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The gut-derived peptide hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important physiological roles. Stabilized agonists of the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) for the management of diabetes and obesity have generated widespread enthusiasm and have become blockbuster drugs. These therapeutics are refractory to the action of dipeptidyl peptidase-4 (DPP4), that catalyzes rapid removal of the two N-terminal residues of the native peptides, in turn severely diminishing their activity profiles. Here we report that a single atom change from carbon to nitrogen in the backbone of the entire peptide make them refractory to DPP4 action while still retaining full potency and efficacy at their respective receptors. This was accomplished by use of aza-amino acids, that are bioisosteric replacements for a-amino acids that perturb the structural backbone and local side chain conformations. Molecular dynamics simulations reveal that aza-amino acid can populate the same conformational space that GLP-1 adopts when bound to the GLP-1R. The insertion of an aza-amino acid at the second position from the N-terminus in semaglutide and in a dual agonist of GLP-1R and GIPR further demonstrates its capability as a viable alternative to current DPP4 resistance strategies while offering additional structural variety.
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Bacterial biofilms are a significant concern in various medical contexts due to their resilience to our immune system as well as antibiotic therapy. Biofilms often require surgical removal and frequently lead to recurrent or chronic infections. Therefore, there is an urgent need for improved strategies to treat biofilm infections. Ultrasound-mediated drug delivery is a technique that combines ultrasound application, often with the administration of acoustically-active agents, to enhance drug delivery to specific target tissues or cells within the body. This method involves using ultrasound waves to assist in the transportation or activation of medications, improving their penetration, distribution, and efficacy at the desired site. The advantages of ultrasound-mediated drug delivery include targeted and localized delivery, reduced systemic side effects, and improved efficacy of the drug at lower doses. This review scrutinizes recent advances in the application of ultrasound-mediated drug delivery for treating biofilm infections, focusing on in vivo studies. We examine the strengths and limitations of this technology in the context of wound infections, device-associated infections, lung infections and abscesses, and discuss current gaps in knowledge and clinical translation considerations.
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The Selux Next-Generation Phenotyping (NGP) system (Charlestown, MA) is a new antimicrobial susceptibility testing system that utilizes two sequential assays performed on all wells of doubling dilution series to determine MICs. A multicenter evaluation of the performance of the Selux NGP system compared with reference broth microdilution was conducted following FDA recommendations and using FDA-defined breakpoints. A total of 2,488 clinical and challenge isolates were included; gram-negative isolates were tested against 24 antimicrobials, and gram-positive isolates were tested against 15 antimicrobials. Data is provided for all organism-antimicrobial combinations evaluated, including those that did and did not meet FDA performance requirements. Overall very major error and major error rates were less than 1% (31/3,805 and 107/15,606, respectively), essential agreement and categorical agreement were >95%, reproducibility was ≥95%, and the average time-to-result (from time of assay start to time of MIC result) was 5.65 hours.
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Antibacterianos , Antiinfecciosos , Humanos , Antibacterianos/farmacología , Reproducibilidad de los Resultados , Pruebas de Sensibilidad MicrobianaRESUMEN
Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cß3 (PLCß3), with a possible contribution of PLCß2, whereas signaling through PLCß4 interferes with synergy. We here show that synergy can be induced by the combination of Gßγ and Gα(q) activation of a single PLCß isoform. Synergy was absent in macrophages lacking both PLCß2 and PLCß3, but it was fully reconstituted following transduction with PLCß3 alone. Mechanisms of PLCß-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCß2. RNAi-mediated knockdown of endogenous PLCßs demonstrated that synergy in these cells was dependent on PLCß3, but PLCß1 and PLCß4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCß3, it could be reconstituted by expression of either human PLCß3 or mouse PLCß2. In contrast, it could not be reconstituted by human PLCß3 with a mutation of the Y box, which disrupted activation by Gßγ, and it was only partially restored by human PLCß3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gßγ and Gα(q) contribute to activation of PLCß3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gßγ and Gα(q) on PLCß and is mediated primarily by PLCß3, although PLCß2 is also competent.
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Señalización del Calcio/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Fosfolipasa C beta/metabolismo , Animales , Complemento C5a/metabolismo , Humanos , Macrófagos/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Mutagénesis , Células 3T3 NIH , Fosfolipasa C beta/genética , ARN Interferente Pequeño , Receptores Purinérgicos P2/metabolismo , Uridina Difosfato/metabolismoRESUMEN
The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E(2), that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase-anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor-alpha, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor kappaB kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates.
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Proteínas de Anclaje a la Quinasa A/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , AMP Cíclico/inmunología , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Subunidad p50 de NF-kappa B/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Línea Celular , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dinoprostona/genética , Dinoprostona/inmunología , Dinoprostona/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Macrófagos/metabolismo , Ratones , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Interferencia de ARN , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Cellular signal transduction machinery integrates information from multiple inputs to actuate discrete cellular behaviors. Interaction complexity exists when an input modulates the output behavior that results from other inputs. To address whether this machinery is iteratively complex--that is, whether increasing numbers of inputs produce exponential increases in discrete cellular behaviors--we examined the modulated secretion of six cytokines from macrophages in response to up to five-way combinations of an agonist of Toll-like receptor 4, three cytokines, and conditions that activated the cyclic adenosine monophosphate pathway. Although all of the selected ligands showed synergy in paired combinations, few examples of nonadditive outputs were found in response to higher-order combinations. This suggests that most potential interactions are not realized and that unique cellular responses are limited to discrete subsets of ligands and pathways that enhance specific cellular functions.
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Citocinas/metabolismo , Macrófagos/metabolismo , Transducción de Señal/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Línea Celular , Quimiocina CCL3/metabolismo , Quimiocina CCL5/metabolismo , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Interferón beta/farmacología , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Interleucina-6/farmacología , Isoproterenol/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Azúcares Ácidos/farmacología , Factores de Tiempo , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems. RESULTS: We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription. CONCLUSION: We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.
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Silenciador del Gen , MicroARNs , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Línea Celular , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Vectores Genéticos , Humanos , Isoenzimas/metabolismo , Riñón/citología , Lentivirus/genética , Macrófagos/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Plásmidos , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , Recombinación Genética , Retroviridae/genética , Transcripción Genética , TransfecciónRESUMEN
The use of RNA interference to knock down protein phosphatases has proven to be a valuable approach to understanding the functions of these enzymes in mammalian cells. Many protein phosphatases exist as multisubunit and multigene families, which has made it difficult to assess their physiological functions using traditional approaches. The ability to selectively knock down specific subunits and individual isoforms with RNA interference has begun to make it possible to determine the contributions of individual phosphatase proteins to cellular signaling. This chapter describes methods for knocking down protein phosphatases with small interfering RNAs in easily transfectable cells and by the introduction of short-hairpin RNAs into less tractable cells using lentivirus vectors.
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Fosfoproteínas Fosfatasas/metabolismo , Interferencia de ARN , Secuencia de Bases , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos/genética , Células HeLa , Humanos , Lentivirus/genética , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , ARN Interferente Pequeño/genéticaRESUMEN
Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.
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Proteínas Quinasas/metabolismo , Proteómica , Animales , Línea Celular , Clonación Molecular , ADN Complementario/genética , Bases de Datos Factuales , Ratones , Sistemas de Lectura Abierta/genética , Plásmidos , Proteínas Quinasas/genética , Transducción de SeñalRESUMEN
RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.