RESUMEN
Zinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. However, the probiotic bacterium Escherichia coli Nissle 1917 (or "Nissle") exhibits appreciable growth in zinc-limited media even when these transporters are deleted. Here, we show that Nissle utilizes the siderophore yersiniabactin as a zincophore, enabling Nissle to grow in zinc-limited media, to tolerate calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. We also show that yersiniabactin's affinity for iron or zinc changes in a pH-dependent manner, with increased relative zinc binding as the pH increases. Thus, our results indicate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to commensal and pathogenic Enterobacteriaceae.
Asunto(s)
Enterobacteriaceae/metabolismo , Sideróforos/metabolismo , Zinc/metabolismo , Transportadoras de Casetes de Unión a ATP , Animales , Proteínas Bacterianas/metabolismo , Proteínas Portadoras , Colon/microbiología , Colon/patología , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Femenino , Complejo de Antígeno L1 de Leucocito , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Fenoles , Salmonella typhi , TiazolesRESUMEN
BACKGROUND: The regulation of cerebral blood flow is critical for normal brain functioning, and many physiological and pathological conditions can have long-term impacts on cerebral blood flow. However, minimally invasive tools to study chronic changes in animal models are limited. NEW METHOD: We developed a minimally invasive surgical technique (cyanoacrylate skull, CAS) allowing us to image cerebral blood flow longitudinally through the intact mouse skull using laser speckle imaging. RESULTS: With CAS we were able to detect acute changes in cerebral blood flow induced by hypercapnic challenge. We were also able to image cerebral blood flow dynamics with laser speckle imaging for over 100 days. Furthermore, the relative cerebral blood flow remained stable in mice from 30 days to greater than 100 days after the surgery. COMPARISON WITH EXISTING METHODS: Previously, achieving continuous long-term optical access to measure cerebral blood flow in individual vessels in a mouse model involved invasive surgery. In contrast, the CAS technique presented here is relatively non-invasive, as it allows stable optical access through an intact mouse skull. CONCLUSIONS: The CAS technique allows researcher to chronically measure cerebral blood flow dynamics for a significant portion of a mouse's lifespan. This approach may be useful for studying changes in blood flow due to cerebral pathology or for examining the therapeutic effects of modifying cerebral blood flow in mouse models relevant to human disease.
Asunto(s)
Circulación Cerebrovascular , Imagen Óptica , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/cirugía , Hemodinámica , Ratones , Cráneo/diagnóstico por imagen , Cráneo/cirugíaRESUMEN
Brain infection by the parasite Toxoplasma gondii in mice is thought to generate vulnerability to predation by mechanisms that remain elusive. Monocytes play a key role in host defense and inflammation and are critical for controlling T. gondii However, the dynamic and regional relationship between brain-infiltrating monocytes and parasites is unknown. We report the mobilization of inflammatory (CCR2+Ly6Chi) and patrolling (CX3CR1+Ly6Clo) monocytes into the blood and brain during T. gondii infection of C57BL/6J and CCR2RFP/+CX3CR1GFP/+ mice. Longitudinal analysis of mice using 2-photon intravital imaging of the brain through cranial windows revealed that CCR2-RFP monocytes were recruited to the blood-brain barrier (BBB) within 2 wk of T. gondii infection, exhibited distinct rolling and crawling behavior, and accumulated within the vessel lumen before entering the parenchyma. Optical clearing of intact T. gondii-infected brains using iDISCO+ and light-sheet microscopy enabled global 3D detection of monocytes. Clusters of T. gondii and individual monocytes across the brain were identified using an automated cell segmentation pipeline, and monocytes were found to be significantly correlated with sites of T. gondii clusters. Computational alignment of brains to the Allen annotated reference atlas [E. S. Lein et al., Nature 445:168-176 (2007)] indicated a consistent pattern of monocyte infiltration during T. gondii infection to the olfactory tubercle, in contrast to LPS treatment of mice, which resulted in a diffuse distribution of monocytes across multiple brain regions. These data provide insights into the dynamics of monocyte recruitment to the BBB and the highly regionalized localization of monocytes in the brain during T. gondii CNS infection.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Monocitos/metabolismo , Toxoplasmosis/diagnóstico por imagen , Toxoplasmosis/metabolismo , Animales , Antígenos Ly/metabolismo , Barrera Hematoencefálica/diagnóstico por imagen , Receptor 1 de Quimiocinas CX3C/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores CCR2/metabolismoRESUMEN
Alzheimer's disease (AD) is the leading cause of age-related neurodegeneration and is characterized neuropathologically by the accumulation of insoluble beta-amyloid (Aß) peptides. In AD brains, plaque-associated myeloid (PAM) cells cluster around Aß plaques but fail to effectively clear Aß by phagocytosis. PAM cells were originally thought to be brain-resident microglia. However, several studies have also suggested that Aß-induced inflammation causes peripheral monocytes to enter the otherwise immune-privileged brain. The relationship between AD progression and inflammation in the brain remains ambiguous because microglia and monocyte-derived macrophages are extremely difficult to distinguish from one another in an inflamed brain. Whether PAM cells are microglia, peripheral macrophages, or a mixture of both remains unclear. CD11a is a component of the ß2 integrin LFA1. We have determined that CD11a is highly expressed on peripheral immune cells, including macrophages, but is not expressed by mouse microglia. These expression patterns remain consistent in LPS-treated inflamed mice, as well as in two mouse models of AD. Thus, CD11a can be used as a marker to distinguish murine microglia from infiltrating peripheral immune cells. Using CD11a, we show that PAM cells in AD transgenic brains are comprised entirely of microglia. We also demonstrate a novel fluorescence-assisted quantification technique (FAQT), which reveals a significant increase in T lymphocytes, especially in the brains of female AD mice. Our findings support the notion that microglia are the lead myeloid players in AD and that rejuvenating their phagocytic potential may be an important therapeutic strategy.
Asunto(s)
Enfermedad de Alzheimer/patología , Antígeno CD11a/metabolismo , Microglía/metabolismo , Microglía/patología , Células Mieloides/metabolismo , Algoritmos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/cirugía , Animales , Animales Recién Nacidos , Trasplante de Médula Ósea , Encéfalo/metabolismo , Encéfalo/patología , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Inflamación/etiología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Células Mieloides/efectos de los fármacos , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , Toxoplasmosis/complicacionesRESUMEN
Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, in most cases, it is not known whether the compounds tested alter the timing of particular cell cycle transitions. Here, we evaluated an FDA-approved drug library to identify pharmaceuticals that alter cell cycle progression in yeast, using DNA content measurements by flow cytometry. This approach revealed strong cell cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. Based on their effects on cell cycle progression, we also examined cell proliferation in the presence of both compounds. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. Combinations of interest among diverse pharmaceuticals are difficult to identify, due to the daunting number of possible combinations that must be evaluated. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Fluoxetina/farmacología , Gemfibrozilo/farmacología , Proliferación Celular/efectos de los fármacos , Interacciones Farmacológicas , Citometría de Flujo , Mitosis/efectos de los fármacosRESUMEN
Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.