Nanospray desorption electrospray ionization (nano‐DESI) is an ambient ionization mass spectrometry imaging (MSI) approach that enables spatial mapping of biological and environmental samples with high spatial resolution and throughput. Because nano‐DESI has not yet been commercialized, researchers develop their own sources and interface them with different commercial mass spectrometers. Previously, several protocols focusing on the fabrication of nano‐DESI probes have been reported. In this tutorial, we discuss different hardware requirements for coupling the nano‐DESI source to commercial mass spectrometers, such as the safety interlock, inlet extension, and contact closure. In addition, we describe the structure of our custom software for controlling the nano‐DESI MSI platform and provide detailed instructions for its usage. With this tutorial, interested researchers should be able to implement nano‐DESI experiments in their labs.
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Background: It is a major clinical challenge to ensure the long-term function of transplanted kidneys. Specifically, the injury associated with cold storage of kidneys compromises the long-term function of the grafts after transplantation. Therefore, the molecular mechanisms underlying cold-storage–related kidney injury are attractive therapeutic targets to prevent injury and improve long-term graft function. Previously, we found that constitutive proteasome function was compromised in rat kidneys after cold storage followed by transplantation. Here, we evaluated the role of the immunoproteasome (
i proteasome), a proteasome variant, during cold storage (CS) followed by transplantation.Methods: Established in vivo rat kidney transplant model with or without CS containing vehicle or iproteasome inhibitor (ONX 0914) was used in this study. The
i proteasome function was performed using rat kidney homogenates and fluorescent-based peptide substrate specific to β5i subunit. Western blotting and quantitative RT-PCR were used to assess the subunit expression/level of thei proteasome (β5i) subunit.Results: We demonstrated a decrease in the abundance of the β5i subunit of the
i proteasome in kidneys during CS, but β5i levels increased in kidneys after CS and transplant. Despite the increase in β5i levels and its peptidase activity within kidneys, inhibiting β5i during CS did not improve graft function after transplantation.Summary: These results suggest that the pharmacological inhibition of immunoproteasome function during CS does not improve graft function or outcome. In light of these findings, future studies targeting immunoproteasomes during both CS and transplantation may define the role of immunoproteasomes on short- and long-term kidney transplant outcomes.
Free, publicly-accessible full text available February 2, 2025 -
Glioblastoma ranks among the most lethal of primary brain malignancies, with glioblastoma stem cells (GSCs) at the apex of tumor cellular hierarchies. Here, to discover novel therapeutic GSC targets, we interrogated gene expression profiles from GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs), revealing EYA2 as preferentially expressed by GSCs. Targeting EYA2 impaired GSC maintenance and induced cell cycle arrest, apoptosis, and loss of self-renewal. EYA2 displayed novel localization to centrosomes in GSCs, and EYA2 tyrosine (Tyr) phosphatase activity was essential for proper mitotic spindle assembly and survival of GSCs. Inhibition of the EYA2 Tyr phosphatase activity, via genetic or pharmacological means, mimicked EYA2 loss in GSCs in vitro and extended the survival of tumor-bearing mice. Supporting the clinical relevance of these findings, EYA2 portends poor patient prognosis in glioblastoma. Collectively, our data indicate that EYA2 phosphatase function plays selective critical roles in the growth and survival of GSCs, potentially offering a high therapeutic index for EYA2 inhibitors.more » « less
