RESUMO
In 2013, we adopted MALDI-TOF MS using the Bruker Biotyper system for identification of anaerobic bacteria into our routine clinical practice. Here, we describe our experience with the use of MALDI-TOF MS for anaerobic bacterial identification, highlighting its value in replacing the more costly and time-consuming 16S ribosomal RNA gene PCR plus sequencing-based approach as the primary method of anaerobic bacterial identification. We also describe our more recent experience with the use of early/rapid MALDI-TOF MS for identification of anaerobic bacteria performed on short incubation (4-6â¯h) plated aerobic media from anaerobic blood culture bottles positive for Gram-negative bacilli.
Assuntos
Bactérias Anaeróbias/isolamento & purificação , Infecções Bacterianas/microbiologia , Técnicas de Tipagem Bacteriana/métodos , Testes Diagnósticos de Rotina/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Bactérias Anaeróbias/química , Bactérias Anaeróbias/classificação , Bactérias Anaeróbias/genética , Infecções Bacterianas/diagnóstico , DNA Bacteriano/genética , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genéticaRESUMO
The human cytomegalovirus tegument protein UL69 has been shown to be required for efficient viral replication at low multiplicities of infection. Several functions have been associated with UL69, including its ability to regulate cell cycle progression, translation, and the export of viral transcripts from the nucleus to the cytoplasm. However, it remains unclear which, if any, of these activities contribute to the phenotype observed with the UL69 deletion mutant. UL69 has been shown to interact with the cellular protein SPT6. The functional significance of this interaction has never been examined in the context of an infection. To address this, we generated UL69 mutant viruses that were unable to interact with SPT6 and determined what effect these mutations had on virus replication. Abolishing UL69's ability to interact with the SPT6 protein inhibited virus replication to levels indistinguishable from those observed following infection with the UL69 deletion mutant. Surprisingly, abolishing UL69's interaction with SPT6 also resulted in the impairment of UL69 shuttling activity. Finally, we demonstrate that inhibition of SPT6 expression by short hairpin RNA (shRNA) knockdown inhibits wild-type virus replication. Taken together, our results demonstrate that UL69's ability to interact with SPT6 plays a critical role in viral replication.
Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Fatores de Transcrição/metabolismo , Replicação Viral , Linhagem Celular , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Regulação Viral da Expressão Gênica , Humanos , Ligação Proteica , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
The human cytomegalovirus (HCMV) tegument protein UL69 is important for efficient viral replication at low multiplicities of infection. Several molecular mechanisms by which UL69 contributes to HCMV replication have been proposed, including UL69's ability to interact with the mRNA export factors UAP56 and URH49 to facilitate the shuttling of viral mRNAs from the nuclei of infected cells. Using a UL69 viral mutant that is unable to bind UAP56 and URH49, we demonstrated that UL69's interaction with UAP56 or URH49 does not contribute to the growth phenotype associated with the UL69 deletion mutant.
Assuntos
Citomegalovirus/fisiologia , RNA Helicases DEAD-box/metabolismo , Interações Hospedeiro-Patógeno , Transativadores/metabolismo , Replicação Viral , Animais , Linhagem Celular , Deleção de Genes , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Ligação Proteica , Transativadores/genéticaRESUMO
Caloric restriction enhances N-methyl-D-aspartate (NMDA) receptor binding and upregulates messenger RNA expression of the GluN1 subunit during aging. Old growth hormone receptor knockout mice resemble old calorically restricted rodents in enhanced life span and brain function, as compared with aged controls. This study examined whether aged growth hormone receptor knockout mice also show enhanced expression of NMDA receptors. Six or 23- to 24-month-old male normal-sized control or dwarf growth hormone receptor knockout mice were assayed for NMDA-displaceable [(3)H]glutamate binding (autoradiography) and GluN1 subunit messenger RNA (in situ hybridization). There was slight sparing of NMDA receptor binding densities within aged medial prefrontal and motor cortices, similar to caloric restriction, but there were greater age-related declines in GluN1 messenger RNA in growth hormone receptor knockout versus control mice. These results suggest that some of the functional improvements in aged mice with altered growth hormone signaling may be due to enhancement of NMDA receptors, but not through the upregulation of messenger RNA for the GluN1 subunit.