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1.
Arch Gynecol Obstet ; 310(1): 229-235, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38649500

RESUMO

BACKGROUND: Cervical cerclage is the only effective treatment for cervical insufficiency, effectively preventing late miscarriage and preterm birth. The effectiveness and safety of emergency cervical cerclage (ECC) as an emergency treatment when the cervix is already dilated or when there is protrusion of the fetal membranes into the vagina remain controversial, especially in pregnancies at 24-28 weeks when the fetus is viable. There is still no consensus on whether emergency cervical cerclage should be performed in such cases. PURPOSE: To investigate the effectiveness and safety of emergency cervical cerclage in singleton pregnant women at 24-28 weeks of gestation. METHODS: This study employed a single-center prospective cohort design, enrolling singleton pregnant women at 24-28 weeks of gestation with ultrasound or physical examination indicating cervical dilation or even membrane protrusion. Emergency cervical cerclage was compared with conservative treatment. The primary endpoints included a comprehensive assessment of perinatal pregnancy loss, significant neonatal morbidity, and adverse neonatal outcomes. Secondary endpoints included prolonged gestational age, preterm birth, neonatal hospitalization rate, premature rupture of membranes, and intrauterine infection/chorioamnionitis. RESULTS: From June 2021 to March 2023, a total of 133 pregnant women participated in this study, with 125 completing the trial, and were allocated to either the Emergency Cervical Cerclage (ECC) group (72 cases) or the conservative treatment group (53 cases) based on informed consent from the pregnant women. The rate of adverse neonatal outcomes was 8.33% in the ECC group and 26.42% in the conservative treatment (CT) group, with a statistically significant difference (P = 0.06). There were no significant differences between the two groups in terms of perinatal pregnancy loss and significant neonatal morbidity. The conservative treatment group had a mean prolonged gestational age of 63.0 (23.0, 79.5) days, while the ECC group had 84.0 (72.5, 89.0) days, with a statistically significant difference between the two groups (P < 0.001). Compared with CT group, the ECC group showed a significantly reduced incidence of preterm birth before 28 weeks, 32 weeks, and 34 weeks, with statistical significance (P = 0.046, 0.007, 0.001), as well as a significantly decreased neonatal hospitalization rate (P = 0.013, 0.031). Additionally, ECC treatment did not increase the risk of preterm premature rupture of membranes or intrauterine infection/chorioamnionitis, with no statistically significant differences (P = 0.406, 0.397). CONCLUSION: In singleton pregnant women with cervical insufficiency at 24-28 weeks of gestation, emergency cervical cerclage can reduce adverse neonatal pregnancy outcomes, effectively prolong gestational age, decrease preterm births before 28 weeks, 32 weeks, and 34 weeks, lower neonatal hospitalization rates, and does not increase the risk of preterm premature rupture of membranes or intrauterine infection/chorioamnionitis.


Assuntos
Cerclagem Cervical , Nascimento Prematuro , Incompetência do Colo do Útero , Humanos , Feminino , Gravidez , Adulto , Estudos Prospectivos , Nascimento Prematuro/prevenção & controle , Nascimento Prematuro/epidemiologia , Incompetência do Colo do Útero/cirurgia , Idade Gestacional , Resultado da Gravidez , Recém-Nascido , Segundo Trimestre da Gravidez , Ruptura Prematura de Membranas Fetais/epidemiologia , Emergências , Aborto Espontâneo/prevenção & controle , Aborto Espontâneo/epidemiologia , Tratamento de Emergência/estatística & dados numéricos
2.
mBio ; 13(1): e0362121, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35038896

RESUMO

Quorum sensing (QS) is a chemical communication process in which bacteria produce, release, and detect extracellular signaling molecules called autoinducers. Via combined transcriptional and posttranscriptional regulatory mechanisms, QS allows bacteria to collectively alter gene expression on a population-wide scale. Recently, the TetR family transcriptional regulator LuxT was shown to control Vibrio harveyi qrr1, encoding the Qrr1 small RNA that functions at the core of the QS regulatory cascade. Here, we use RNA sequencing to reveal that, beyond the control of qrr1, LuxT is a global regulator of 414 V. harveyi genes, including those involved in type III secretion, siderophore production, and aerolysin toxin biosynthesis. Importantly, LuxT directly represses swrZ, encoding a GntR family transcriptional regulator, and LuxT control of type III secretion, siderophore, and aerolysin genes occurs by two mechanisms, one that is SwrZ dependent and one that is SwrZ independent. All of these target genes specify QS-controlled behaviors that are enacted when V. harveyi is at low cell density. Thus, LuxT and SwrZ function in parallel with QS to drive particular low-cell-density behaviors. Phylogenetic analyses reveal that luxT is highly conserved among Vibrionaceae, but swrZ is less well conserved. In a test case, we find that in Aliivibrio fischeri, LuxT also represses swrZ. SwrZ is a repressor of A. fischeri siderophore production genes. Thus, LuxT repression of swrZ drives the activation of A. fischeri siderophore gene expression. Our results indicate that LuxT is a major regulator among Vibrionaceae, and in the species that also possess swrZ, LuxT functions with SwrZ to control gene expression. IMPORTANCE Bacteria precisely tune gene expression patterns to successfully react to changes that occur in the environment. Defining the mechanisms that enable bacteria to thrive in diverse and fluctuating habitats, including in host organisms, is crucial for a deep understanding of the microbial world and also for the development of effective applications to promote or combat particular bacteria. In this study, we show that a regulator called LuxT controls over 400 genes in the marine bacterium Vibrio harveyi and that LuxT is highly conserved among Vibrionaceae species, ubiquitous marine bacteria that often cause disease. We characterize the mechanisms by which LuxT controls genes involved in virulence and nutrient acquisition. We show that LuxT functions in parallel with a set of regulators of the bacterial cell-to-cell communication process called quorum sensing to promote V. harveyi behaviors at low cell density.


Assuntos
Sideróforos , Vibrio , Sideróforos/metabolismo , Filogenia , Vibrio/genética , Percepção de Quorum/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica
3.
Environ Res ; 204(Pt A): 111974, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34480945

RESUMO

BACKGROUND: Recent research attention has been paid to anthropogenic heat emissions (AE), temperature increase generated by human activity such as lighting, transportation, manufacturing, construction, and building climate controls. However, there is no epidemiological data available to investigate the association between anthropogenic heat emissions and metabolic syndrome (MetS), a cluster of conditions that increase risk of stroke, heart disease and diabetes. OBJECTIVE: To explore the relationships between AE and MetS in China. METHODS: We recruited 15,477 adults from the 33 Communities Chinese Health Study, a cross-sectional study in northeastern China. We retrieved anthropogenic heat flux by collecting socio-economic and energy consumption data as well as satellite-based nighttime light and Normalized Difference Vegetation Index datasets, including emissions from buildings, transportation, human metabolism, and industries. We also measured MetS components consisting of triglycerides, high density lipoprotein cholesterol, fasting glucose, systolic blood pressure, and diastolic blood pressure, and waist circumference. Restricted cubic spline models were applied to assess the associations between AE and MetS. RESULTS: The median flux of total AE was 30.98 W/m2 and industrial AE was the dominant contributor (87.64%). The adjusted odds ratio and 95% confidence interval (CI) of MetS for the 75th and 95th percentiles of the total AE against the threshold were 1.29 (95% CI: 1.21, 1.38) and 1.65 (95% CI: 1.47, 1.85). Greater AE was associated with higher odds of MetS in a dose-response pattern, and the lowest point of U-shape curve indicated the threshold effect. Participants who are young and middle-aged exhibited stronger associations between AE and MetS. CONCLUSIONS: Our novel findings reveal that AE are positively associated with MetS and that associations are modified by age. Further investigations into the mechanisms of the effects are needed.


Assuntos
Síndrome Metabólica , Adulto , Glicemia , China/epidemiologia , Estudos Transversais , Temperatura Alta , Humanos , Síndrome Metabólica/epidemiologia , Pessoa de Meia-Idade , Fatores de Risco , Circunferência da Cintura
4.
Environ Int ; 155: 106596, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33940391

RESUMO

BACKGROUND: Chlorinated polyfluorinated ether sulfonic acids (Cl-PFESAs), a group of perfluorooctane sulfonate (PFOS) alternatives, can be widely observed in humans and environmental matrices. However, associations between exposure to Cl-PFESAs and serum lipid levels in adults are unknown. OBJECTIVE: To explore the relationships between Cl-PFESA levels and serum lipid levels in adults. METHODS: We analyzed 1238 adults from the Isomers of C8 Health Project, a cross-sectional study conducted in China from July 2015 to October 2016. The average age of the participants was 61.98 ± 14.40 years. We quantified two select legacy per- and perfluoroalkyl substances [perfluorooctanoic acid (PFOA) and PFOS] and their alternatives (6:2 and 8:2 Cl-PFESAs). We also measured four serum lipids: low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglycerides (TG). We used generalized linear models to estimate the associations between PFASs and serum lipids, with PFASs defined as either a categorical variable divided into quartiles or as a continuous variable. RESULTS: We found that 6:2 Cl-PFESA was positively associated with serum TC and LDL-C. For instance, LDL-C levels in the highest quartile of 6:2 Cl-PFESA exposure (Q4) were significantly higher than those in the lowest quartile (Q1) [ß: 0.19, 95% confidence interval (CI): 0.08, 0.30]. Further analysis showed that one ln-ng/mL increase in 6:2 Cl-PFESA exposure corresponded to a 0.10 mmol/L (95% CI: 0.05, 0.16) LDL-C increase, and that exposure to 8:2 Cl-PFESA was negatively correlated with HDL-C (ß: -0.03, 95% CI: -0.05, -0.01). TC had a similar relationship with both 6:2 Cl-PFESA and legacy PFASs. Participants with a BMI ≥ 25 kg/m2 exhibited a stronger association between 6:2 Cl-PFESA and TC. CONCLUSIONS: Our findings make the novel suggestion that exposure to Cl-PFESAs are adversely associated with serum lipid levels, and that such associations are also observed in legacy PFASs. Increased investigation into the effects of Cl-PFESAs exposure on human health is warranted.


Assuntos
Ácidos Alcanossulfônicos , Fluorocarbonos , Adulto , Idoso , China , Estudos Transversais , Fluorocarbonos/análise , Humanos , Pessoa de Meia-Idade
5.
J Biol Chem ; 295(10): 2916-2931, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31964715

RESUMO

Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae, the quorum-sensing autoinducer 3,5-dimethyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR, encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it with existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2'-position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N-alanyl-aminoacetone (Ala-AA), also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure-function analysis identifies key features required for VqmA activation and DNA binding and establishes that, whereas VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity.


Assuntos
Proteínas de Bactérias/metabolismo , Pirazóis/metabolismo , Percepção de Quorum , Transdução de Sinais , Fatores de Transcrição/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Ligantes , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Pirazóis/química , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética
6.
PLoS Pathog ; 15(6): e1007820, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194839

RESUMO

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. Pseudomonas aeruginosa, an opportunistic pathogen, employs multiple quorum-sensing systems to control behaviors including virulence factor production and biofilm formation. One P. aeruginosa quorum-sensing receptor, called RhlR, binds the cognate autoinducer N-butryl-homoserine lactone (C4HSL), and the RhlR:C4HSL complex activates transcription of target quorum-sensing genes. Here, we use a genetic screen to identify RhlR mutants that function independently of the autoinducer. The RhlR Y64F W68F V133F triple mutant, which we call RhlR*, exhibits ligand-independent activity in vitro and in vivo. RhlR* can drive wildtype biofilm formation and infection in a nematode animal model. The ability of RhlR* to properly regulate quorum-sensing-controlled genes in vivo depends on the quorum-sensing regulator RsaL keeping RhlR* activity in check. RhlR is known to function together with PqsE to control production of the virulence factor called pyocyanin. Likewise, RhlR* requires PqsE for pyocyanin production in planktonic cultures, however, PqsE is dispensable for RhlR*-driven pyocyanin production on surfaces. Finally, wildtype RhlR protein is not sufficiently stabilized by C4HSL to allow purification. However, wildtype RhlR can be stabilized by the synthetic ligand mBTL (meta-bromo-thiolactone) and RhlR* is stable without a ligand. These features enabled purification of the RhlR:mBTL complex and of RhlR* for in vitro examination of their biochemical activities. To our knowledge, this work reports the first RhlR protein purification.


Assuntos
Proteínas de Bactérias , Pseudomonas aeruginosa , Percepção de Quorum/fisiologia , Receptores de Superfície Celular , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans , Mutação de Sentido Incorreto , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/química , Piocianina/genética , Piocianina/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
7.
ACS Chem Biol ; 14(3): 378-389, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30763066

RESUMO

Bacteria use a cell-cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.


Assuntos
4-Butirolactona/análogos & derivados , Proteínas de Bactérias/metabolismo , Percepção de Quorum/efeitos dos fármacos , Transativadores/metabolismo , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Aminoácidos/química , Escherichia coli/efeitos dos fármacos , Ligantes , Estrutura Molecular , Mutação , Ligação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Transdução de Sinais , Relação Estrutura-Atividade
10.
Nat Chem Biol ; 13(5): 551-557, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28319101

RESUMO

Quorum sensing (QS) is a cell-cell communication process that enables bacteria to track cell population density and orchestrate collective behaviors. QS relies on the production and detection of, and the response to, extracellular signal molecules called autoinducers. In Vibrio cholerae, multiple QS circuits control pathogenesis and biofilm formation. Here, we identify and characterize a new QS autoinducer-receptor pair. The autoinducer is 3,5-dimethylpyrazin-2-ol (DPO). DPO is made from threonine and alanine, and its synthesis depends on threonine dehydrogenase (Tdh). DPO binds to and activates a transcription factor, VqmA. The VqmA-DPO complex activates expression of vqmR, which encodes a small regulatory RNA. VqmR represses genes required for biofilm formation and toxin production. We propose that DPO allows V. cholerae to regulate collective behaviors to, among other possible roles, diversify its QS output during colonization of the human host.


Assuntos
Biofilmes/crescimento & desenvolvimento , Pirazóis/metabolismo , Proteínas Repressoras/metabolismo , Vibrio cholerae/metabolismo , Regulação Bacteriana da Expressão Gênica , Pirazóis/química , Percepção de Quorum , Proteínas Repressoras/química , Proteínas Repressoras/genética , Vibrio cholerae/genética , Vibrio cholerae/crescimento & desenvolvimento
11.
J Biol Chem ; 292(10): 4064-4076, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28119451

RESUMO

Quorum sensing is a process of cell-cell communication that bacteria use to regulate collective behaviors. Quorum sensing depends on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. In many bacterial species, quorum sensing controls virulence factor production. Thus, disrupting quorum sensing is considered a promising strategy to combat bacterial pathogenicity. Several members of a family of naturally produced plant metabolites called flavonoids inhibit Pseudomonas aeruginosa biofilm formation by an unknown mechanism. Here, we explore this family of molecules further, and we demonstrate that flavonoids specifically inhibit quorum sensing via antagonism of the autoinducer-binding receptors, LasR and RhlR. Structure-activity relationship analyses demonstrate that the presence of two hydroxyl moieties in the flavone A-ring backbone are essential for potent inhibition of LasR/RhlR. Biochemical analyses reveal that the flavonoids function non-competitively to prevent LasR/RhlR DNA binding. Administration of the flavonoids to P. aeruginosa alters transcription of quorum sensing-controlled target promoters and suppresses virulence factor production, confirming their potential as anti-infectives that do not function by traditional bacteriocidal or bacteriostatic mechanisms.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Flavonoides/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/fisiologia , Transativadores/antagonistas & inibidores , Virulência/efeitos dos fármacos , Regulação Alostérica , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Pseudomonas aeruginosa/crescimento & desenvolvimento , Percepção de Quorum/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
12.
Proc Natl Acad Sci U S A ; 112(7): E766-75, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25646441

RESUMO

Quorum sensing (QS) is a process of cell-to-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence and biofilm formation in Vibrio cholerae, the causative agent of cholera disease. Differential RNA sequencing (RNA-seq) of wild-type V. cholerae and a locked low-cell-density QS-mutant strain identified 7,240 transcriptional start sites with ∼ 47% initiated in the antisense direction. A total of 107 of the transcripts do not appear to encode proteins, suggesting they specify regulatory RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene encoding a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription, that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtx (repeats in toxin) toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide to our knowledege the first global annotation of the transcriptional start sites in V. cholerae and highlight the importance of posttranscriptional regulation for collective behaviors in this human pathogen.


Assuntos
Biofilmes , RNA Viral/genética , Análise de Sequência de RNA , Vibrio cholerae/genética , Sequência de Bases , Perfilação da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
13.
J Bacteriol ; 197(1): 73-80, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25313392

RESUMO

Bacteria use a chemical communication process called quorum sensing to monitor cell density and to alter behavior in response to fluctuations in population numbers. Previous studies with Vibrio harveyi have shown that LuxR, the master quorum-sensing regulator, activates and represses >600 genes. These include six genes that encode homologs of the Escherichia coli Bet and ProU systems for synthesis and transport, respectively, of glycine betaine, an osmoprotectant used during osmotic stress. Here we show that LuxR activates expression of the glycine betaine operon betIBA-proXWV, which enhances growth recovery under osmotic stress conditions. BetI, an autorepressor of the V. harveyi betIBA-proXWV operon, activates the expression of genes encoding regulatory small RNAs that control quorum-sensing transitions. Connecting quorum-sensing and glycine betaine pathways presumably enables V. harveyi to tune its execution of collective behaviors to its tolerance to stress.


Assuntos
Pressão Osmótica/fisiologia , Percepção de Quorum/fisiologia , Vibrio/fisiologia , Betaína/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estresse Fisiológico , Transativadores/genética , Transativadores/metabolismo , Vibrio/genética
14.
PLoS Pathog ; 8(6): e1002767, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22761573

RESUMO

Quorum sensing (QS) is a bacterial cell-cell communication process that relies on the production and detection of extracellular signal molecules called autoinducers. QS allows bacteria to perform collective activities. Vibrio cholerae, a pathogen that causes an acute disease, uses QS to repress virulence factor production and biofilm formation. Thus, molecules that activate QS in V. cholerae have the potential to control pathogenicity in this globally important bacterium. Using a whole-cell high-throughput screen, we identified eleven molecules that activate V. cholerae QS: eight molecules are receptor agonists and three molecules are antagonists of LuxO, the central NtrC-type response regulator that controls the global V. cholerae QS cascade. The LuxO inhibitors act by an uncompetitive mechanism by binding to the pre-formed LuxO-ATP complex to inhibit ATP hydrolysis. Genetic analyses suggest that the inhibitors bind in close proximity to the Walker B motif. The inhibitors display broad-spectrum capability in activation of QS in Vibrio species that employ LuxO. To the best of our knowledge, these are the first molecules identified that inhibit the ATPase activity of a NtrC-type response regulator. Our discovery supports the idea that exploiting pro-QS molecules is a promising strategy for the development of novel anti-infectives.


Assuntos
Proteínas de Bactérias/metabolismo , Percepção de Quorum/fisiologia , Vibrio cholerae/fisiologia , Vibrio cholerae/patogenicidade , Biofilmes/crescimento & desenvolvimento , Western Blotting , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Relação Estrutura-Atividade , Virulência/fisiologia
15.
Proc Natl Acad Sci U S A ; 109(31): 12746-51, 2012 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-22802636

RESUMO

Cyclic di-GMP (c-di-GMP) is a second messenger molecule that regulates the transition between sessile and motile lifestyles in bacteria. Bacteria often encode multiple diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that produce and degrade c-di-GMP, respectively. Because of multiple inputs into the c-di-GMP-signaling network, it is unclear whether this system functions via high or low specificity. High-specificity signaling is characterized by individual DGCs or PDEs that are specifically associated with downstream c-di-GMP-mediated responses. In contrast, low-specificity signaling is characterized by DGCs or PDEs that modulate a general signal pool, which, in turn, controls a global c-di-GMP-mediated response. To determine whether c-di-GMP functions via high or low specificity in Vibrio cholerae, we correlated the in vivo c-di-GMP concentration generated by seven DGCs, each expressed at eight different levels, to the c-di-GMP-mediated induction of biofilm formation and transcription. There was no correlation between total intracellular c-di-GMP levels and biofilm formation or gene expression when considering all states. However, individual DGCs showed a significant correlation between c-di-GMP production and c-di-GMP-mediated responses. Moreover, the rate of phenotypic change versus c-di-GMP concentration was significantly different between DGCs, suggesting that bacteria can optimize phenotypic output to c-di-GMP levels via expression or activation of specific DGCs. Our results conclusively demonstrate that c-di-GMP does not function via a simple, low-specificity signaling pathway in V. cholerae.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Vibrio cholerae/fisiologia , Proteínas de Bactérias/genética , GMP Cíclico/genética , GMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Fósforo-Oxigênio Liases/genética
16.
Mol Microbiol ; 83(6): 1095-108, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22295878

RESUMO

Quorum sensing, a bacterial cell-cell communication process, controls biofilm formation and virulence factor production in Vibrio cholerae, a human pathogen that causes the disease cholera. The major V. cholerae autoinducer is (S)-3-hydroxytridecan-4-one (CAI-1). A membrane bound two-component sensor histidine kinase called CqsS detects CAI-1, and the CqsS → LuxU → LuxO phosphorelay cascade transduces the information encoded in CAI-1 into the cell. Because the CAI-1 ligand is known and because the signalling circuit is simple, consisting of only three proteins, this system is ideal for analysing ligand regulation of a sensor histidine kinase. Here we reconstitute the CqsS → LuxU → LuxO phosphorylation cascade in vitro. We find that CAI-1 inhibits the initial auto-phosphorylation of CqsS whereas subsequent phosphotransfer steps and CqsS phosphatase activity are not CAI-1-controlled. CAI-1 binding to CqsS causes a conformational change that renders His194 in CqsS inaccessible to the CqsS catalytic domain. CqsS mutants with altered ligand detection specificities are faithfully controlled by their corresponding modified ligands in vitro. Likewise, pairing of agonists and antagonists allows in vitro assessment of their opposing activities. Our data are consistent with a two-state model for ligand control of histidine kinases.


Assuntos
Proteínas de Bactérias/metabolismo , Cólera/microbiologia , Regulação Bacteriana da Expressão Gênica , Cetonas/metabolismo , Proteínas Quinases/metabolismo , Vibrio cholerae/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Histidina Quinase , Humanos , Ligantes , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Percepção de Quorum , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Vibrio cholerae/química , Vibrio cholerae/genética , Vibrio cholerae/fisiologia
17.
Int J Oncol ; 40(4): 1196-202, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22179544

RESUMO

LeY (Lewis Y) is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Elevation of LeY is frequently observed in epithelial-derived cancers and is correlated to pathological staging and prognosis. To study the role of LeY on cancer cells, a stably LeY-overexpressing cell line, RMG-I-H, was developed previously by transfection of the α1,2-fucosyltransferase gene, a key enzyme that catalyzes the synthesis of LeY, into ovarian carcinoma-derived RMG-I cells. Our studies have shown that LeY is involved in the changes in biological behavior of RMG-I-H cells. However, the mechanism is still largely unknown. In this study, we determined the structural relationship and co-localization between LeY and TßRI/TßRII, respectively, and the potential cellular signaling mechanism was also investigated. We found that both TßRI and TßRII contain the LeY structure, and the level of LeY in TßRI and TßRII in RMG-I-H cells was significantly increased. Overexpression of LeY up-regulates the phosphorylation of ERK, Akt and down-regulates the phosphorylation of Smad2/3. In addition, the phosphorylation intensity was attenuated significantly by LeY monoantibody. These findings suggest that LeY is involved in the changes in biological behavior through TGF­ß receptors via Smad, ERK/MAPK and PI3K/Akt signaling pathways. We suggest that LeY may be an important composition of growth factor receptors and could be an attractive candidate for cancer diagnosis and treatment.


Assuntos
Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Neoplasias Ovarianas/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Antígenos do Grupo Sanguíneo de Lewis/genética , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transdução de Sinais , Transfecção
18.
Int J Mol Sci ; 11(10): 3748-59, 2010 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-21152298

RESUMO

Lewis y (LeY) antigen is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Overexpression of LeY is frequently observed in epithelial-derived cancers and has been correlated to the pathological staging and prognosis. However, the effects of LeY on ovarian cancer are not yet clear. Previously, we transfected the ovarian cancer cell line RMG-I with the α1,2-fucosyltransferase gene to obtain stable transfectants, RMG-I-H, that highly express LeY. In the present study, we examined the proliferation, tumorigenesis, adhesion and invasion of the cell lines with treatment of LeY monoclonal antibody (mAb). Additionally, we examined the expression of TGF-ß1, VEGF and b-FGF in xenograft tumors. The results showed that the proliferation and adhesion in vitro were significantly inhibited by treatment of RMG-I-H cells with LeY mAb. When subcutaneously inoculated in nude mice, RMG-I-H cells produced large tumors, while mock-transfected cells RMG-I-C and the parental cells RMG-I produced small tumors. Moreover, the tumor formation by RMG-I-H cells was inhibited by preincubating the cells with LeY mAb. Notably, the expression of TGF-ß1, VEGF and b-FGF all increased in RMG-I-H cells. In conclusion, LeY plays an important role in promoting cell proliferation, tumorigenecity and adhesion, and these effects may be related to increased levels of growth factors. The LeY antibody shows potential application in the treatment of LeY-positive tumors.


Assuntos
Carcinogênese , Carcinoma/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Neoplasias Ovarianas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Carcinoma/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Fatores de Crescimento de Fibroblastos/genética , Humanos , Antígenos do Grupo Sanguíneo de Lewis/genética , Camundongos , Camundongos Nus , Neoplasias Ovarianas/patologia , Fator de Crescimento Transformador beta/genética , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética
19.
Proc Natl Acad Sci U S A ; 107(12): 5575-80, 2010 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-20212168

RESUMO

Bacterial histidine kinases transduce extracellular signals into the cytoplasm. Most stimuli are chemically undefined; therefore, despite intensive study, signal recognition mechanisms remain mysterious. We exploit the fact that quorum-sensing signals are known molecules to identify mutants in the Vibrio cholerae quorum-sensing receptor CqsS that display altered responses to natural and synthetic ligands. Using this chemical-genetics approach, we assign particular amino acids of the CqsS sensor to particular roles in recognition of the native ligand, CAI-1 (S-3 hydroxytridecan-4-one) as well as ligand analogues. Amino acids W104 and S107 dictate receptor preference for the carbon-3 moiety. Residues F162 and C170 specify ligand head size and tail length, respectively. By combining mutations, we can build CqsS receptors responsive to ligand analogues altered at both the head and tail. We suggest that rationally designed ligands can be employed to study, and ultimately to control, histidine kinase activity.


Assuntos
Proteínas de Bactérias/fisiologia , Proteínas Quinases/fisiologia , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/fisiologia , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Genes Bacterianos , Histidina Quinase , Cetonas/metabolismo , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Modelos Moleculares , Mutagênese , Mutação , Proteínas Quinases/genética , Percepção de Quorum/efeitos dos fármacos , Percepção de Quorum/genética , Percepção de Quorum/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Vibrio cholerae/genética
20.
J Exp Clin Cancer Res ; 28: 154, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20003467

RESUMO

BACKGROUND: Lewis y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cell surface. Elevated expression of Lewis y has been found in 75% of ovarian tumor, and the high expression level is correlated to the tumor's pathological staging and prognosis. This study was to investigate the effect and the possible mechanism of Lewis y on the proliferation of human ovarian cancer cells. METHODS: We constructed a plasmid encoding alpha1,2-fucosyltransferase (alpha1,2-FT) gene and then transfected it into ovarian carcinoma-derived RMG-I cells with lowest Lewis y antigen expression level. Effect of Lewis y on cell proliferation was assessed after transfection. Changes in cell survival and signal transduction were evaluated after alpha-L-fucosidase, anti-Lewis y antibody and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment. RESULTS: Our results showed that the levels of alpha1,2-FT gene and Lewis y increased significantly after transfection. The cell proliferation of ovarian carcinoma-derived RMG-I cells sped up as the Lewis y antigen was increased. Both of alpha-L-fucosidase and anti-Lewis y antibody inhibited the cell proliferation. The phosphorylation level of Akt was apparently elevated in Lewis y-overexpressing cells and the inhibitor of PI3K, LY294002, dramatically inhibited the growth of Lewis y-overexpressing cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of alpha1,2-FT were attenuated significantly by the monoantibody to Lewis y and by the PI3K inhibitor LY294002. CONCLUSIONS: Increased expression of Lewis y antigen plays an important role in promoting cell proliferation through activating PI3K/Akt signaling pathway in ovarian carcinoma-derived RMG-I cells. Inhibition of Lewis y expression may provide a new therapeutic approach for Lewis y positive ovarian cancer.


Assuntos
Fucosiltransferases/metabolismo , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Neoplasias Ovarianas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transfecção , Galactosídeo 2-alfa-L-Fucosiltransferase
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