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Bacterial infections still pose a severe threat to public health, necessitating novel tools for real-time analysis of microbial behaviors in living organisms. While genetically engineered strains with fluorescent or luminescent reporters are commonly used in tracking bacteria, their inâ vivo uses are often limited. Here, we report a near-infrared fluorescent D-amino acid (FDAA) probe, Cy7ADA, for inâ situ labeling and intravital imaging of bacterial infections in mice. Cy7ADA probe effectively labels various bacteria inâ vitro and pathogenic Staphylococcus aureus in mice after intraperitoneal injection. Because of Cy7's high tissue penetration and the quick excretion of free probes via urine, real-time visualization of the pathogens in a liver abscess model via intravital confocal microscopy is achieved. The biodistributions, including their intracellular localization within Kupffer cells, are revealed. Monitoring bacterial responses to antibiotics also demonstrates Cy7ADA's capability to reflect the bacterial load dynamics within the host. Furthermore, Cy7ADA facilitates three-dimensional pathogen imaging in tissue-cleared liver samples, showcasing its potential for studying the biogeography of microbes in different organs. Integrating near-infrared FDAA probes with intravital microscopy holds promise for wide applications in studying bacterial infections inâ vivo.
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Colorantes Fluorescentes , Staphylococcus aureus , Animales , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Ratones , Carbocianinas/química , Aminoácidos/química , Infecciones Estafilocócicas/diagnóstico por imagen , Infecciones Estafilocócicas/microbiología , Microscopía Intravital/métodos , Imagen Óptica , Infecciones Bacterianas/diagnóstico por imagen , Infecciones Bacterianas/microbiología , Rayos InfrarrojosRESUMEN
The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.
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Dodecanol/análogos & derivados , Atractivos Sexuales/biosíntesis , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Proteínas de Transporte de Ácidos Grasos/genética , Proteínas de Transporte de Ácidos Grasos/metabolismo , Femenino , Cromatografía de Gases y Espectrometría de Masas , Mariposas Nocturnas , Ácido Mirístico/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Células Sf9/metabolismo , TranscriptomaRESUMEN
Increased vascular permeability is an important hallmark of many diseases, including cancer, cerebral ischemia, and severe inflammatory disorders. In this regard, the noninvasive assessment of pathologically increased vascular permeability in vivo is of great interest. In this study, the potential of albumin- and transthyretin-binding radioligands was evaluated for imaging of vascular hyperpermeability. For this purpose, the bleomycin-induced lung injury model was used as a model of inflammation-associated vascular leakage. The plasma protein-binding ligands, which bind to albumin (DOTA-PPB-01) and transthyretin (DOTA-PPB-03), were radiolabeled and used for nuclear imaging and biodistribution studies. In this regard, 177Lu was employed as a surrogate nuclide for detailed preclinical investigations, including single-photon emission computed tomography (SPECT) studies, whereas 44Sc was proposed as a radionuclide for positron emission tomography (PET), which may be relevant for future clinical translation. Mice were administered with these radioligands 6-9 days after intratracheal instillation of bleomycin or saline. Bleomycin-treated mice developed pronounced lung inflammation with enhanced vascular permeability that was reflected in significantly increased lung size and weight due to edema and infiltration with inflammatory cells. Biodistribution studies revealed significantly higher accumulation of 177Lu-DOTA-PPB-01 in injured lungs as compared to lungs of control animals at all investigated time points (4-48 h p.i.). The best contrast was achieved at late time points (16.1 ± 2.91% IA/g vs 2.03 ± 1.22% IA/g, 48 h p.i.) when the blood activity levels were â¼7.5% IA/g. Injection of 177Lu-DOTA-PPB-03 also resulted in increased lung accumulation in bleomycin-treated mice at all investigated time points (2-8 h p.i.). The pharmacokinetics was significantly faster, however, resulting in good contrast already at 8 h p.i. (4.32 ± 0.85% IA/g vs 1.06 ± 0.10% IA/g) when blood activity levels were â¼2% IA/g. The absolute lung accumulation of 177Lu-DOTA-PPB-03 was significantly lower than that of 177Lu-DOTA-PPB-01. PET/CT scans performed with 44Sc-DOTA-PPB-01 distinguished injured from healthy lungs only at late time points (20 h p.i.), whereas 44Sc-DOTA-PPB-03 already allowed the differentiation at 4 h p.i. due to its faster clearance. The investigated radioligands, 44Sc/177Lu-DOTA-PPB-01 and 44Sc/177Lu-DOTA-PPB-03, hold promise for the visualization of vascular leakage in a variety of pathological conditions. 44Sc would be the radionuclide of choice for clinical application as it can be stably coordinated with a DOTA chelator and enables PET imaging over extended periods.
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Lesión Pulmonar Aguda/diagnóstico por imagen , Imagen Molecular/métodos , Prealbúmina/metabolismo , Radiofármacos/administración & dosificación , Albúmina Sérica Humana/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Animales , Compuestos Aza/química , Bleomicina/administración & dosificación , Bleomicina/toxicidad , Permeabilidad Capilar , Modelos Animales de Enfermedad , Femenino , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Pulmón/irrigación sanguínea , Pulmón/diagnóstico por imagen , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Lutecio/administración & dosificación , Lutecio/química , Lutecio/farmacocinética , Ratones , Ratones Endogámicos C57BL , Tomografía de Emisión de Positrones/métodos , Prealbúmina/química , Radioisótopos/administración & dosificación , Radioisótopos/química , Radioisótopos/farmacocinética , Radiofármacos/química , Radiofármacos/farmacocinética , Escandio/administración & dosificación , Escandio/química , Escandio/farmacocinética , Albúmina Sérica Humana/química , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único/métodosRESUMEN
Mammalian alpha/beta hydrolase domain (ABHD) family of proteins have emerged as key regulators of lipid metabolism and are found to be associated with human diseases. Human α/ß-hydrolase domain containing protein 11 (ABHD11) has recently been predicted as a potential biomarker for human lung adenocarcinoma. In silico analyses of the ABHD11 protein sequence revealed the presence of a conserved lipase motif GXSXG. However, the role of ABHD11 in lipid metabolism is not known. To understand the biological function of ABHD11, we heterologously expressed the human ABHD11 in budding yeast, Saccharomyces cerevisiae. In vivo [14C]acetate labeling of cellular lipids in yeast cells overexpressing ABHD11 showed a decrease in triacylglycerol content. Overexpression of ABHD11 also alters the molecular species of triacylglycerol in yeast. Similar activity was observed in its yeast homolog, Ygr031w. The role of the conserved lipase motif in the hydrolase activity was proven by the mutation of all conserved amino acid residues of GXSXG motif. Collectively, our results demonstrate that human ABHD11 and its yeast homolog YGR031W have a pivotal role in the lipid metabolism.
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Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Serina Proteasas/metabolismo , Humanos , Metabolismo de los Lípidos , Saccharomyces cerevisiae/citologíaRESUMEN
BACKGROUND: Curcumin, derived from the rhizome Curcuma longa, is a natural anti-cancer agent and has been shown to inhibit proliferation and survival of tumor cells. Although the anti-cancer effects of curcumin are well established, detailed understanding of the signaling pathways altered by curcumin is still lacking. In this study, we carried out SILAC-based quantitative proteomic analysis of a HNSCC cell line (CAL 27) to investigate tyrosine signaling in response to curcumin. RESULTS: Using high resolution Orbitrap Fusion Tribrid Fourier transform mass spectrometer, we identified 627 phosphotyrosine sites mapping to 359 proteins. We observed alterations in the level of phosphorylation of 304 sites corresponding to 197 proteins upon curcumin treatment. We report here for the first time, curcumin-induced alterations in the phosphorylation of several kinases including TNK2, FRK, AXL, MAPK12 and phosphatases such as PTPN6, PTPRK, and INPPL1 among others. Pathway analysis revealed that the proteins differentially phosphorylated in response to curcumin are known to be involved in focal adhesion kinase signaling and actin cytoskeleton reorganization. CONCLUSIONS: The study indicates that curcumin may regulate cellular processes such as proliferation and migration through perturbation of the focal adhesion kinase pathway. This is the first quantitative phosphoproteomics-based study demonstrating the signaling events that are altered in response to curcumin. Considering the importance of curcumin as an anti-cancer agent, this study will significantly improve the current knowledge of curcumin-mediated signaling in cancer.
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Esophageal squamous-cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early-stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non-neoplastic Het-1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry-based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA-based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation.
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Carcinoma de Células Escamosas/metabolismo , Efrina-A2/metabolismo , Neoplasias Esofágicas/metabolismo , Fosfotirosina/análisis , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular , Línea Celular Tumoral , Efrina-A2/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Esófago/metabolismo , Esófago/patología , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Espectrometría de Masas , Fosforilación , Fosfotirosina/genética , Fosfotirosina/metabolismoRESUMEN
Bacteria have evolved a diverse set of enzymes that enable them to subvert host defense mechanisms as well as to form part of the prokaryotic immune system. Due to their unique and varied biochemical activities, these bacterial enzymes have emerged as key tools for understanding and investigating biological systems. In this review, we summarize and discuss some of the most prominent bacterial enzymes used for the site-specific modification of proteins, in vivo protein labeling, proximity labeling, interactome mapping, signaling pathway manipulation, and therapeutic discovery. Finally, we provide a perspective on the complementary advantages and limitations of using bacterial enzymes compared with chemical probes for exploring biological systems.
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Despite the fast progress in our understanding of the complex functions of gut microbiota, it is still challenging to directly investigate the in vivo microbial activities and processes on an individual cell basis. To gain knowledge of the indigenous growth/division patterns of the diverse mouse gut bacteria with a relatively high throughput, here, we propose an integrative strategy, which combines the use of fluorescent probe labeling, confocal imaging with single-cell sorting, and sequencing. Mouse gut bacteria sequentially labeled by two fluorescent d-amino acid probes in vivo were first imaged by confocal microscopy to visualize their growth patterns, which can be unveiled by the distribution of the two fluorescence signals on each bacterium. Bacterial cells of interest on the imaging slide were then sorted using a laser ejection equipment, and the collected cells were then sequenced individually to identify their taxa. Our strategy allows integrated acquirement of the growth pattern knowledge of a variety of gut bacteria and their genomic information on a single-cell basis, which should also have great potential in studying many other complex bacterial systems.
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Much of our current knowledge on nonribosomal peptide synthetases (NRPSs) is based on studies in which the full NRPS system or each protein domain is expressed in heterologous hosts. Consequently, methods to detect the endogenous activity of NRPSs, under natural cellular conditions, are needed for the study of NRPS cell biology. Here, we describe the in vivo activity-based protein profiling (ABPP) for endogenous NRPSs and its applications to the study of their activities in bacteria. Remarkably, in vitro and in vivo ABPP in the context of the surfactin producer Bacillus subtilis enabled the visualization, tracking, and imaging of an endogenous SrfAB-NRPS with remarkable selectivity and sensitivity. Furthermore, in vivo, ABPP allowed the discovery of the degradation processes of the endogenous SrfAB-NRPS in the context of its native producer bacteria. Overall, this study deepens our understanding of the properties of NRPSs that cannot be addressed by conventional methods.
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Bacillus subtilis/enzimología , Lipopéptidos/biosíntesis , Péptido Sintasas/metabolismo , Proteómica , Bacillus subtilis/citología , Lipopéptidos/química , Conformación ProteicaRESUMEN
Retrograde dyes are often used in basic research to investigate neuronal innervations of an organ. This article describes the experimental data on the application of retrograde dyes on the mouse heart in vivo and on the cardiac or neuronal cultures in vitro. By providing this information, cardiac or inneinnervations can be evaluated in vivo. Therefore, unknown cellular and molecular mechanisms and systemic interactions in the body can be investigated. In particular, we provided practical tips to lower mortality risks following the cardiac surgery and evaluated the staining capacity and fluorescent characteristics of the Di-8-ANEPPQ dye in the cardiac tissue and cell cultures. First, primary cultures of mouse nodose ganglia (NG) neurons and mouse neonatal cardiomyocytes were stained with Di-8-ANEPPQ. The Di-8-ANEPPQ signal from live cultures were visualized using spinning disk confocal microscopy to verify the lipophilic and fluorescent labeling capacity of Di-8-ANEPPQ. Next, the excitation and emission data of Di-8-ANEPPQ were collected between 415 nm and 690 nm using power spectrum module of confocal microscopy. This spectrum analysis could be useful for the researchers who plan to use Di-8-ANEPPQ in combination with other fluorescent dyes to eliminate any florescent overlap. In order to label the heart tissue with tracer dyes Di-8-ANEPPQ or DiI in vivo, the heart was exposed without damaging lungs or other tissues following anesthetization, then the retrograde dye was applied as a paste for DiI or injected to the apex of the heart for Di-8-ANEPPQ and the operation area was sutured. The surgical procedure required intubation to control the respiratory reflex without the need to perform a tracheotomy and yielded high viability. Following labeling the heart in vivo, the heart was dissected, and images of injection area were captured using confocal microscopy. All fluorescent images of Di-8-ANEPPQ labeled cells were analyzed by using the Fiji software. Overall, these data provide applicable data to other investigators to trace the sensory neurons innervating not only the heart but also other organs using Di-8-ANEPPQ. These data support the original research article titled "Evaluation of bilateral cardiac afferent distribution at the spinal and vagal ganglia by retrograde labeling" that was accepted for publication in Brain Research Journal [1].
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The discovery that the RNA guided bacterial endonuclease Cas9 can be harnessed to target and manipulate user-defined genomic sequences has greatly influenced the field of genome engineering. Interestingly, a catalytically dead Cas9 (dCas9) can be employed as a targeted DNA-binding platform to alter gene expression. By fusing this dCas9 to eGFP, we and others could show that the CRISPR/Cas9 system can be further expanded to label and trace genomic loci in living cells. We demonstrated that by exchanging the sgRNA, dCas9-eGFP could be specifically directed to various heterochromatic sequences within the nucleus. Here, we provide a basic protocol for this versatile tool and describe how to verify new dCas9-eGFP targets.
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Sistemas CRISPR-Cas , Mapeo Cromosómico , Sitios Genéticos , Genoma , Animales , Células Cultivadas , Mapeo Cromosómico/métodos , Clonación Molecular , Expresión Génica , Marcación de Gen , Genes Reporteros , Hibridación Fluorescente in Situ , Ratones , Microscopía Fluorescente , ARN Guía de Kinetoplastida , Secuencias Repetitivas de Ácidos Nucleicos , TransfecciónRESUMEN
Recruitment of leukocytes into arteries is a hallmark event throughout all stages of atherosclerosis and hence stands out as a primary therapeutic target. To understand the molecular mechanisms of arterial leukocyte subset infiltration, real-time visualization of recruitment processes of leukocyte subsets at high resolution is a prerequisite. In this review we provide a balanced overview of optical imaging modalities in the more commonly used experimental models for atherosclerosis (e.g., mouse models) allowing for in vivo display of recruitment processes in large arteries and further detail strategies to overcome hurdles inherent to arterial imaging. We further provide a synopsis of techniques allowing for non-toxic, photostable labeling of target structures. Finally, we deliver a short summary of ongoing developments including the emergence of novel labeling approaches, the use of superresolution microscopy, and the potentials of opto-acoustic microscopy and intravascular 2-dimensional near-infrared fluorescence microscopy.
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Arterias/inmunología , Aterosclerosis/inmunología , Quimiotaxis de Leucocito , Inflamación/inmunología , Microscopía Intravital/métodos , Leucocitos/inmunología , Animales , Arterias/patología , Aterosclerosis/patología , Modelos Animales de Enfermedad , Colorantes Fluorescentes , Inflamación/patología , Leucocitos/patología , Ratones , Microscopía Fluorescente , Imagen Óptica , Técnicas Fotoacústicas , Espectroscopía Infrarroja Corta , Factores de TiempoRESUMEN
UNLABELLED: The purpose of this study was to develop a novel in vivo albumin-labeling method to allow PET of cardiac function after myocardial infarction and vascular leakage and increased permeability in inflammatory diseases and malignant tumors. METHODS: To label albumin in vivo, we synthesized a NOTA (1,4,7-triazacyclononane-N,N',Nâ³-triacetic acid)-conjugated truncated form of Evans blue (NEB). (18)F labeling was achieved by the formation of an (18)F-aluminum fluoride ((18)F-AlF) complex, and (64)Cu labeling was obtained by a standard chelation method. Sixty-minute dynamic PET imaging was performed on normal mice to evaluate the distribution of (18)F-AlF-NEB, which was compared with in vitro-labeled mouse serum albumin ((18)F-fluorobenzyl-MSA). Electrocardiography-gated PET imaging was performed in a mouse model of myocardial infarction. Both dynamic and static PET scans were obtained in a mouse inflammation model induced by local injection of turpentine to evaluate vascular leakage. Tumor permeability was studied by dynamic and late-point static PET using (64)Cu-NEB in a UM-22B xenograft model. RESULTS: NEB was successfully synthesized, and (18)F labeling including work-up took about 20-30 min, with a radiochemical purity greater than 95% without the need for high-performance liquid chromatography purification. Most of the radioactivity was retained in the circulation system at 60 min after injection (26.35 ± 1.52 percentage injected dose per gram [%ID/g]). With electrocardiography-gated PET, ventricles of the heart and major arteries were clearly visualized. The myocardial infarction mice showed much lower left ventricular ejection fraction than the control mice. Inflammatory muscles showed significantly higher tracer accumulation than the contralateral healthy ones. UM-22B tumor uptake of (64)Cu-NEB gradually increased with time (5.73 ± 1.11 %ID/g at 1 h and 8.03 ± 0.77 %ID/g at 2 h after injection). CONCLUSION: The distribution and local accumulation of serum albumin can be noninvasively visualized and quantified by (18)F-AlF-NEB and (64)Cu-NEB PET. The simple labeling and broad applications make these imaging probes attractive for clinical translation.
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Marcaje Isotópico/métodos , Tomografía de Emisión de Positrones , Albúmina Sérica , Animales , Permeabilidad Capilar , Electrocardiografía , Corazón/diagnóstico por imagen , Corazón/fisiología , Masculino , Ratones , Neovascularización Patológica/diagnóstico por imagen , RadioquímicaRESUMEN
Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals.