No abstract

3-Deoxy-d--oct-2-ulosonic acid (Kdo) is an essential component of LPS in the outer leaflet of the Gram-negative bacterial outer membrane. Although labeling of with the chemicalreporter 8-azido-3,8-dideoxy-d--oct-2-ulosonic acid (Kdo-N) has been reported, its incorporation into LPS has not been directly shown. We have now verified Kdo-N incorporation into LPS at the molecular level. Using microscopy and PAGE analysis, we show that Kdo-N is localized to the outer membrane and specifically incorporates into rough and deep-rough LPS. In an strain lacking endogenous Kdo biosynthesis, supplementation with exogenous Kdo restored full-length core-LPS, which suggests that the Kdo biosynthetic pathways might not be essential in the presence of sufficient exogenous Kdo. In contrast, exogenous Kdo-N only restored a small fraction of core LPS with the majority incorporated into truncated LPS. The truncated LPS were identified as Kdo-N-lipid IV and (Kdo-N)-lipid IV by MS analysis. The low level of Kdo-N incorporation could be partly explained by a 6-fold reduction in the specificity constant of the CMP-Kdo synthetase KdsB with Kdo-N compared with Kdo. These results indicate that the azido moiety in Kdo-N interferes with its utilization and may limit its utility as a tracer of LPS biosynthesis and transport in We propose that our findings will be helpful for researchers using Kdo and its chemical derivatives for investigating LPS biosynthesis, transport, and assembly in Gram-negative bacteria.

A new sample preparation method for MALDI tissue imaging has been developed for the analysis of low molecular weight compounds that employs matrix pre-coated MALDI targets. Tissue sections need only to be transferred onto the pre-coated target before analysis for fast and easy sample preparation. Pre-coated targets have a homogenous matrix coating with uniform crystals of approximately 1–2 μm and do not require solvents that may lead to analyte delocalization within a tissue section. We report here the use of matrix pre-coated targets for imaging of lipids, peptides, and pharmaceuticals in tissues.

The JAK kinases JAK1, JAK2, JAK3, and TYK2 play key roles in cytokine signaling. Activation of the JAK/STAT pathways is linked to many diseases involving the immune system, including atopic dermatitis. As systemic JAK inhibitor pharmacology is associated with side effects, topical administration to the skin has been considered to locally restrict the site of action. Several orally bioavailable JAK inhibitors repurposed for topical use have been recently approved or are in clinical development. Here, we disclose our clinical candidate CEE321, which is a potent pan JAK inhibitor in enzyme and cellular assays. In contrast to repurposed oral drugs, CEE321 does not display high potency in blood and has a high clearance in vivo. Therefore, we consider CEE321 to be a “soft drug”. When applied topically to human skin that was stimulated with the cytokines IL4 and IL13 ex vivo, CEE321 potently inhibited biomarkers relevant to atopic dermatitis.

In the analysis of biological tissue by imaging mass spectrometry (IMS), the limit of detection and dynamic range are of paramount importance in obtaining experimental results that provide insight into underlying biological processes. Many important biomolecules are present in the tissue milieu in low concentrations and in complex mixtures with other compounds of widely ranging abundances, challenging the limits of analytical technologies. In many IMS experiments, the ion signal can be dominated by a few highly abundant ion species. On trap-based instrument platforms that accumulate ions prior to mass analysis, these high abundance ions can diminish the detection and dynamic range of lower abundance ions. Herein, we characterize two strategies for combating these challenges during IMS experiments on a hybrid QqFT-ICR MS. In one iteration, the mass resolving capabilities of a quadrupole mass filter are used to selectively enrich for ions of interest via a technique previously termed continuous accumulation of selected ions (CASI). Secondly, we have introduced a supplemental dipolar AC waveform to the quadrupole mass filter of a commercial QqFT-ICR mass spectrometer to perform selected ion ejection prior to the ion accumulation region. This setup allows the selective ejection of the most abundant ion species prior to ion accumulation, thereby greatly improving the molecular depth with which IMS can probe tissue samples.