RESUMO
The United States is facing an unprecedented epidemic of opioid addiction and death due to opioid overdose. In an effort to improve patient knowledge and safe use about opioids, an Electronic Medication Complete Communication (EMC2) opioid strategy was developed targeting opioid naïve patients in the Emergency Department (ED). We conducted pre and post sociotechnical systems analyses to evaluate the variance between the process before the intervention and whether or not the process changed as expected with the new intervention. Results were analyzed using thematic qualitative analysis. Sociotechnical systems modeling illustrates the complexity of designing interventions for emergency medicine that affect multiple patients, providers, work systems, technologies, and processes. The post work systems model illustrates that several elements in the external ED environment can affect the effectiveness of the intervention. Sociotechnical systems analysis is an effective tool to illustrate the opportunities for designing health system interventions and evaluating the fidelity of such interventions.
Assuntos
Analgésicos Opioides , Medicina de Emergência , Serviço Hospitalar de Emergência , Humanos , Padrões de Prática Médica , Análise de Sistemas , Estados UnidosRESUMO
OBJECTIVE: The aim of this study was to determine the frequency and nature of physician, nurse, and pharmacist verbal counseling at the time of a new prescription for an opioid-acetaminophen containing medication as recalled by patients. DESIGN: A mixed methods approach with data from cross sectional, structured interviews was used. SETTING: The settings were one academic emergency department in Chicago, IL and one outpatient pharmacy at a public hospital in Atlanta, GA. PATIENTS: One hundred forty-nine patients receiving a new prescription for an opioid-acetaminophen medication were enrolled. METHODS: Interviews assessed patient recall of counseling they received from their physician, nurse, and pharmacist upon receiving the new prescription. Their responses were unitized and assigned to categories. RESULTS: One hundred forty-nine patients were enrolled; 61.1% African American and 58.4% female. Seven major categories of responses were noted; frequencies of patient recall for counseling in these categories were reported. Four categories related to the content of the counseling discussion were (1) details of administration (patient recall counseling from: physician/nurse only 44.3%, pharmacist only 5.4%, both providers 12.8%); (2) activities to avoid and side effects (36.2%, 4.7%, 8.7%); (3) medication indication (32.9%, 4%, 4%); and (4) addictive potential (9.3%, 1.3%, 0%). Three categories describe patients' recall of the interaction in broad terms: (5) being referred to print informational material accompanying the prescription (MD/RN only 7.4%, pharmacist only 20.1%, both providers 2.7%); (6) having questions solicited (0%, 11.4%, 0%); (7) having no interaction relating to medication counseling (3.4%, 32.2%, 1.3%). CONCLUSIONS: Patients infrequently recall counseling from providers on topics that are important to prevent harm from opioid-acetaminophen prescriptions. Future patient-centered clinical research should target identifying optimal strategies to convey these critical messages.
Assuntos
Acetaminofen/efeitos adversos , Analgésicos Opioides/efeitos adversos , Aconselhamento/estatística & dados numéricos , Pessoal de Saúde , Adulto , Estudos Transversais , Combinação de Medicamentos , Feminino , Humanos , Masculino , Rememoração Mental , Pessoa de Meia-Idade , Transtornos Relacionados ao Uso de Opioides/prevenção & controle , Dor/tratamento farmacológico , PrescriçõesRESUMO
LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1)O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1)O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1)O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1)O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of (1)O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.
Assuntos
Antivirais/farmacologia , Oxazóis/farmacologia , Rodanina/análogos & derivados , Vírus da Febre do Vale do Rift/efeitos dos fármacos , Tionas/farmacologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/química , Disponibilidade Biológica , Membrana Celular/efeitos dos fármacos , Membrana Celular/virologia , Camundongos , Camundongos Endogâmicos BALB C , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Rodanina/farmacologia , Febre do Vale de Rift/tratamento farmacológico , Oxigênio Singlete/química , Relação Estrutura-AtividadeRESUMO
Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M) protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV) is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4) pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS) and the leucine-rich nuclear export signal (NES) found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors) resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50) of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Infecções por Henipavirus/virologia , Vírus Nipah/patogenicidade , Ubiquitina/metabolismo , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Ácidos Borônicos/farmacologia , Bortezomib , Núcleo Celular/efeitos dos fármacos , Chlorocebus aethiops , Citoplasma/efeitos dos fármacos , Imunofluorescência , Células HeLa , Infecções por Henipavirus/genética , Infecções por Henipavirus/metabolismo , Humanos , Imunoprecipitação , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Dados de Sequência Molecular , Mutação/genética , Sinais de Localização Nuclear , Inibidores de Proteases/farmacologia , Processamento de Proteína Pós-Traducional , Transporte Proteico , Pirazinas/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Células Vero , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/genética , Montagem de Vírus/efeitos dos fármacos , Eliminação de Partículas ViraisRESUMO
We describe an antiviral small molecule, LJ001, effective against numerous enveloped viruses including Influenza A, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses, and HIV-1. In sharp contrast, the compound had no effect on the infection of nonenveloped viruses. In vitro and in vivo assays showed no overt toxicity. LJ001 specifically intercalated into viral membranes, irreversibly inactivated virions while leaving functionally intact envelope proteins, and inhibited viral entry at a step after virus binding but before virus-cell fusion. LJ001 pretreatment also prevented virus-induced mortality from Ebola and Rift Valley fever viruses. Structure-activity relationship analyses of LJ001, a rhodanine derivative, implicated both the polar and nonpolar ends of LJ001 in its antiviral activity. LJ001 specifically inhibited virus-cell but not cell-cell fusion, and further studies with lipid biosynthesis inhibitors indicated that LJ001 exploits the therapeutic window that exists between static viral membranes and biogenic cellular membranes with reparative capacity. In sum, our data reveal a class of broad-spectrum antivirals effective against enveloped viruses that target the viral lipid membrane and compromises its ability to mediate virus-cell fusion.
Assuntos
Antivirais/farmacologia , Rodanina/análogos & derivados , Viroses/tratamento farmacológico , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Rodanina/química , Rodanina/farmacologia , Rodanina/uso terapêutico , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismoRESUMO
Nipah virus (NiV) and Hendra virus (HeV) are the only paramyxoviruses requiring Biosafety Level 4 (BSL-4) containment. Thus, study of henipavirus entry at less than BSL-4 conditions necessitates the use of cell-cell fusion or pseudotyped reporter virus assays. Yet, these surrogate assays may not fully emulate the biological properties unique to the virus being studied. Thus, we developed a henipaviral entry assay based on a beta-lactamase-Nipah Matrix (betala-M) fusion protein. We first codon-optimized the bacterial betala and the NiV-M genes to ensure efficient expression in mammalian cells. The betala-M construct was able to bud and form virus-like particles (VLPs) that morphologically resembled paramyxoviruses. betala-M efficiently incorporated both NiV and HeV fusion and attachment glycoproteins. Entry of these VLPs was detected by cytosolic delivery of betala-M, resulting in enzymatic and fluorescent conversion of the pre-loaded CCF2-AM substrate. Soluble henipavirus receptors (ephrinB2) or antibodies against the F and/or G proteins blocked VLP entry. Additionally, a Y105W mutation engineered into the catalytic site of betala increased the sensitivity of our betala-M based infection assays by 2-fold. In toto, these methods will provide a more biologically relevant assay for studying henipavirus entry at less than BSL-4 conditions.
Assuntos
Fluoresceínas/metabolismo , Henipavirus/fisiologia , Lactamas/metabolismo , Proteínas da Matriz Viral/metabolismo , Virossomos/metabolismo , Internalização do Vírus , beta-Lactamases/metabolismo , Animais , Contenção de Riscos Biológicos/economia , Contenção de Riscos Biológicos/métodos , Genes Reporter , Proteínas Recombinantes de Fusão/genética , Proteínas da Matriz Viral/genética , Virossomos/genética , beta-Lactamases/genéticaRESUMO
Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes several days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.
Assuntos
Anticorpos Antivirais/sangue , Vírus Hendra/imunologia , Infecções por Henipavirus/imunologia , Testes de Neutralização/métodos , Vírus Nipah/imunologia , Virologia/métodos , Animais , Antígenos Virais , Humanos , Testes de Neutralização/economia , Vesiculovirus/genética , Proteínas Virais , Virologia/economiaRESUMO
Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.
Assuntos
Fusão de Membrana/fisiologia , Vírus Nipah/fisiologia , Polissacarídeos/fisiologia , Proteínas do Envelope Viral/fisiologia , Proteínas Virais de Fusão/fisiologia , Animais , Linhagem Celular , Cães , Glicosilação , Células HeLa , Infecções por Henipavirus/prevenção & controle , Infecções por Henipavirus/virologia , Humanos , Fusão de Membrana/genética , Testes de Neutralização , Vírus Nipah/genética , Vírus Nipah/patogenicidade , Polissacarídeos/genética , Polissacarídeos/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/biossíntese , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismoRESUMO
EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 10(-4) s(-1) for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.