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Self-assembled quantum dots (QDs) based on III-V semiconductors have excellent properties for applications in quantum optics. However, the presence of a 2D wetting layer (WL) which forms during the Stranski-Krastanov growth of QDs can limit their performance. Here, we investigate WL formation during QD growth by the droplet epitaxy technique. We use a combination of photoluminescence excitation spectroscopy, lifetime measurements, and transmission electron microscopy to identify the presence of an InGaAs WL in these droplet epitaxy QDs, even in the absence of distinguishable WL luminescence. We observe that increasing the amount of Ga deposited on a GaAs (100) surface prior to the growth of InGaAs QDs leads to a significant reduction in the emission wavelength of the WL to the point where it can no longer be distinguished from the GaAs acceptor peak emission in photoluminescence measurements. However increasing the amount of Ga deposited does not suppress the formation of a WL under the growth conditions used here.
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Arsenicais , Gálio , Pontos Quânticos , Arsenicais/química , Luminescência , Gálio/químicaRESUMO
We demonstrate that the ubiquitous laboratory magnetic stirrer provides a simple passive method of magnetic levitation, in which the so-called "flea" levitates indefinitely. We study the onset of levitation and quantify the flea's motion (a combination of vertical oscillation, spinning and "waggling"), finding excellent agreement with a mechanical analytical model. The waggling motion drives recirculating flow, producing a centripetal reaction force that stabilized the flea. Our findings have implications for the locomotion of artificial swimmers and the development of bidirectional microfluidic pumps, and they provide an alternative to sophisticated commercial levitators.
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In the evaporation of microlitre liquid droplets, the accepted view is that surface tension dominates and the effect of gravity is negligible. We report, through the first use of rotating optical coherence tomography, that a change in the flow pattern and speed occurs when evaporating binary liquid droplets are tilted, conclusively showing that gravitational effects dominate the flow. We use gas chromatography to show that these flows are solutal in nature, and we establish a flow phase diagram demonstrating the conditions under which different flow mechanisms occur.
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INTRODUCTION: Ineffective coping may lead to impaired job performance and burnout, with adverse consequences to staff well-being and patient outcomes. We examined the relationship between coping styles and burnout in emergency physicians, nurses and support staff at seven small, medium and large emergency departments (ED) in a Canadian health region (population 500,000). METHODS: Linear regression with the Coping Inventory for Stressful Situations (CISS) and Maslach Burnout Inventory (MBI) was used to evaluate the effect of coping style on levels of burnout in a cross-sectional survey of 616 ED staff members. CISS measures coping style in three categories: task-oriented, emotion-oriented and avoidance-oriented coping; MBI, in use for 30â years, assesses the level of burnout in healthcare workers. RESULTS: Task-oriented coping was associated with decreased risk of burnout, while emotion-oriented coping was associated with increased risk of burnout. DISCUSSION: Specific coping styles are associated with varied risk of burnout in ED staff across several different types of hospitals in a regional network. Coping style intervention may reduce burnout, while leading to improvement in staff well-being and patient outcomes. Further studies should focus on building and sustaining task-oriented coping, along with alternatives to emotion-oriented coping.
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Adaptação Psicológica , Esgotamento Profissional/prevenção & controle , Esgotamento Profissional/psicologia , Serviço Hospitalar de Emergência , Corpo Clínico Hospitalar/psicologia , Recursos Humanos de Enfermagem Hospitalar/psicologia , Adulto , Canadá , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos e Questionários , Adulto JovemRESUMO
In this Letter, we present narrow line width (7 µeV), nearly background-free single-photon emission (g((2))(0) = 0.02) and highly indistinguishable photons (V = 0.73) from site-controlled In(Ga)As/GaAs quantum dots. These excellent properties have been achieved by combining overgrowth on ex situ pit-patterned substrates with vertical stacking of spectrally distinct quantum dot layers. Our study paves the way for large-scale integration of quantum dots into quantum photonic circuits as indistinguishable single-photon sources.
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DESIGN: This was a prospective observational study. BACKGROUND AND AIMS: The characteristics of cannabis-involved motor vehicle collisions are poorly understood. This study of injured drivers identifies demographic and collision characteristics associated with high tetrahydrocannabinol (THC) concentrations. SETTING: The study was conducted in 15 Canadian trauma centres between January 2018 and December 2021. CASES: The cases (n = 6956) comprised injured drivers who required blood testing as part of routine trauma care. MEASUREMENTS: We quantified whole blood THC and blood alcohol concentration (BAC) and recorded driver sex, age and postal code, time of crash, crash type and injury severity. We defined three driver groups: high THC (THC ≥ 5 ng/ml and BAC = 0), high alcohol (BAC ≥ 0.08% and THC = 0) and THC/BAC-negative (THC = 0 = BAC). We used logistic regression techniques to identify factors associated with group membership. FINDINGS: Most injured drivers (70.2%) were THC/BAC-negative; 1274 (18.3%) had THC > 0, including 186 (2.7%) in the high THC group; 1161 (16.7%) had BAC > 0, including 606 (8.7%) in the high BAC group. Males and drivers aged less than 45 years had higher adjusted odds of being in the high THC group (versus the THC/BAC-negative group). Importantly, 4.6% of drivers aged less than 19 years had THC ≥ 5 ng/ml, and drivers aged less than 19 years had higher unadjusted odds of being in the high THC group than drivers aged 45-54 years. Males, drivers aged 19-44 years, rural drivers, seriously injured drivers and drivers injured in single-vehicle, night-time or weekend collisions had higher adjusted odds ratios (aORs) for being in the high alcohol group (versus THC/BAC-negative). Drivers aged less than 35 or more than 65 years and drivers involved in multi-vehicle, daytime or weekday collisions had higher adjusted odds for being in the high THC group (versus the high BAC group). CONCLUSIONS: In Canada, risk factors for cannabis-related motor vehicle collisions appear to differ from those for alcohol-related motor vehicle collisions. The collision factors associated with alcohol (single-vehicle, night-time, weekend, rural, serious injury) are not associated with cannabis-related collisions. Demographic factors (young drivers, male drivers) are associated with both alcohol and cannabis-related collisions, but are more strongly associated with cannabis-related collisions.
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Acidentes de Trânsito , Consumo de Bebidas Alcoólicas , Dronabinol , Fumar Maconha , Ferimentos e Lesões , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Acidentes de Trânsito/estatística & dados numéricos , Fatores Etários , Consumo de Bebidas Alcoólicas/sangue , Dronabinol/sangue , Fumar Maconha/sangue , Medição de Risco , Fatores de Risco , Fatores Sexuais , Ferimentos e Lesões/epidemiologiaRESUMO
PURPOSE: Point-of-care ultrasonography (POCUS) is an established tool in the management of hypotensive patients in the emergency department (ED). We compared the diagnostic accuracy of a POCUS protocol versus standard assessment without POCUS in patients with undifferentiated hypotension. METHODS: This was an international, multicenter randomized controlled trial included three EDs in North America and three in South Africa from September 2012 to December 2016. Hypotensive patients were randomized to early POCUS protocol plus standard care (POCUS group) or standard care without POCUS (control group). Initial and secondary diagnoses were recorded at 0 and 60 min. The main outcome was measures of diagnostic accuracy of a POCUS protocol in differentiating between cardiogenic and non-cardiogenic shock. Secondary outcomes were diagnostic performance for shock sub-types, as well as changes in perceived category of shock and overall diagnosis. RESULTS: Follow-up was completed for 270 of 273 patients. For cardiogenic shock, the POCUS-based diagnostic approach (POCUS) performed similarly to the non-POCUS approach (control) for specificity [95.5% (89.9-98.5) vs.93.8% (87.7-97.5)]; positive likelihood ratio (17.92 vs 14.80); negative likelihood ratio (0.21 vs 0.09) and diagnostic odds ratio (85.6 vs 166.57), with a similar overall diagnostic accuracy between the two approaches [93.7% (88-97.2) vs 93.6% (87.8-97.2)]. Diagnostic performance measures were similar across sub-categories of shock. CONCLUSION: This is the first randomized controlled trial to compare diagnostic performance of a POCUS protocol to standard care without POCUS in undifferentiated hypotensive ED patients. POCUS performed well diagnostically in undifferentiated hypotensive patients, especially as a rule-in test; however, performance did not differ meaningfully from standard assessment.
RéSUMé: OBJECTIF: L'échographie au point d'intervention (POCUS) est un outil bien établi dans la gestion des patients hypotendus dans le service des urgences. Nous avons comparé la précision diagnostique d'un protocole POCUS par rapport à une évaluation standard sans POCUS chez des patients présentant une hypotension indifférenciée. MéTHODES: Il s'agissait d'un essai contrôlé randomisé international multicentrique incluant 3 services d'urgence en Amérique du Nord et 3 en Afrique du Sud de septembre 2012 à décembre 2016. Les patients hypotenseurs ont été répartis par randomisation selon le protocole POCUS précoce plus les soins standard (groupe POCUS) ou les soins standard sans POCUS (groupe témoin). Les diagnostics initiaux et secondaires ont été enregistrés à 0 et 60 minutes. Le principal résultat était la mesure de la précision diagnostique d'un protocole POCUS pour différencier le choc cardiogénique du choc non cardiogénique. Les résultats secondaires étaient la performance diagnostique pour les sous-types de chocs, ainsi que les changements dans la perception de la catégorie de choc et du diagnostic global. RéSULTATS: Le suivi a été complété pour 270 des 273 patients. Pour le choc cardiogénique, l'approche diagnostique basée sur le POCUS (POCUS) a donné des résultats similaires à l'approche non-POCUS (Contrôle) pour la spécificité (95,5 % (89,998,5) vs 93,8 % (87,797,5)) ; Rapport de vraisemblance positif (17,92 vs 14,80) ; Le rapport de vraisemblance négatif (0,21 vs 0,09) et le rapport de cotes diagnostiques (85,6 vs 166,57), avec une précision diagnostique globale similaire entre les deux approches (93,7 % (8897,2) vs 93,6 % (87,897,2). Les mesures de performance diagnostique étaient similaires dans toutes les sous-catégories de choc. CONCLUSION: Il s'agit du premier essai contrôlé randomisé visant à comparer la performance diagnostique d'un protocole POCUS aux soins standard sans POCUS chez des patients hypotendus indifférenciés aux urgences. La POCUS a donné de bons résultats diagnostiques chez les patients hypotendus indifférenciés, surtout en tant que test de référence ; cependant, les performances ne diffèrent pas de manière significative de l'évaluation standard.
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Hipotensão , Choque , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Ultrassonografia/métodos , Hipotensão/diagnóstico por imagem , Choque/diagnóstico por imagem , Serviço Hospitalar de Emergência , Choque CardiogênicoRESUMO
The lack of structural symmetry which usually characterizes semiconductor quantum dots lifts the energetic degeneracy of the bright excitonic states and hampers severely their use as high-fidelity sources of entangled photons. We demonstrate experimentally and theoretically that it is always possible to restore the excitonic degeneracy by the simultaneous application of large strain and electric fields. This is achieved by using one external perturbation to align the polarization of the exciton emission along the axis of the second perturbation, which then erases completely the energy splitting of the states. This result, which holds for any quantum dot structure, highlights the potential of combining complementary external fields to create artificial atoms meeting the stringent requirements posed by scalable semiconductor-based quantum technology.
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The fabrication of a cross-wire p-i-n light emitting diode (LED) by molecular beam epitaxial overgrowth on mesa-patterned GaAs(100) substrates is presented. Micron-wide mesa stripes fabricated by standard photolithography are subsequently narrowed to sub-micron dimensions by GaAs overgrowth due to net migration towards the mesa top. Chains of InAs quantum dots (QDs) can then be grown in a self-aligned manner on top of the narrow GaAs ridge mesa, forming the active region of the QD-chain LED. The kinetics of the overgrowth is discussed and the electroluminescence operation of the LED is presented.
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Entangled photon pairs are an important resource in quantum optics, and are essential for quantum information applications such as quantum key distribution and controlled quantum logic operations. The radiative decay of biexcitons-that is, states consisting of two bound electron-hole pairs-in a quantum dot has been proposed as a source of triggered polarization-entangled photon pairs. To date, however, experiments have indicated that a splitting of the intermediate exciton energy yields only classically correlated emission. Here we demonstrate triggered photon pair emission from single quantum dots suggestive of polarization entanglement. We achieve this by tuning the splitting to zero, through either application of an in-plane magnetic field or careful control of growth conditions. Entangled photon pairs generated 'on demand' have significant fundamental advantages over other schemes, which can suffer from multiple pair emission, or require post-selection techniques or the use of photon-number discriminating detectors. Furthermore, control over the pair generation time is essential for scaling many quantum information schemes beyond a few gates. Our results suggest that a triggered entangled photon pair source could be implemented by a simple semiconductor light-emitting diode.
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BACKGROUND: Emergency physicians were trained to perform echo in life support (ELS)--that is, limited transthoracic echocardiography during advanced life support (ALS) management of cardiac arrest. METHODS: Data were collected on the adequacy of views obtained and timing of the scan, as well as the clinical findings of pericardial effusion and ventricular wall motion. Any intervention performed as a result of the scan was also noted. ELS was performed on 50 patients during cardiac arrest. RESULTS: Adequate views were obtained in 47 (94%) scans, and 45 (90%) were obtained within the 10 s rhythm check. Twenty patients (40%) had ventricular wall motion (VWM), three (6%) had pericardial effusions and six patients (12%) had an intervention performed as a direct result of the scan. These included pericardiocentesis, thrombolysis and insertion of a chest drain. The presence of VWM had a positive predictive value of 55%. The absence of VWM resulted in a negative predictive value of 97% for predicting return of spontaneous circulation (ROSC). CONCLUSION: It is concluded that ELS is feasible and that the scan findings may guide further interventions.
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Suporte Vital Cardíaco Avançado , Ecocardiografia , Serviço Hospitalar de Emergência , Parada Cardíaca/diagnóstico por imagem , Adulto , Estudos de Coortes , Estudos de Viabilidade , Parada Cardíaca/etiologia , Parada Cardíaca/terapia , Humanos , Valor Preditivo dos Testes , Estudos RetrospectivosRESUMO
State readout is a key requirement for a quantum computer. For semiconductor-based qubit devices it is usually accomplished using a separate mesoscopic electrometer. Here we demonstrate a simple detection scheme in which a radio frequency resonant circuit coupled to a semiconductor double quantum dot is used to probe its charge and spin states. These results demonstrate a new noninvasive technique for measuring charge and spin states in quantum dot systems without requiring a separate mesoscopic detector.
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The UK government response to COVID-19 has been heavily criticised. We report witnesses' perceptions of what has shaped UK policies and how these policies have been received by healthcare workers. Such studies are usually affected by hindsight. Here we deploy a novel prospective approach to capture real-time information. We are historians, social scientists and biomedical researchers who study how societies cope with infectious disease. In February 2020 we began regular semi-structured calls with prominent members of policy communities, and health care professionals, to elicit their roles in, and reactions to, the pandemic response. We report witnesses' perceptions that personal protective equipment (PPE) stocks were too small, early warnings have not led to sufficiently rapid policy decisions, and a lack of transparency is sapping public trust. Significant successes include research mobilisation. The early experiences and reactions of our witnesses suggest important issues for investigation, notably a perception of delay in decision making.
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COVID-19/epidemiologia , Política de Saúde , Vigilância da População/métodos , Medicina Estatal/organização & administração , Pesquisa Biomédica/organização & administração , Comunicação , Planejamento em Desastres/organização & administração , Humanos , Entrevistas como Assunto , Pandemias , SARS-CoV-2 , Fatores de Tempo , Reino Unido/epidemiologiaRESUMO
We surveyed published reports on about 50 glycoproteins whose amino acid sequence, glycosylation sites, and type of glycosylation at a particular site have been established. We note that high-mannose substances were rarely found at the N-terminal side of a previously glycosylated complex site. There was a very definite distribution of complex sites about the N-terminal region. Furthermore, secreted glycoproteins usually contained only complex oligosaccharides whereas membrane proteins contained both types. We suggest that the position of the glycosylation site with respect to the N-terminus affects the extent of oligosaccharide processing and subsequent presentation of complex or high-mannose structures in the mature glycoprotein. This review relates glycosylation type to its position in the known sequence of given proteins and discusses these observations in light of known glycosylation processing reactions.
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Glicoproteínas , Sequência de Aminoácidos , Asparagina , Fenômenos Químicos , Química , Manose , Proteínas de Membrana , Oligossacarídeos , Conformação Proteica , SolubilidadeRESUMO
Rotavirus VP7 is a membrane-associated protein of the endoplasmic reticulum (ER). It is the product of rotavirus gene 9 which potentially encodes a protein of 326 amino acids that contains two amino terminal hydrophobic domains, h1 and h2, each preceded by an initiation codon. Comparison of the size of products derived from altered genes containing coding sequences for both h1 and h2 with those lacking the h1 sequence ('dhl' mutants), indicates that initiation takes place at M30 immediately preceding h2 (residues F32 to L48) and that h2 is cleaved, confirming the studies of others (Stirzaker, S.C., P.L. Whitfeld, D.L. Christie, A.R. Bellamy, and G.W. Both. 1987. J. Cell Biol. 105:2897-2903). Our previous work had shown that deletions in the carboxy end of h2, extending to amino acid 61 in the open reading frame, resulted in secretion of VP7. The region from amino acid number 51-61, present in wild-type VP7 but missing in the secreted mutant delta 47-61, was thus implicated to have a role in ER retention. To test this, a series of chimeric genes were constructed by fusing the first 63 codons of wild-type VP7, delta 1-14 or delta 51-61/dhl, to the mouse salivary alpha-amylase gene, a secretory protein, such that the fusion junction was located at the exact mature terminus of amylase. The chimeric proteins VP7(63)/amylase, delta 1-14(63)/amylase and delta 51-61(63)/dhl/amylase were secreted when expressed in cells and the h2 domain was cleaved when mRNA was translated in vitro. These results imply that the sequence 51-61 is necessary but not sufficient for ER retention. When a second series of VP7/amylase chimera were constructed extending the VP7 contribution to amino acid 111, the product expressed by delta 1-14(111)/amylase was not secreted whereas that of delta 47-61(111)/amylase was. Significantly, the intracellular delta 1-14(111)/amylase product exhibited an amylase enzymatic specific activity that was similar to that of the wild-type amylase product. We conclude that two regions of VP7 mediate its retention in the ER, the first lies within the sequence 51-61 and the second within the sequence 62-111, which contains the glycosylation site for VP7. Both regions are necessary for retention, though neither is sufficient alone.
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Retículo Endoplasmático/microbiologia , Proteínas de Membrana/metabolismo , Rotavirus/fisiologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Aminoácidos/fisiologia , Animais , Sequência de Bases , Transporte Biológico , Clonagem Molecular , DNA Viral , Retículo Endoplasmático/metabolismo , Vetores Genéticos , Técnicas In Vitro , Cinética , Proteínas de Membrana/genética , Metionina , Dados de Sequência Molecular , Mutação , Plasmídeos , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Rotavirus/genética , Transfecção , Tunicamicina/farmacologia , Proteínas Virais/genética , alfa-Amilases/genética , alfa-Amilases/metabolismoRESUMO
The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Ma104 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins. VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a time-dependent manner. For VP7, Man8GlcNAc2 and Man6GlcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man8GlcNAc2. VP7 on mature virus was processed to Man5GlcNAc2. It is suggested that the alpha-mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man8GlcNAc2. After a 20-min chase of [3H]mannose-labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins.
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Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Rotavirus/metabolismo , Proteínas Virais/metabolismo , Animais , Transporte Biológico , Capsídeo/metabolismo , Metabolismo dos Carboidratos , Sequência de Carboidratos , Linhagem Celular , Cães , Macaca mulatta , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Coelhos , Proteínas Estruturais ViraisRESUMO
Membrane bound polysomes were prepared from HeLa cells infected with vesicular stomatitis virus (VSV), after pulse labeling with [3H]mannose for various times from 15 to 90 min. Oligosaccharides on nascent chains were released from peptides by treatment with endoglycosidase H and sized by high resolution Biogel P4 chromatography. Processing on some nascent chains proceeded to the removal of all three types of alpha-linked glucose and one alpha-1,2-mannose from the Glc3Man9GlcNAc precursor showing that the enzymes responsible were not only active on nascent chains but were present in the rough endoplasmic reticulum (RER). Incubation of cells for various times in cycloheximide, where chain elongation had ceased, made no difference to the profile of oligosaccharides on the nascent chains, and trimming proceeded no further than Man8GlcNAc2Asn . Carbonyl cyanide m-chlorophenylhydrazone (CCCP), an energy inhibitor reportedly able to block the transfer of glycoproteins from the RER, increases the amount of Man8-oligosaccharides on the nascent chains and also the amount of Glc3Man9GlcNAc precursor. On completed G protein in the RER fraction from which membrane bound polysomes were prepared, processing occurred to Man6 - but not to Man5GlcNAc sized oligosaccharides in the CCCP-treated cells. By contrast, processing to Man5GlcNAc oligosaccharides was observed in unfractionated control cells.
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Glucose/metabolismo , Manose/metabolismo , Glicoproteínas de Membrana , Biossíntese de Proteínas , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral , Proteínas Virais/genética , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Transformação Celular Viral , Cromatografia em Gel , Células HeLa/metabolismo , Humanos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Virais/isolamento & purificaçãoRESUMO
Maturation of rotavirus occurs in the ER. The virus transiently acquires an ER-derived membrane surrounding the virus particle before the eventual formation of double-shelled particles. The maturation process includes the retention and selective loss of specific viral protein(s) as well as the ER-derived membrane during formation of the outer capsid of the mature virus. When infected cells were depleted of Ca++ by use of the ionophore A23187 in calcium-free medium, membrane-enveloped intermediates were seen to accumulate. When Mn++, an efficient Ca++ competitor, was used to replace Ca++ in the medium, the accumulation of the enveloped intermediate was again observed, pointing to an absolute requirement of Ca++ in the maturation process. It was previously demonstrated in this laboratory that a hetero-oligomeric complex of NS28, VP7, and VP4 exists which may participate in the budding of the single-shelled particle into the ER (Maass, D. R., and P. H. Atkinson, 1990. J. Virol. 64:2632-2641). The present study demonstrates that either in the absence of Ca++ or in the presence of tunicamycin, a glycosylation inhibitor, VP7 is excluded from these hetero-oligomers. In the presence of Mn++, VP4 was blocked in forming a hetero-oligomeric complex with NS28 and VP7. The electrophoretic mobility of the viral glycoproteins synthesized in the presence of the ionophore were found to be altered. This size difference was attributed to altered N-linked glycosylation and carbohydrate processing of the viral glycoproteins. These results imply a major role for calcium and the state of glycosylation of NS28 in the assembly and acquisition of specific viral protein conformations necessary for the correct association of proteins during virus maturation in the ER.
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Calcimicina/farmacologia , Cálcio/fisiologia , Ácido Egtázico/farmacologia , Retículo Endoplasmático/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Rotavirus/fisiologia , Tunicamicina/farmacologia , Proteínas Virais/genética , Animais , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Substâncias Macromoleculares , Microscopia Eletrônica , Rotavirus/efeitos dos fármacos , Rotavirus/genética , Rotavirus/ultraestrutura , Proteínas Virais/biossíntese , Proteínas Virais/isolamento & purificaçãoRESUMO
A plasma membrane fraction of HeLa S3 cells, consisting of ghosts, is characterized more fully. A simple procedure is described which permits light and electron microscope study of the plasma membrane fraction through the entire depth of the final product pellet and through large areas parallel to the surface. Contamination by nuclei is 0.14%, too little for DNA detection by the diphenylamine reaction. Contamination by rough endoplasmic reticulum and ribosomes is small, a single ghost containing about 3% of the RNA in a single cell. Mitochondria were not encountered. Electron microscopy also shows (a) small vesicles associated with the outer surface of the ghosts, and (b) a filamentous web at the inner face of the ghost membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis shows that of the many Coomassie Blue-stained bands two were prominent. One, 43,000 daltons, co-migrated with purified rabbit muscle actin and constituted about 7.5% of the plasma membrane protein. The other major band, 34,000 daltons, was concentrated in the plasma membrane fraction. Two major glycoproteins detected by autoradiography of [14C]fucose-labeled glycoproteins on the gels, had apparent molecular weights of 35,000 daltons and 32,000 daltons. These major bands did not stain with Coomassie Blue. There were many other minor glycoprotein bands in the 200,000- to 80,000-dalton range. Ouabain-sensitive, Na+, K+-adenosine triphosphatase (ATPase) activity of the ghost fraction is purified 9.1 (+/- 2.2) times over the homogenate; recover of the activity is 12.0 (+/- 3.8%) of the homogenate. Enrichment and recovery of fucosylglycoprotein parallel those for ouabain-sensitive Na+, K+-ATPase activity. Fucosyl glycoprotein is recovered more than the enzyme activity in a smooth membrane vesicle fraction probably containing the bulk of plasma membrane not recovered as ghosts.
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Membrana Celular/ultraestrutura , Células HeLa/ultraestrutura , Actinas/análise , Adenosina Trifosfatases/análise , DNA de Neoplasias/análise , Glicoproteínas/análise , Proteínas de Membrana/análise , Peso Molecular , Miosinas/análise , Proteínas de Neoplasias/análise , RNA Neoplásico/análise , Frações SubcelularesRESUMO
Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.