生物物理学

Tracking macrophages by non-invasive molecular imaging can provide useful insights into the immunobiology of inflammatory disorders in preclinical disease models. Perfluorocarbon nanoemulsions (PFC-NEs) have been well documented in their ability to be taken up by macrophages through phagocytosis and serve as ¹⁹F magnetic resonance imaging (MRI) tracers of inflammation in vivo and ex vivo. Incorporation of near-infrared fluorescent (NIRF) dyes in PFC-NEs can help monitor the spatiotemporal distribution of macrophages in vivo during inflammatory processes, using NIRF imaging as a complementary methodology to MRI. Here, we discuss in depth how both colloidal and fluorescence stabilities of the PFC-NEs are essential for successful and reliable macrophage tracking in vivo and for their detection in excised tissues ex vivo by NIRF imaging. Furthermore, PFC-NE quality assures NIRF imaging reproducibility and reliability across preclinical studies, providing insights into inflammation progression and therapeutic response. Previous studies focused on assessments of colloidal property changes in response to stress and during storage as a means of quality control. We recently focused on the joint evaluation of both colloidal and fluorescence properties and their relationship to NIRF imaging outcomes. In this protocol, we summarize the key assessments of the fluorescent dye–labeled nanoemulsions, which include long-term particle size distribution monitoring as the measure of colloidal stability and monitoring of the fluorescence signal. Due to its simplicity and reproducibility, our protocols are easy to adopt for researchers to assess the quality of PFC-NEs for in vivo NIRF imaging applications.

Translational work in rodents elucidates basic mechanisms that drive complex behaviors relevant to psychiatric and neurological conditions. Nonetheless, numerous promising studies in rodents later fail in clinical trials, highlighting the need for improving the translational utility of preclinical studies in rodents. Imaging of small rodents provides an important strategy to address this challenge, as it enables a whole-brain unbiased search for structural and dynamic changes that can be directly compared to human imaging. The functional significance of structural changes identified using imaging can then be further investigated using molecular and genetic tools available for the mouse. Here, we describe a pipeline for unbiased search and characterization of structural changes and network properties, based on diffusion MRI data covering the entire mouse brain at an isotropic resolution of 100 µm. We first used unbiased whole-brain voxel-based analyses to identify volumetric and microstructural alterations in the brain of adult mice exposed to unpredictable postnatal stress (UPS), which is a mouse model of complex early life stress (ELS). Brain regions showing structural abnormalities were used as nodes to generate a grid for assessing structural connectivity and network properties based on graph theory. The technique described here can be broadly applied to understand brain connectivity in other mouse models of human disorders, as well as in genetically modified mouse strains.

Graphic abstract:

Pipeline for characterizing structural connectome in the mouse brain using diffusion magnetic resonance imaging. Scale bar = 1 mm.

In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains, particularly in the conformational transition and dynamics study area where a much higher quantity of the receptors and G proteins is required even in comparison to X-ray and cryo-EM (5 mg/ml, 3 μl/sample) when NMR spectroscopy (5 mg/ml, 250 μl /sample) is applied. As a result, the expression levels of the insect and mammalian systems are also difficult to meet this demand, not to mention the prohibitive cost of producing GPCRs and G proteins using these systems for a vast majority of laboratories. Therefore, exploration of an effective, affordable, and practical approach with broad applicability is demanded. Pichia pastoris expression system has shown its promise in the GPCR preparation with many merits that other eukaryotic expression systems can’t compete with. GPCRs expressed in this system are inexpensive, easy-to-manipulate, and capable of isotopically labeling. Herein, we present related protocols recently developed and upgraded in our lab, including expressions and purifications of P. pastoris derived GPCR along with Gα and Gβγ proteins. We anticipate that these protocols will advance the conformational transition and dynamics studies of the GPCR and its complexes.

In drug development programmes, multiple assays are needed for the determination of protein-compound interactions and evaluation of potential use in assays with protein-protein interactions. In this protocol we describe the waterLOGSY NMR method for confirming protein-ligand binding events.

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological importance of post-transcriptional RNA modifications is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. To obtain information on the tRNA maturation process, we have developed a methodology, using NMR as a tool to monitor tRNA maturation in a non-disruptive and continuous fashion in cellular extracts. By following the maturation of a model yeast tRNA with time-resolved NMR, we showed that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. The implementation of this method requires the production for NMR spectroscopy of tRNA samples with different modification status, in order to identify the NMR signature of individual modifications. The production of tRNA samples for the analysis of modification pathways with NMR spectroscopy will be presented here and examplified on the yeast tRNA^Phe, but can be extended to any other tRNA by changing the sequence of the construct. The protocol describes the production of unmodified tRNA samples by in vitro transcription, and the production of modified tRNA samples by recombinant expression of tRNAs in E. coli.

We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (³¹P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid medium).

In this protocol, we describe a method to visualize and map dural lymphatic vessels in-vivo using magnetic resonance imaging (MRI) and ex-vivo using histopathological techniques. While MRI protocols for routine imaging of meningeal lymphatics include contrast-enhanced T2-FLAIR and T1-weighted black-blood imaging, a more specific 3D mapping of the lymphatic system can be obtained by administering two distinct gadolinium-based MRI contrast agents on different days (gadofosveset and gadobutrol) and subsequently processing images acquired before and after administration of each type of contrast. In addition, we introduce methods for optimal immunostaining of lymphatic and blood vessel markers in human dura mater ex-vivo.