RESUMEN
Chemical exchange saturation transfer (CEST) MRI has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non-existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide, was designed to be 198 amino acids. SuperCESTide was expressed in E. coli and purified with size exclusion chromatography. The magnetic transfer ratio asymmetry generated by superCESTide was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine) and human protamine. These data show that novel peptides with sequences optimized in silico for CEST contrast that utilize a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments.
Asunto(s)
Técnicas Biosensibles , Escherichia coli , Humanos , Imagen por Resonancia Magnética/métodos , Péptidos , Protaminas , Aminoácidos , Medios de Contraste/químicaRESUMEN
Calcium-responsive contrast agents for magnetic resonance imaging (MRI) offer a promising approach for noninvasive brain-wide monitoring of neural activity at any arbitrary depth. Current examples of MRI-based calcium probes involve synthetic molecules and nanoparticles, which cannot be used to examine calcium signaling in a genetically encoded form. Here, we describe a new MRI sensor for calcium, based entirely on a naturally occurring calcium-binding protein known as calprotectin. Calcium-binding causes calprotectin to sequester manganese ions, thereby limiting Mn2+ enhanced paramagnetic relaxation of nearby water molecules. We demonstrate that this mechanism allows calprotectin to alter T1 and T2 based MRI signals in response to biologically relevant calcium concentrations. The resulting response amplitude, i.e., change in relaxation time, is comparable to existing MRI-based calcium sensors as well as other reported protein-based MRI sensors. As a preliminary demonstration of its biological applicability, we used calprotectin to detect calcium in a lysed hippocampal cell preparation as well as in intact Chinese hamster ovary cells treated with a calcium ionophore. Calprotectin thus represents a promising path toward noninvasive imaging of calcium signaling by combining the molecular and cellular specificity of genetically encodable tools with the ability of MRI to image through scattering tissue of any size and depth.
Asunto(s)
Técnicas Biosensibles , Calcio , Animales , Células CHO , Cricetinae , Cricetulus , Imagen por Resonancia MagnéticaRESUMEN
Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.
Asunto(s)
Colon/microbiología , Disentería Bacilar/microbiología , Shigella flexneri/fisiología , Sistemas de Secreción Tipo III/fisiología , Animales , Biomarcadores , Modelos Animales de Enfermedad , Femenino , Genes Reporteros , Cobayas , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Especificidad de Órganos , Distribución TisularRESUMEN
A hypothesis and the experiments to test it propose that very long-term memories, such as fear conditioning, are stored as the pattern of holes in the perineuronal net (PNN), a specialized ECM that envelops mature neurons and restricts synapse formation. The 3D intertwining of PNN and synapses would be imaged by serial-section EM. Lifetimes of PNN vs. intrasynaptic components would be compared with pulse-chase (15)N labeling in mice and (14)C content in human cadaver brains. Genetically encoded indicators and antineoepitope antibodies should improve spatial and temporal resolution of the in vivo activity of proteases that locally erode PNN. Further techniques suggested include genetic KOs, better pharmacological inhibitors, and a genetically encoded snapshot reporter, which will capture the pattern of activity throughout a large ensemble of neurons at a time precisely defined by the triggering illumination, drive expression of effector genes to mark those cells, and allow selective excitation, inhibition, or ablation to test their functional importance. The snapshot reporter should enable more precise inhibition or potentiation of PNN erosion to compare with behavioral consequences. Finally, biosynthesis of PNN components and proteases would be imaged.