RESUMEN
We developed a method in which the NS3 cis-protease from hepatitis C virus can be used as a ligand-inducible connection to control the function and localization of engineered proteins in mammalian cells. To demonstrate the versatility of this approach, we designed drug-sensitive transcription factors and transmembrane signaling proteins, the activities of which can be tightly and reversibly controlled through the use of clinically tested antiviral protease inhibitors.
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Antivirales/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Células CHO , Cricetulus , ADN/genética , ADN/metabolismo , Transducción de Señal , Proteínas no Estructurales Virales/metabolismoRESUMEN
EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.
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Química Clic , ADN/análisis , Lípidos/análisis , Microscopía Electrónica/métodos , Peptidoglicano/análisis , ARN/análisis , Aminobutiratos/química , ADN/química , ADN/metabolismo , Colorantes Fluorescentes/química , Células HEK293 , Células HeLa , Humanos , Lípidos/química , Listeria monocytogenes/metabolismo , Estructura Molecular , Neuronas/química , Neuronas/metabolismo , Peptidoglicano/biosíntesis , ARN/química , ARN/metabolismo , Oxígeno Singlete/químicaRESUMEN
Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.
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Proteínas de Caenorhabditis elegans/biosíntesis , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica/fisiología , Proteoma/biosíntesis , Proteómica/métodos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Marcaje Isotópico/métodos , Mutación , Proteoma/genéticaRESUMEN
Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.
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Aminoácidos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas Microbiológicas , Antibacterianos/química , Células HeLa , Humanos , Espectrometría de Masas , Metionina-ARNt Ligasa/química , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Mutación , Norleucina/química , Proteoma , Proteómica/métodos , Factores de Tiempo , Factores de Virulencia , Yersinia enterocolitica/metabolismoRESUMEN
Synthesizing, localizing, and stabilizing new protein copies at synapses are crucial factors in maintaining the synaptic changes required for storing long-term memories. PKMζ recently emerged as a molecule putatively responsible for maintaining encoded memories over time because its presence correlates with late LTP and because its inhibition disrupts LTP in vitro and long-term memory storage in vivo. Here we investigated PKMζ stability in rat neurons to better understand its role during information encoding and storage. We used TimeSTAMP reporters to track the synthesis and degradation of PKMζ as well as a related atypical PKC, PKCλ. These reporters revealed that both PKMζ and PKCλ were upregulated after chemical LTP induction; however, these new PKMζ copies exhibited more rapid turnover than basally produced PKMζ, particularly in dendritic spines. In contrast to PKMζ, new PKCλ copies exhibited elevated stability. Stable information storage over long periods of time is more challenging the shorter the metabolic lifetime of the candidate molecules.
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Espinas Dendríticas/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Proteolisis , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Espinas Dendríticas/fisiología , Estabilidad de Enzimas , Células HEK293 , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Potenciación a Largo Plazo , Datos de Secuencia Molecular , Proteína Quinasa C/genética , Ratas , Ratas Sprague-Dawley , Sinapsis/fisiología , Regulación hacia ArribaRESUMEN
Newly synthesized cellular proteins can be tagged with a variety of metabolic labels that distinguish them from preexisting proteins and allow them to be identified and tracked. Many such labels are incorporated into proteins via the endogenous cellular machinery and can be used in numerous cell types and organisms. Though broad applicability has advantages, we aimed to develop a strategy to restrict protein labeling to specified mammalian cells that express a transgene. Here we report that heterologous expression of a mutant methionyl-tRNA synthetase from Escherichia coli permits incorporation of azidonorleucine (Anl) into proteins made in mammalian (HEK293) cells. Anl is incorporated site-selectively at N-terminal positions (in competition with initiator methionines) and is not found at internal sites. Site selectivity is enabled by the fact that the bacterial synthetase aminoacylates mammalian initiator tRNA, but not elongator tRNA. N-terminally labeled proteins can be selectively conjugated to a variety of useful probes; here we demonstrate use of this system in enrichment and visualization of proteins made during various stages of the cell cycle. N-terminal incorporation of Anl may also be used to engineer modified proteins for therapeutic and other applications.
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Proteínas de Escherichia coli/biosíntesis , Escherichia coli/enzimología , Leucina/metabolismo , Metionina-ARNt Ligasa/biosíntesis , Iniciación de la Cadena Peptídica Traduccional , ARN de Transferencia de Metionina/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Leucina/análogos & derivados , Leucina/genética , Metionina-ARNt Ligasa/genética , ARN de Transferencia de Metionina/genéticaRESUMEN
INTRODUCTION AND IMPORTANCE: Lunate fractures without associated dislocations are rare injuries, combination scaphoid-lunate fractures without an associated dislocation are even more rare of which few are reported in the literature. CASE PRESENTATION: This case report describes a 16-year-old otherwise healthy male with ipsilateral scaphoid and lunate fractures after punching a goalpost with his left hand, for which he had surgical management. This patient had a successful treatment with two headless compression screws through a single extended carpal tunnel approach. Surgery was performed within two weeks of injury after initially immobilized with a thumb spica splint. The postoperative period was complicated by noncompliance with weight-bearing status and missed three-month followup. However, by six months, subsequent visits demonstrated radiographic and clinical healing as well as full wrist range of motion without any other sequelae. CLINICAL DISCUSSION: This case offers more evidence regarding this rare injury. Lunate fractures and scaphoid fractures can both be treated with open reduction and internal fixation using headless compression screws, however little evidence exists when it comes to treating them in combination. CONCLUSION: The use of headless compression screws through a single extended carpal tunnel approach led to clinical and radiographic healing in a 16 year-old-male with combined scaphoid and lunate fractures at 6 month follow up.
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We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o -nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.
RESUMEN
Cells interpret mechanical stimuli from their environments and neighbors, but the ability to engineer customized mechanosensing capabilities has remained a synthetic and mechanobiology challenge. Here we introduce tension-tuned synthetic Notch (SynNotch) receptors to convert extracellular and intercellular forces into specifiable gene expression changes. By elevating the tension requirements of SynNotch activation, in combination with structure-guided mutagenesis, we designed a set of receptors with mechanical sensitivities spanning the physiologically relevant picoNewton range. Cells expressing these receptors can distinguish between varying tensile forces and respond by enacting customizable transcriptional programs. We applied these tools to design a decision-making circuit, through which fibroblasts differentiate into myoblasts upon stimulation with distinct tension magnitudes. We also characterize cell-generated forces transmitted between cells during Notch signaling. Overall, this work provides insight into how mechanically induced changes in protein structure can be used to transduce physical forces into biochemical signals. The system should facilitate the further programming and dissection of force-related phenomena in biological systems.
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Mecanotransducción Celular , Transducción de Señal , ProteínasRESUMEN
Chemical control of protein activity is a powerful tool for scientific study, synthetic biology, and cell therapy; however, for broad use, effective chemical inducer systems must minimally crosstalk with endogenous processes and exhibit desirable drug delivery properties. Accordingly, the drug-controllable proteolytic activity of hepatitis C cis- protease NS3 and its associated antiviral drugs have been used to regulate protein activity and gene modulation. These tools advantageously exploit non-eukaryotic/prokaryotic proteins and clinically approved inhibitors. Here we expand the toolkit by utilizing catalytically inactive NS3 protease as a high affinity binder to genetically encoded, antiviral peptides. Through our approach, we create NS3-peptide complexes that can be displaced by FDA-approved drugs to modulate transcription, cell signaling, split-protein complementation. With our developed system, we discover a new mechanism to allosterically regulate Cre recombinase. Allosteric Cre regulation with NS3 ligands enables orthogonal recombination tools in eukaryotic cells and functions in divergent organisms to control prokaryotic recombinase activity.
RESUMEN
Chemical control of protein activity is a powerful tool for scientific study, synthetic biology, and cell therapy; however, for broad use, effective chemical inducer systems must minimally crosstalk with endogenous processes and exhibit desirable drug delivery properties. Accordingly, the drug-controllable proteolytic activity of hepatitis C cis-protease NS3 and its associated antiviral drugs have been used to regulate protein activity and gene modulation. These tools advantageously exploit non-eukaryotic and non-prokaryotic proteins and clinically approved inhibitors. Here, we expand the toolkit by utilizing catalytically inactive NS3 protease as a high affinity binder to genetically encoded, antiviral peptides. Through our approach, we create NS3-peptide complexes that can be displaced by FDA-approved drugs to modulate transcription, cell signaling, and split-protein complementation. With our developed system, we invented a new mechanism to allosterically regulate Cre recombinase. Allosteric Cre regulation with NS3 ligands enables orthogonal recombination tools in eukaryotic cells and functions in divergent organisms to control prokaryotic recombinase activity.
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Antivirales , Proteasas Virales , Antivirales/farmacología , Antivirales/química , Hepacivirus , Péptido Hidrolasas , Péptidos/farmacología , Péptidos/química , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAML's catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3'-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.
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Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein's gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a "pulse" in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Noncanonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. In this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology.
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Aminoácidos/metabolismo , Biosíntesis de Proteínas , Proteínas/metabolismo , Alquenos/química , Alquinos/química , Alquinos/metabolismo , Aminoácidos/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Azidas/química , Catálisis , Cobre/química , Colorantes Fluorescentes/química , Glicina/análogos & derivados , Glicina/química , Glicina/metabolismo , Espectrometría de Masas , Microscopía Fluorescente , Mutación , Proteínas/químicaRESUMEN
INTRODUCTION: Flexor pollicis longus (FPL) tendon rupture is a known complication after a distal radius (DR) fracture and subsequent fixation with a volar plate. A commonly accepted theory is the attrition of the flexor tendon by the prominent volar plate or theoretical injury to the tendon during the initial injury. An increasingly rare complication of distal radius open reduction internal fixation (ORIF) with volar plate fixation is stenosing tenosynovitis, more commonly known as trigger finger. PRESENTATION OF CASE: We present a case of FPL rupture 7 years after volar plate fixation for DR fracture with thumb triggering in an elderly patient. To treat her trigger thumb, a corticosteroid injection was administered for symptomatic relief. Without resolution of her symptoms, she was scheduled for hardware removal and A1 pulley release. At her preoperative visit, she was found to have a rupture of her FPL tendon. DISCUSSION/CONCLUSION: This case report provides insight into an atypical presentation of delayed-onset FPL rupture and preceding trigger thumb. Especially in individuals with no inciting events, the patient's trigger thumb after volar plate distal radius ORIF may have been a warning sign for impending FPL rupture. This underscores the importance in considering potential tendon attrition as part of a differential diagnosis in a patient presenting with trigger thumb after distal radius ORIF with a volar plate. In assessing for FPL tendon rupture, diagnostic imaging modalities such as ultrasound may be utilized in evaluating this condition to prevent potential loss of function.
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Chimeric antigen receptor (CAR) T cells can revolutionize cancer medicine. However, overactivation, lack of tumor-specific surface markers, and antigen escape have hampered CAR T cell development. A multi-antigen targeting CAR system regulated by clinically approved pharmaceutical agents is needed. Here, we present VIPER CARs (versatile protease regulatable CARs), a collection of inducible ON and OFF switch CAR circuits engineered with a viral protease domain. We established their controllability using FDA-approved antiviral protease inhibitors in a xenograft tumor and a cytokine release syndrome mouse model. Furthermore, we benchmarked VIPER CARs against other drug-gated systems and demonstrated best-in-class performance. We showed their orthogonality in vivo using the ON VIPER CAR and OFF lenalidomide-CAR systems. Finally, we engineered several VIPER CAR circuits by combining various CAR technologies. Our multiplexed, drug-gated CAR circuits represent the next progression in CAR design capable of advanced logic and regulation for enhancing the safety of CAR T cell therapy.
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Neoplasias , Receptores Quiméricos de Antígenos , Ratones , Animales , Humanos , Receptores Quiméricos de Antígenos/genética , Linfocitos T , Inmunoterapia Adoptiva , Lenalidomida , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Metabolic labeling of proteins with the methionine surrogate azidonorleucine can be targeted exclusively to specified cells through expression of a mutant methionyl-tRNA synthetase (MetRS). In complex cellular mixtures, proteins made in cells that express the mutant synthetase can be tagged with affinity reagents (for detection or enrichment) or fluorescent dyes (for imaging). Proteins made in cells that do not express the mutant synthetase are neither labeled nor detected.
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Marcadores de Afinidad/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Metionina-ARNt Ligasa/metabolismo , Proteínas/metabolismo , Alanina/análogos & derivados , Alanina/metabolismo , Aminoacil-ARNt Sintetasas/genética , Animales , Línea Celular , Técnicas de Cocultivo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiología , Metionina-ARNt Ligasa/genética , Ratones , Mutagénesis Sitio-Dirigida , Mutación , Norleucina/análogos & derivados , Norleucina/metabolismo , Biosíntesis de ProteínasRESUMEN
The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na(2)S(2)O(4), 2% HOCH(2)CH(2)SH, 10% HCO(2)H, 95% CF(3)CO(2)H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO(2)H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.
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Alquinos/química , Azidas/química , Biotina/química , Sondas Moleculares/química , Proteínas/química , Ciclización , Dapsona , Proteínas Fluorescentes Verdes/química , Sondas Moleculares/síntesis química , Estructura Secundaria de ProteínaRESUMEN
The circadian clock, which consists of endogenous self-sustained and cell-autonomous oscillations in mammalian cells, is known to regulate a wide range of peripheral tissues. The unique upregulation of a clock gene, neuronal PAS domain protein 2 (Npas2), observed along with fibroblast aging prompted us to investigate the role of Npas2 in the homeostasis of dermal structure using in vivo and in vitro wound healing models. Time-course healing of a full-thickness skin punched wound exhibited significantly faster wound closure in Npas2-/- mice than wild-type (WT) C57Bl/6J mice. Dorsal skin fibroblasts isolated from WT, Npas2+/-, and Npas2-/- mice exhibited consistent profiles of core clock gene expression except for Npas2 and Per2. In vitro behavioral characterizations of dermal fibroblasts revealed that Npas2-/- mutation was associated with increased proliferation, migration, and cell contraction measured by floating collagen gel contraction and single-cell force contraction assays. Npas2 knockout fibroblasts carrying sustained the high expression level of type XII and XIV FAICT collagens and synthesized dermis-like thick collagen fibers in vitro. Confocal laser scanning microscopy demonstrated the reconstruction of dermis-like collagen architecture in the wound healing area of Npas2-/- mice. This study indicates that the induced Npas2 expression in fibroblasts may interfere with skin homeostasis, wound healing, and dermal tissue reconstruction, providing a basis for novel therapeutic target and strategy. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Piel/metabolismo , Cicatrización de Heridas/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genéticaRESUMEN
PURPOSE: To determine the effect of rescheduling on prescription practices in a large academic hospital-based multidisciplinary practice comprising anesthesiologist-trained pain physicians. PATIENTS AND METHODS: We examined the number of HCP prescriptions written and quantity of tablets prescribed during a 6-month period prior to rescheduling and compared this with a 6-month period 1 year after rescheduling. We also examined the changes in prescription of tramadol and acetaminophen with codeine from one period to the next. RESULTS: Our pain clinic conducted 3,320 office visits during the 6-month period prior to HCP rescheduling and 6,003 office visits in the 6-month period 1 year after rescheduling. The charted data from each of these visits were used for our analysis. The mean number of tablets of HCPs prescribed per patient decreased from 318.48 in the pre-period to 242.27 tablets in the post-period, while the mean number of HCP prescriptions per patient decreased from 2.24 to 1.84. The mean number of acetaminophen with codeine tablets prescribed per patient increased from 3.46 to 15.27 in the pre- and post-period. Similarly, the mean number of tramadol tablets per patient increased from 47.33 to 61.97 in the pre- and post-period. The mean number of acetaminophen with codeine and tramadol prescriptions per patient increased from 0.02 to 0.15 and 0.38 to 0.51 in the pre- and post-period, respectively. In the 6-month post-period, fewer new patients were started on opioids compared to the 6-month pre-period, 16% and 27%, respectively. CONCLUSION: Our study showed a significant decrease in the mean number of HCP prescriptions written per patient, as well as a decrease in the mean number of HCP tablets prescribed. Pain physicians in our clinic increased the number of prescriptions for the non-HCPs. The number of acetaminophen with codeine and tramadol tablets prescribed significantly increased. Therefore, the rescheduling of HCPs has profoundly impacted practices within this academic pain clinic.