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1.
J Infect Dis ; 221(Suppl 1): S109-S112, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32134476

RESUMO

Cytomegalovirus (CMV) remains an important pathogen in the transplant population. As such, the US Food and Drug Administration has published a guidance to encourage and inform the development of therapeutics for the treatment and prevention of CMV disease in this population. This review summarizes important phase 3 trial design considerations for industry and provides rationale for some of the recommendations included in the guidance.


Assuntos
Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/prevenção & controle , Citomegalovirus , Transplante de Órgãos/efeitos adversos , Antivirais/farmacologia , Antivirais/uso terapêutico , Ensaios Clínicos Fase III como Assunto , Citomegalovirus/imunologia , Infecções por Citomegalovirus/etiologia , Humanos , Hospedeiro Imunocomprometido , Transplante de Órgãos/métodos , Projetos de Pesquisa , Resultado do Tratamento , Carga Viral
2.
Mol Cell Proteomics ; 15(6): 2186-202, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27099342

RESUMO

Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.


Assuntos
Proteínas de Bactérias/metabolismo , Desulfovibrio vulgaris/metabolismo , Escherichia coli/metabolismo , Proteômica/métodos , Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas
3.
Mol Cell Proteomics ; 15(5): 1539-55, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26873250

RESUMO

Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.


Assuntos
Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Desulfovibrio vulgaris/metabolismo , Escherichia coli/metabolismo , Cromatografia de Afinidade , Bases de Dados de Proteínas , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Técnicas do Sistema de Duplo-Híbrido
4.
Int J Clin Pharmacol Ther ; 52(6): 446-53, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24755134

RESUMO

OBJECTIVES: Literature reports regarding the efficacy of efavirenz (EFV) 600 mg with rifampin (RIF) are not consistent. Evaluation of a drug-drug interaction (DDI) study and supportive semi-mechanistic population pharmacokinetic (PK) analyses were undertaken to help delineate this issue. DESIGN/METHODS: DDI study and supportive semi-mechanistic population PK analyses were provided by BMS. Population PK analysis was based on six studies with intensive EFV PK sampling. An ACTG study with sparse PK sampling was used for model evaluation. Simulations compared EFV exposure at various doses in combination with RIF to EFV exposures at 600 mg once daily (QD). Effects of CYP2B6 genotypes on the magnitude of EFV-RIF interaction were also explored. RESULTS: In DDI study, co-administering EFV 600 mg QD and RIF reduced mean EFV exposure by ~ 30%. Population PK model provided acceptable predictive performance of central tendency and variability for EFV C0, Cmax, and AUC. Simulations predicted that increasing EFV to 800 mg QD with RIF would result in EFV AUC and Cmax similar to EFV 600 mg QD alone. EFV AUC and Cmax were ~ 2 times higher in subjects with reduced function CYP2B6 genotypes. However, the RIF effect was consistent across all genotypes. EFV dose adjustment to 800 mg QD did not increase the risk of overexposure compared to 600 mg EFV QD within each genotype. CONCLUSION: Dose adjustment based on matching systemic exposure was recommended to mitigate the potential for sub-therapeutic EFV exposures. Our review did not reveal any safety concerns in subjects receiving EFV 800 mg QD with RIF.


Assuntos
Antibióticos Antituberculose/administração & dosagem , Benzoxazinas/administração & dosagem , Aprovação de Drogas , Infecções por HIV/tratamento farmacológico , Inibidores da Transcriptase Reversa/administração & dosagem , Rifampina/administração & dosagem , Tuberculose/tratamento farmacológico , United States Food and Drug Administration , Alcinos , Antibióticos Antituberculose/efeitos adversos , Área Sob a Curva , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Benzoxazinas/efeitos adversos , Benzoxazinas/farmacocinética , Coinfecção , Simulação por Computador , Ciclopropanos , Citocromo P-450 CYP2B6 , Esquema de Medicação , Cálculos da Dosagem de Medicamento , Interações Medicamentosas , Genótipo , Infecções por HIV/diagnóstico , Infecções por HIV/metabolismo , Humanos , Modelos Biológicos , Fenótipo , Polimedicação , Inibidores da Transcriptase Reversa/efeitos adversos , Inibidores da Transcriptase Reversa/farmacocinética , Rifampina/efeitos adversos , Tuberculose/diagnóstico , Tuberculose/metabolismo , Estados Unidos
5.
J Proteome Res ; 11(12): 5720-35, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23098413

RESUMO

Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.


Assuntos
Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Desulfovibrio vulgaris/química , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Membrana/isolamento & purificação , Complexos Multiproteicos/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/química , Membrana Celular/química , Cromatografia por Troca Iônica , Desulfovibrio vulgaris/enzimologia , Detergentes/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Espectrometria de Massas , Proteínas de Membrana/química , Peso Molecular , Complexos Multiproteicos/química , Periplasma/química , Periplasma/enzimologia , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteoma/química , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Solubilidade
6.
Appl Environ Microbiol ; 77(18): 6313-22, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21764955

RESUMO

Environmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.


Assuntos
Biodiversidade , Microbiologia Ambiental , Metagenômica/métodos , Análise em Microsséries/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , DNA Complementar/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade
8.
Antiviral Res ; 169: 104549, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31279814

RESUMO

Letermovir is a human cytomegalovirus (HCMV) terminase inhibitor recently approved in the United States for prophylaxis of HCMV infection or disease in adult HCMV-seropositive recipients [R+] of an allogeneic hematopoietic stem cell transplant. In the registrational trial, the rate of clinically significant HCMV infection, defined as the development of HCMV DNAemia leading to preemptive antiviral therapy or the diagnosis of HCMV end-organ disease, through 24 weeks post-transplant, was significantly lower among subjects who received letermovir prophylaxis through 14 weeks post-transplant compared to those who received placebo. We performed independent analyses of the HCMV nucleotide sequencing data generated by next-generation sequencing from this phase 3 registrational trial of letermovir to identify viral genetic characteristics associated with virologic failure during and following letermovir prophylaxis. The pUL56 substitutions V236M, E237G, and C325W, identified at previously known resistance-associated positions, were detected in the virus of subjects who were treated with letermovir and failed letermovir prophylaxis. Several additional substitutions were detected in pUL56 and pUL89, and further characterization is needed to determine if any of these substitutions are clinically relevant. The analyses reported herein were conducted to confirm sponsor-reported drug-resistance pathways, to assess the frequency of resistance, and to better understand the risk of prophylaxis failures and treatment-emergent drug resistance.


Assuntos
Citomegalovirus/genética , Farmacorresistência Viral/genética , Genômica , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Acetatos/farmacologia , Substituição de Aminoácidos , Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Endodesoxirribonucleases/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Quinazolinas/farmacologia , Transplante de Células-Tronco
9.
J Clin Pharmacol ; 59(6): 784-798, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30586161

RESUMO

This review summarizes the significant impact of cytomegalovirus (CMV) infection on solid organ and hematopoietic stem cell transplant recipients. A discussion of the various CMV prevention and treatment strategies is provided, including a detailed description of each of the available CMV antiviral drugs.


Assuntos
Antivirais/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/prevenção & controle , Transplantados , Antivirais/farmacocinética , Infecções por Citomegalovirus/diagnóstico , Farmacorresistência Viral , Drogas em Investigação/uso terapêutico , Previsões , Humanos
10.
J Microbiol Methods ; 86(2): 204-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21605602

RESUMO

Immunomagnetic separation (IMS) has proved highly efficient for recovering microorganisms from heterogeneous samples. Current investigation targeted the separation of viable cells of the sulfate-reducing bacterium, Desulfovibrio vulgaris. Streptavidin-coupled paramagnetic beads and biotin labeled antibodies raised against surface antigens of this microorganism were used to capture D. vulgaris cells in both bioreactor grown laboratory samples and from extremely low-biomass environmental soil and subsurface drilling samples. Initial studies on detection, recovery efficiency and viability for IMS were performed with laboratory grown D. vulgaris cells using various cell densities. Efficiency of cell isolation and recovery (i.e., release of the microbial cells from the beads following separation) was followed by microscopic imaging and acridine orange direct counts (AODC). Excellent recovery efficiency encouraged the use of IMS to capture Desulfovibrio spp. cells from low-biomass environmental samples. The environmental samples were obtained from a radionuclide-contaminated site in Germany and the chromium (VI)-contaminated Hanford site, an ongoing bioremediation project of the U.S. Department of Energy. Field deployable IMS technology may greatly facilitate environmental sampling and bioremediation process monitoring and enable transcriptomics and proteomics/metabolomics-based studies directly on cells collected from the field.


Assuntos
Técnicas Bacteriológicas/métodos , Desulfovibrio vulgaris/isolamento & purificação , Microbiologia Ambiental , Separação Imunomagnética/métodos , Anticorpos Antibacterianos/imunologia , Desulfovibrio vulgaris/imunologia , Alemanha , Viabilidade Microbiana , Sensibilidade e Especificidade , Estados Unidos
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