Saccharomyces cerevisiae is a traditional D-glucose fermenting strain and could utilize D-xylose and L-arabinose after introducing the initial metabolic pathways. The efficiency and simultaneous coutilization of the two pentoses and D-glucose for ethanol production in S. cerevisiae still need to be optimized. Previously, we constructed an L-arabinose-utilizing S. cerevisiae BSW3AP. In this study, we further introduced the XI and XR-XDH metabolic pathways of D-xylose into BSW3AP to obtain D-glucose, D-xylose, and L-arabinose cofermenting strain. Benefits of evolutionary engineering: the resulting strain BSW4XA3 displayed a simultaneous coutilization of D-xylose and L-arabinose with similar consumption rates, and the D-glucose metabolic capacity was not decreased. After 120 h of fermentation on mixed D-glucose, D-xylose, and L-arabinose, BSW4XA3 consumed 24% more amounts of pentoses and the ethanol yield of mixed sugars was increased by 30% than that of BSW3AP. The resulting strain BSW4XA3 was a useful chassis for further enhancing the coutilization efficiency of mixed sugars for bioethanol production.


Assuntos
Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Monossacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Biocombustíveis , Etanol , Fermentação , Monossacarídeos/análise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
11.
Nucleic Acids Res ; 45(8): 4431-4451, 2017 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-28119420

RESUMO

Here we employ a set of RNA Polymerase II (Pol II) activity mutants to determine the consequences of increased or decreased Pol II catalysis on gene expression in Saccharomyces cerevisiae. We find that alteration of Pol II catalytic rate, either fast or slow, leads to decreased Pol II occupancy and apparent reduction in elongation rate in vivo. However, we also find that determination of elongation rate in vivo by chromatin immunoprecipitation can be confounded by the kinetics and conditions of transcriptional shutoff in the assay. We identify promoter and template-specific effects on severity of gene expression defects for both fast and slow Pol II mutants. We show that mRNA half-lives for a reporter gene are increased in both fast and slow Pol II mutant strains and the magnitude of half-life changes correlate both with mutants' growth and reporter expression defects. Finally, we tested a model that altered Pol II activity sensitizes cells to nucleotide depletion. In contrast to model predictions, mutated Pol II retains normal sensitivity to altered nucleotide levels. Our experiments establish a framework for understanding the diversity of transcription defects derived from altered Pol II activity mutants, essential for their use as probes of transcription mechanisms.


Assuntos
RNA Polimerase II/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Biocatálise , Imunoprecipitação da Cromatina , Regulação Fúngica da Expressão Gênica , Meia-Vida , Mutação , Nucleotídeos/deficiência , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/enzimologia , Elongação da Transcrição Genética , Sítio de Iniciação de Transcrição
12.
PLoS Genet ; 12(11): e1006321, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27898685

RESUMO

The active sites of multisubunit RNA polymerases have a "trigger loop" (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.


Assuntos
Proteínas Mutantes/genética , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Transcrição Genética , Alelos , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Proteínas Mutantes/química , Mutação , Dobramento de Proteína , Estrutura Secundária de Proteína , Transporte Proteico/genética , RNA Polimerase II/química , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato
13.
PLoS One ; 10(3): e0120923, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25793756

RESUMO

The pigmentation of many Aeromonas species has been thought to be due to the production of a L-DOPA (L-3,4-dihydroxyphenylalanine) based melanin. However, in this study we found that although L-DOPA synthesis occurs in the high-melanin-yielding Aeromonas media strain WS, it plays a minor, if any, role in pigmentation. Instead, the pigmentation of A. media strain WS is due to the production of pyomelanin through HGA (homogentisate). Gene products of phhA (encodes phenylalanine hydroxylase), tyrB and aspC (both encode aromatic amino acid aminotransferase), and hppD (encodes 4-hydroxyphenylpyruvate dioxygenase) constitute a linear pathway of converting phenylalanine to HGA and disruption of any one of these genes impairs or blocks pigmentation of A. media strain WS. This HGA biosynthesis pathway is widely distributed in Aeromonas, but HGA is only detectable in the cultures of pigmented Aeromonas species. Heterologous expression of HppD from both pigmented and non-pigmented Aeromonas species in E. coli leads to the production of pyomelanin and thus pigmentation, suggesting that most Aeromonas species have the critical enzymes to produce pyomelanin through HGA. Taken together, we have identified a widely conserved biosynthesis pathway of HGA based pyomelanin in Aeromonas that may be responsible for pigmentation of many Aeromonas species.


Assuntos
Aeromonas/metabolismo , Vias Biossintéticas/genética , Ácido Homogentísico/metabolismo , Melaninas/biossíntese , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Aeromonas/genética , Cromatografia Líquida de Alta Pressão , Elementos de DNA Transponíveis/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Testes Genéticos , Ácido Homogentísico/química , Levodopa/biossíntese , Melaninas/química , Mutagênese/genética , Ácidos Fenilpirúvicos/metabolismo , Pigmentação , Transcrição Genética , Tirosina/metabolismo
14.
Cell ; 154(4): 775-88, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23932120

RESUMO

RNA polymerase II (RNAPII) lies at the core of dynamic control of gene expression. Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ∼60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This analysis enabled functional assignment of RNAPII subdomains and uncovered connections between individual regions and other protein complexes. Using splicing microarrays and mutants that alter elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified fast and slow mutants that favor upstream and downstream start site selection, respectively. The striking coordination of polymerization rate with transcription initiation and splicing suggests that transcription rate is tuned to regulate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multifunctional machines at amino acid resolution.


Assuntos
Epistasia Genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alelos , Estudo de Associação Genômica Ampla , Mutação Puntual , RNA Polimerase II/química , Processamento de RNA , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Genética , Transcriptoma
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