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1.
J Mass Spectrom Adv Clin Lab ; 30: 51-60, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38074293

RESUMO

Introduction: The VeriStrat® test (VS) is a blood-based assay that predicts a patient's response to therapy by analyzing eight features in a spectrum obtained from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of human serum and plasma. In a recent analysis of the INSIGHT clinical trial (NCT03289780), it was found that the VS labels, VS Good and VS Poor, can effectively predict the responsiveness of non-small cell lung cancer (NSCLC) patients to immune checkpoint inhibitor (ICI) therapy. However, while VS measures the intensities of spectral features using MALDI-TOF analysis, the specific proteoforms underlying these features have not been comprehensively identified. Objectives: The objective of this study was to identify the proteoforms that are measured by VS. Methods: To resolve the features obtained from the low-resolution MALDI-TOF procedure used to acquire mass spectra for VS DeepMALDI® analysis of serum was employed. This technique allowed for the identification of finer peaks within these features. Additionally, a combination of reversed-phase fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then used to identify the proteoforms associated with these peaks. Results: The analysis revealed that the primary constituents of the spectrum measured by VS are serum amyloid A1, serum amyloid A2, serum amyloid A4, C-reactive protein, and beta-2 microglobulin. Conclusion: Proteoforms involved in host immunity were identified as significant components of these features. This newly acquired information improves our understanding of how VS can accurately predict patient response to therapy. It opens up additional studies that can expand our understanding even further.

2.
Environ Epidemiol ; 7(4): e254, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37545805

RESUMO

The frequency and severity of wildfires in the Western United States have increased over recent decades, motivating hypotheses that wildfires contribute to the incidence of coccidioidomycosis, an emerging fungal disease in the Western United States with sharp increases in incidence observed since 2000. While coccidioidomycosis outbreaks have occurred among wildland firefighters clearing brush, it remains unknown whether fires are associated with an increased incidence among the general population. Methods: We identified 19 wildfires occurring within California's highly endemic San Joaquin Valley between 2003 and 2015. Using geolocated surveillance records, we applied a synthetic control approach to estimate the effect of each wildfire on the incidence of coccidioidomycosis among residents that lived within a hexagonal buffer of 20 km radii surrounding the fire. Results: We did not detect excess cases due to wildfires in the 12 months (pooled estimated percent change in cases: 2.8%; 95% confidence interval [CI] = -29.0, 85.2), 13-24 months (7.9%; 95% CI = -27.3, 113.9), or 25-36 months (17.4%; 95% CI = -25.1, 157.1) following a wildfire. When examined individually, we detected significant increases in incidence following three of the 19 wildfires, all of which had relatively large adjacent populations, high transmission before the fire, and a burn area exceeding 5,000 acres. Discussion: We find limited evidence that wildfires drive increases in coccidioidomycosis incidence among the general population. Nevertheless, our results raise concerns that large fires in regions with ongoing local transmission of Coccidioides may be associated with increases in incidence, underscoring the need for field studies examining Coccidioides spp. in soils and air pre- and post-wildfires.

3.
Annu Rev Public Health ; 43: 271-291, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-34982587

RESUMO

Emerging evidence supports a link between environmental factors-including air pollution and chemical exposures, climate, and the built environment-and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission and coronavirus disease 2019 (COVID-19) susceptibility and severity. Climate, air pollution, and the built environment have long been recognized to influence viral respiratory infections, and studies have established similar associations with COVID-19 outcomes. More limited evidence links chemical exposures to COVID-19. Environmental factors were found to influence COVID-19 through four major interlinking mechanisms: increased risk of preexisting conditions associated with disease severity; immune system impairment; viral survival and transport; and behaviors that increase viral exposure. Both data and methodologic issues complicate the investigation of these relationships, including reliance on coarse COVID-19 surveillance data; gaps in mechanistic studies; and the predominance of ecological designs. We evaluate the strength of evidence for environment-COVID-19 relationships and discuss environmental actions that might simultaneously address the COVID-19 pandemic, environmental determinants of health, and health disparities.


Assuntos
Poluição do Ar , COVID-19 , Poluição do Ar/efeitos adversos , COVID-19/epidemiologia , Humanos , Incidência , Pandemias , SARS-CoV-2
4.
Ecol Evol ; 10(18): 10207-10218, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33005376

RESUMO

Complex interactions within multitrophic communities are fundamental to the evolution of individual species that reside within them. One common outcome of species interactions are fitness trade-offs, where traits adaptive in some circumstances are maladaptive in others. Here, we identify a fitness trade-off between fecundity and survival in the cynipid wasp Callirhytis quercusbatatoides that induces multichambered galls on the stem of its host plant Quercus virginiana. We first quantified this trade-off in natural populations by documenting two relationships: a positive association between the trait gall size and fecundity, as larger galls contain more offspring, and a negative association between gall size and survival, as larger galls are attacked by birds at a higher rate. Next, we performed a field-based experimental evolution study where birds were excluded from the entire canopy of 11 large host trees for five years. As a result of the five-year release from avian predators, we observed a significant shift to larger galls per tree. Overall, our study demonstrates how two opposing forces of selection can generate stabilizing selection on a critical phenotypic trait in wild populations, and how traits can evolve rapidly in the predicted direction when conditions change.

5.
J Hand Surg Glob Online ; 2(1): 46-54, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35415473

RESUMO

Ray resections have been a viable treatment option for patients with tumors, trauma, infection, vascular insufficiency, or other abnormalities of the hand since the procedure was described in the 1920s. The creation of a functional hand after central ray resection presents unique technical challenges: insufficient closure of the gap between the metacarpals bordering the resected ray can produce an enlarged space between remaining digits and potentially cause digital malrotation, both of which negatively affect hand function. The goal is to make the space between resulting fingers as close to normal as possible. A number of procedures were described to address this issue, but unfortunately, they can be technically onerous and may require prolonged immobilization, the use of internal hardware, or the use of temporary hardware requiring removal. We describe a technique for amputation of the affected ray at the proximal metacarpal metadiaphyseal flare and a concomitant closing wedge osteotomy to allow superior gap closure between the residual fingers while maintaining the structure of the carpus and alignment of the hand. This improves functional and aesthetic outcomes after central ray resection of the hand.

6.
Biol Lett ; 15(9): 20190470, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31480937

RESUMO

Anthropogenic environmental change is predicted to disrupt multitrophic interactions, which may have drastic consequences for population-level processes. Here, we investigate how a large-scale human-mediated disturbance affects the abundance of North America's most venomous caterpillar species, Megalopyge opercularis. Specifically, we used a natural experiment where netting was deployed to cover the entire canopies of a subset of mature southern live oak trees (Quercus virginiana) to exclude urban pest birds (grackles and pigeons), throughout an 8.1 km2 area encompassing a medical centre in Houston, Texas. We used this experimental exclusion to test the following hypothesis: release from avian predators increases caterpillar abundance to outbreak levels, which increases the risk to human health. Results from a multi-year survey show that caterpillar abundance increased, on average, more than 7300% on netted versus non-netted trees. Thus, increases in caterpillar abundance due to anthropogenic enemy release increase human exposure to this venomous pest, and should be considered a health threat in the area. This study emphasizes the unforeseen consequences of ecological disturbance for species interactions and highlights the importance of considering ecology in urban planning.


Assuntos
Mariposas , Peçonhas , Animais , Surtos de Doenças , Humanos , Texas , Árvores
7.
Front Pharmacol ; 10: 752, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354477

RESUMO

Conjunctival inflammation disturbs the blood-tear barrier and thus affects the tear film stability and composition. We aimed to develop a non-invasive and reliable method to induce conjunctivitis in dogs, a large animal model for translational work on ocular surface disease in humans. Six beagle dogs underwent a randomized, vehicle-controlled, balanced crossover trial-on six separate days, one eye received topical artificial tears (vehicle), while the other eye received one of six concentrations of histamine solution (0.005-500 mg/ml). At sequential times after eyedrop administration, a conjunctivitis score was given to each eye based on the degree of palpebral and bulbar conjunctival hyperemia and chemosis, ocular pruritus, and discharge. Total protein content (TPC) and serum albumin were quantified in tear fluid at baseline and 20 min. Additionally, 13 dogs presenting for various ophthalmic diseases with associated conjunctivitis were examined. Experimentally induced conjunctivitis developed rapidly (<1 min) following topical histamine administration and lasted for 1-3 h (four lowest doses) to 6-8 h (two highest doses). The severity of conjunctivitis was dose-dependent. Histamine was overall well tolerated, although transient blepharitis, aqueous flare, and ocular hypertension occurred in a few dogs receiving histamine ≥375 mg/ml. TPC and serum albumin levels increased in tears of eyes receiving histamine ≥1.0 mg/ml, being significantly higher than vehicle and baseline in eyes receiving histamine ≥375 mg/ml. Lacrimal albumin levels were also increased in 13 dogs with naturally acquired conjunctivitis, up 2.7-14.9 fold compared to contralateral healthy eyes. Histamine-induced conjunctivitis represents a robust model for translational work on the ocular surface given the low cost, non-invasiveness, self-resolving nature, ability to adjust the duration and severity of the disease, and shared features with naturally occurring ocular diseases. Histamine solutions of 1, 10, and 375 mg/ml induce mild, moderate, and severe conjunctivitis in dogs, respectively. Leakage of serum albumin in tear fluid of eyes with conjunctivitis suggests a breakdown of the blood-tear barrier.

8.
JAAPA ; 32(2): 27-29, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30694948

RESUMO

Emphysematous gastritis is a rare disease caused by gas-forming organisms. This article describes how a CT scan and aggressive IV drug therapy helped clinicians beat the odds for a patient with this high-mortality disease.


Assuntos
Enfisema/diagnóstico por imagem , Enfisema/tratamento farmacológico , Gastrite/diagnóstico por imagem , Gastrite/tratamento farmacológico , Tomografia Computadorizada por Raios X/métodos , Administração Intravenosa , Idoso , Humanos , Levofloxacino/administração & dosagem , Masculino , Metronidazol/administração & dosagem
9.
J Mol Diagn ; 19(3): 404-416, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28433077

RESUMO

Nearly 80% of cancer patients do not have genetic mutation results available at initial oncology consultation; up to 25% of patients begin treatment before receiving their results. These factors hinder the ability to pursue optimal treatment strategies. This study validates a blood-based genome-testing service that provides accurate results within 72 hours. We focused on targetable variants in advanced non-small cell lung carcinoma-epidermal growth factor receptor gene (EGFR) variant L858R, exon 19 deletion (ΔE746-A750), and T790M; GTPase Kirsten ras gene (KRAS) variants G12C/D/V; and echinoderm microtubule associated protein like and 4 anaplastic lymphoma receptor tyrosine kinase fusion (EML4-ALK) transcripts 1/2/3. Test development included method and clinical validation using samples from donors with (n = 219) or without (n = 30) cancer. Clinical sensitivity and specificity for each variant ranged from 78.6% to 100% and 94.2% to 100%, respectively. We also report on 1643 non-small cell lung carcinoma samples processed in our CLIA-certified laboratory. Mutation results were available within 72 hours for 94% of the tests evaluated. We detected 10.5% mutations for EGFR sensitizing (n = 2801 samples tested), 13.8% mutations for EGFR resistance (n = 1055), 13.2% mutations in KRAS (n = 3477), and 2% mutations for EML4-ALK fusion (n = 304). This rapid, highly sensitive, and actionable blood-based assay service expands testing options and supports faster treatment decisions.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Análise Mutacional de DNA/métodos , Quinase do Linfoma Anaplásico , Proteínas de Ciclo Celular/genética , Receptores ErbB/genética , Éxons/genética , Humanos , Neoplasias Pulmonares/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores Proteína Tirosina Quinases/genética , Serina Endopeptidases/genética
10.
Cytometry B Clin Cytom ; 92(6): 451-455, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-25917935

RESUMO

BACKGROUND: Point-of-care (POC) CD4 T-cell counting is increasingly recognized as providing improved linkage-to-care during management of HIV infection, particularly in resource-limited settings where disease burden is highest. This study evaluated prototype POC CD4 T-cell counters from MBio Diagnostics in the context of low CD4 count, hospitalized patients in Mozambique. This study measured system performance when presented with challenging, low count samples from HIV/AIDS patients with acute illnesses resulting in hospitalization. METHODS: Forty whole blood samples were collected from donors on the medical service at Maputo Central Hospital and absolute CD4 counts were generated on the MBio CD4 system and a reference laboratory using flow cytometry. RESULTS: The mean and median CD4 counts by the flow cytometry reference were 173 and 80 cells/µL, respectively. Correlation between the MBio CD4 System and the reference was good. Bland-Altman analysis showed a mean bias of +15 cells/µL (+9 to +21 cells/µL, 95% CI), and limits of agreement of -47 to 77 cells/µL. For samples with counts >100 cells/µL (N = 14), the mean coefficient of variation was 7.3%. For samples with counts <50 cells/µL, mean absolute bias of replicate samples was 4.8 cells/µL. When two MBio readers were compared, Bland-Altman bias was -4 cells/µL (-13 to +6 cells/µL, 95% CI), and limits of agreement of -63 and +55 cells/µL. CONCLUSIONS: The MBio System holds promise as a POC system for quantitation of CD4 T cells in resource-limited settings given system throughput (80-100 cartridges/day), design simplicity, and ease-of-use. © 2015 International Clinical Cytometry Society.


Assuntos
Contagem de Linfócito CD4/instrumentação , Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo/instrumentação , Infecções por HIV/diagnóstico , Imunofenotipagem/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/virologia , Estudos de Casos e Controles , Computadores de Mão/economia , Computadores de Mão/provisão & distribuição , Países em Desenvolvimento , Citometria de Fluxo/economia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Imunofenotipagem/instrumentação , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/virologia , Aplicativos Móveis/economia , Aplicativos Móveis/provisão & distribuição , Moçambique , Padrões de Referência , Reprodutibilidade dos Testes
11.
Nano Lett ; 14(1): 353-8, 2014 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-24328385

RESUMO

We report a systematic investigation of the size dependence of negative trion (T(-)) Auger recombination rates in free-standing colloidal CdSe nanocrystals. Colloidal n-type CdSe nanocrystals of various radii have been prepared photochemically, and their trion decay dynamics have been measured using time-resolved photoluminescence spectroscopy. Trion Auger time constants spanning 3 orders of magnitude are observed, ranging from 57 ps (radius R = 1.4 nm) to 2.2 ns (R = 3.2 nm). The data reveal a substantially stronger size dependence than found for bi- or multiexciton Auger recombination in CdSe or other semiconductor nanocrystals, scaling in proportion to R(4.3).

12.
J Am Chem Soc ; 135(50): 18782-5, 2013 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-24289732

RESUMO

A method for electronic doping of colloidal CdSe nanocrystals (NCs) is reported. Anaerobic photoexcitation of CdSe NCs in the presence of a borohydride hole quencher, Li[Et3BH], yields colloidal n-type CdSe NCs possessing extra conduction-band electrons compensated by cations deposited by the hydride hole quencher. The photodoped NCs possess excellent optical quality and display the key spectroscopic signatures associated with NC n-doping, including a bleach at the absorption edge, appearance of a new IR absorption band, and Auger quenching of the excitonic photoluminescence. Although stable under anaerobic conditions, these spectroscopic changes are all reversed completely upon exposure of the n-doped NCs to air. Chemical titration of the added electrons confirms previous correlations between absorption bleach and electron accumulation and provides a means of quantifying the extent of electron trapping in some NCs. The generality of this photodoping method is demonstrated by initial results on colloidal CdE (E = S, Te) NCs as well as on CdSe quantum dot films.

13.
N Z Med J ; 126(1372): 12-24, 2013 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-23793173

RESUMO

AIMS: A prospective analysis was undertaken of the workload of prehospital triage and treatment facilities established in Wellington for the 2011 and 2012 International Rugby Sevens, and the Rugby World Cup 2011 (RWC). The introduction of an alcohol intoxication pathway, the impact of the initiative on ambulance and Emergency Department (ED) workload, and its cost effectiveness were assessed. METHODS: A log of patients seen and their diagnoses and treatment was maintained. An alcohol questionnaire was completed when applicable. Patients intoxicated with alcohol were managed in accordance with a flowchart designed for paramedic use. Costs and savings were calculated. RESULTS: Half the patients were New Zealanders. The average age was 25 years with a slight female preponderance (52.9% female). 30% were students. Alcohol was a contributory or causative factor for the patient's attendance in 80-90% of cases. Approximately 60% of the 121 patients seen at the last two events would have had to be transferred to the ED in the absence of the treatment centre. Cost savings for the ambulance service and ED for the RWC and 2012 Sevens are estimated to be NZ$70,000. No adverse clinical event was identified. CONCLUSIONS: With minimal supervision, event medics and paramedics can safely care for the majority of patients attending large rugby events in New Zealand, easing the pressure on ambulances and the ED, and generating significant cost savings for those services.


Assuntos
Intoxicação Alcoólica/terapia , Ambulâncias/estatística & dados numéricos , Serviço Hospitalar de Emergência/estatística & dados numéricos , Triagem/métodos , Adulto , Ambulâncias/economia , Análise Custo-Benefício , Serviços Médicos de Emergência/métodos , Serviço Hospitalar de Emergência/economia , Futebol Americano , Humanos , Nova Zelândia , Transferência de Pacientes/economia , Transferência de Pacientes/estatística & dados numéricos , Estudos Prospectivos , Carga de Trabalho
14.
PLoS One ; 7(11): e49371, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23173058

RESUMO

Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn²âºbased on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn²âº is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn²âº levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn²âº. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn²âº uptake simultaneously in two compartments, revealing that nuclear Zn²âº rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn²âº more slowly and with a delay of 600-700 sec. Lastly, these studies provide the first glimpse of nuclear Zn²âº and reveal that nuclear Zn²âº is buffered at a higher level than cytosolic Zn²âº.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Zinco/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Cor , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Fatores de Tempo
15.
J Am Chem Soc ; 134(39): 16175-7, 2012 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-22985258

RESUMO

Chemical reductants of sub-conduction-band potentials are demonstrated to induce large photoluminescence enhancement in colloidal ZnSe-based nanocrystals. The photoluminescence quantum yield of colloidal Mn(2+)-doped ZnSe nanocrystals has been improved from 14% to 80% simply by addition of an outer-sphere reductant. Up to 48-fold redox brightening is observed for nanocrystals with lower starting quantum yields. These increases are quickly reversed upon exposure to air and are temporary even under anaerobic conditions. This redox brightening process offers a new and systematic approach to understanding redox-active surface "trap states" and their contributions to the physical properties of colloidal semiconductor nanocrystals.

16.
J Am Chem Soc ; 134(15): 6819-25, 2012 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-22417458

RESUMO

Spectroelectrochemical experiments on wide-gap semiconductor nanocrystals (ZnSe and Mn(2+)-doped ZnSe) have allowed the influence of trap electrochemistry on nanocrystal photoluminescence to be examined in the absence of semiconductor band filling. Large photoluminescence electrobrightening is observed in both materials upon application of a reducing potential and is reversed upon return to the equilibrium potential. Electrobrightening is correlated with the transfer of electrons into nanocrystal films, implicating reductive passivation of midgap surface electron traps. Analysis indicates that the electrobrightening magnitude is determined by competition between electron trapping and photoluminescence (ZnSe) or energy transfer (Mn(2+)-doped ZnSe) dynamics within the excitonic excited state, and that electron trapping is extremely fast (k(trap) ≈ 10(11) s(-1)). These results shed new light on the complex surface chemistries of semiconductor nanocrystals.

17.
ACS Nano ; 5(5): 4158-68, 2011 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-21452880

RESUMO

Auger processes in colloidal semiconductor nanocrystals have been scrutinized extensively in recent years. Whether involving electron-exciton, hole-exciton, or exciton-exciton interactions, such Auger processes are generally fast and hence have been considered prominent candidates for interpreting fast processes relevant to photoluminescence blinking and multiexciton decay. With recent advances in the chemistries of nanocrystal doping, increasing attention is now being paid to analogous photophysical properties of colloidal-doped semiconductor nanocrystals. Here, we report the first investigation of the effects of electron-dopant exchange interactions on dopant luminescence in doped semiconductor nanocrystals. Using electrochemical techniques, electrical control of charge-carrier densities in films of colloidal Mn(2+)-doped CdS quantum dots has been achieved and used to demonstrate remarkably effective Auger de-excitation of photoexcited Mn(2+). The doped nanocrystals are found to be substantially more sensitive to Auger de-excitation than their undoped analogues, a result shown to arise primarily from the long Mn(2+) excited-state lifetime. This observation of exceptionally effective Auger quenching has broader implications in areas of high-power, single-particle, or electrically driven luminescence of doped semiconductor nanocrystals, and also suggests interesting opportunities for modulating Mn(2+) photoluminescence intensities on sublifetime time scales, or for imaging charge carriers in nanocrystal-based devices.


Assuntos
Medições Luminescentes/métodos , Manganês/química , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Semicondutores , Eletroquímica , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Propriedades de Superfície
18.
Nat Immunol ; 12(4): 335-43, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21399639

RESUMO

NF-κB is a major gene regulator in immune responses, and ribosomal protein S3 (RPS3) is an NF-κB subunit that directs specific gene transcription. However, it is unknown how nuclear translocation of RPS3 is regulated. Here we report that phosphorylation of RPS3 Ser209 by the kinase IKKß was crucial for nuclear localization of RPS3 in response to activating stimuli. Moreover, virulence protein NleH1 of the foodborne pathogen Escherichia coli strain O157:H7 specifically inhibited phosphorylation of RPS3 Ser209 and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but resulting in less mortality in a gnotobiotic piglet-infection model. Thus, the IKKß-dependent modification of a specific amino acid in RPS3 promoted specific NF-κB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.


Assuntos
Proteínas de Escherichia coli/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteínas Ribossômicas/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/virologia , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Escherichia coli O157/fisiologia , Proteínas de Escherichia coli/genética , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Quinase I-kappa B/genética , Immunoblotting , Células Jurkat , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , Interferência de RNA , Proteínas Ribossômicas/genética , Homologia de Sequência de Aminoácidos , Serina/genética , Serina/metabolismo , Suínos
19.
Mol Endocrinol ; 22(8): 1781-96, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18550772

RESUMO

17beta-Estradiol (E2) acts through the estrogen receptor alpha (ERalpha) to stimulate breast cancer proliferation. Here, we investigated the functional relationship between ERalpha and signal transducer and activator of transcription (STAT)5b activity in ER+ MCF-7 and T47D human breast cancer cells after specific knockdown of STAT5b. STAT5b small interfering RNA (siRNA) inhibited E2-induced bromodeoxyuridine (BrdU) incorporation in both cell lines, as well as the E2-induced increase in MCF-7 cell number, cyclin D1 and c-myc mRNA, and cyclin D1 protein expression, indicating that STAT5b is required for E2-stimulated breast cancer proliferation. E2 treatment stimulated STAT5b tyrosine phosphorylation at the activating tyrosine Y699, resulting in increased STAT5-mediated transcriptional activity, which was inhibited by a Y669F STAT5b mutant. E2-induced STAT5-mediated transcriptional activity was inhibited by overexpressing a kinase-defective epidermal growth factor receptor (EGFR), or the EGFR tyrosine kinase inhibitor tyrphostin AG1478, indicating a requirement for EGFR kinase activity. Both E2-induced STAT5b tyrosine phosphorylation and STAT5-mediated transcription were also inhibited by the ER antagonist ICI 182,780 and the c-Src inhibitor PP2, indicating additional requirements for the ER and c-Src kinase activity. EGFR and c-Src kinase activities were also required for E2-induced cyclin D1 and c-myc mRNA. Together, these studies demonstrate positive cross talk between ER, c-Src, EGFR, and STAT5b in ER+ breast cancer cells. Increased EGFR and c-Src signaling is associated with tamoxifen resistance in ER+ breast cancer cells. Here we show that constitutively active STAT5b not only increased basal DNA synthesis, but also conferred tamoxifen resistance. Because STAT5b plays an integral role in E2-stimulated proliferation and tamoxifen resistance, it may be an effective therapeutic target in ER+ breast tumors.


Assuntos
Neoplasias da Mama/enzimologia , Receptores ErbB/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Neoplásicos , Humanos , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Fator de Transcrição STAT5/genética , Tamoxifeno/farmacologia , Transcrição Gênica/efeitos dos fármacos
20.
Breast Cancer Res ; 9(6): R79, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17997837

RESUMO

INTRODUCTION: Signal transducers and activators of transcription (STATs) are mediators of cytokine and growth factor signaling. In recent years, STAT5b has emerged as a key regulator of tumorigenesis. STAT5b phosphorylation and activation is mediated by several kinases known to be overexpressed in breast cancer, such as epidermal growth factor receptor, HER2, and c-Src. Breast tumor kinase (Brk), also known as protein tyrosine kinase 6, is a nonreceptor tyrosine kinase expressed in more than 60% of breast cancers. Only a few substrates of the Brk tyrosine kinase have been identified, the most recent being STAT3. In the present article we investigate the potential role of Brk in the phosphorylation and activation STAT5b. METHODS: To determine whether Brk can phosphorylate STAT5b, transient transfection and in vitro kinase assays were performed. Luciferase reporter assays were used to measure Brk-induced STAT5b transcriptional activity. siRNA technology was utilized to investigate the biological significance of Brk-induced activation of STAT5b in breast cancer cell models. RESULTS: Phosphospecific antibodies, mutational analysis, and in vitro kinase assays demonstrated that Brk specifically mediated STAT5b phosphorylation at the activating tyrosine, Y699. Transient transfection of Brk into the Brk-negative BT-549 breast cancer cell line enhanced STAT5b transcriptional activity, as measured by a STAT5-specific luciferase reporter. Furthermore, overexpression of kinase active c-Src enhanced Brk-induced STAT5b transcriptional activity. In Brk-positive breast cancer cell lines BT-20 and SKBr3, knockdown of Brk protein or of STAT5b protein using siRNA methodology resulted in a decrease in DNA synthesis. Knockdown of Brk and STAT5b together did not further decrease DNA synthesis compared with each alone, suggesting that Brk and STAT5b converge on the same pathway, ultimately leading to cellular proliferation. CONCLUSION: Our studies demonstrate that Brk phosphorylates STAT5b on Y699, leading to increased STAT5b transcriptional activity. Furthermore, analysis of DNA synthesis suggests that STAT5b and Brk are converging upon the same proproliferative signaling pathway in breast cancer cells. We propose that Brk, like other tyrosine kinases, signals downstream to STAT5b to mediate proliferation of breast cancer cells. These results further establish STAT5b as well as Brk as potential targets for breast cancer therapy.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteína Tirosina Quinase CSK , Proliferação de Células , Feminino , Humanos , Luciferases , Substâncias Luminescentes , Fosforilação , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transfecção , Quinases da Família src
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