Spatially heterogeneous lipid dysregulation in tuberculous meningitis.

Tuberculous (TB) meningitis is the deadliest form of extrapulmonary TB which disproportionately affects children and immunocompromised individuals. Studies in pulmonary TB have shown that Mycobacterium tuberculosis can alter host lipid metabolism to evade the immune system. Cholesterol lowering drugs (i.

Aberrant N-glycosylation is a therapeutic target in carriers of a common and highly pleiotropic mutation in the manganese transporter ZIP8.

The treatment of defective glycosylation in clinical practice has been limited to patients with rare and severe phenotypes associated with congenital disorders of glycosylation (CDG). Carried by approximately 5% of the human population, the discovery of the highly pleiotropic, missense mutation in a manganese transporter ZIP8 has exposed under-appreciated roles for Mn homeostasis and aberrant Mn-dependent glycosyltransferases activity leading to defective N-glycosylation in complex human diseases. Here, we test the hypothesis that aberrant N-glycosylation contributes to disease pathogenesis of ZIP8 A391T-associated Crohn's disease.

Glutamine transporter SLC38A3 promotes breast cancer metastasis via Gsk3β/β-catenin/EMT pathway.

Breast cancer is the leading cancer-related cause of death in women. Here we show that solute carrier family 38-member 3 (SLC38A3) is overexpressed in breast cancer, particularly in triple-negative breast cancer (TNBC) cells and tissues. Our study reveals that SLC38A3 regulates cellular glutamine, glutamate, asparagine, aspartate, alanine, and glutathione (GSH) levels in breast cancer cells.

Key regulator PNPLA8 drives phospholipid reprogramming induced proliferation and migration in triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC.

Methods: We sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties.

Primary breast tumor induced extracellular matrix remodeling in premetastatic lungs.

The premetastatic niche hypothesis proposes an active priming of the metastatic site by factors secreted from the primary tumor prior to the arrival of the first cancer cells. We investigated several extracellular matrix (ECM) structural proteins, ECM degrading enzymes, and ECM processing proteins involved in the ECM remodeling of the premetastatic niche. Our in vitro model consisted of lung fibroblasts, which were exposed to factors secreted by nonmalignant breast epithelial cells, nonmetastatic breast cancer cells, or metastatic breast cancer cells.

RaMALDI: Enabling simultaneous Raman and MALDI imaging of the same tissue section.

Multimodal tissue imaging techniques that integrate two complementary modalities are powerful discovery tools for unraveling biological processes and identifying biomarkers of disease. Combining Raman spectroscopic imaging (RSI) and matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain fused images with the advantages of both modalities has the potential of providing spatially resolved, sensitive, specific biomolecular information, but has so far involved two separate sample preparations, or even consecutive tissue sections for RSI and MALDI MSI, resulting in images with inherent disparities. We have developed RaMALDI, a streamlined, integrated, multimodal imaging workflow of RSI and MALDI MSI, performed on a single tissue section with one sample preparation protocol.

Generation and multiomic profiling of a double-knockout gastroesophageal junction organoid model.

Inactivation of the tumor suppressor genes tumor protein p53 () and cyclin-dependent kinase inhibitor 2A () occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a paucity of GEJ-specific disease models, cancer-promoting consequences of and inactivation at the GEJ have not been characterized. Here, we report the development of a wild-type primary human GEJ organoid model and a CRISPR-edited transformed GEJ organoid model.

A multimodal pipeline using NMR spectroscopy and MALDI-TOF mass spectrometry imaging from the same tissue sample.

NMR spectroscopy and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) are both commonly used to detect large numbers of metabolites and lipids in metabolomic and lipidomic studies. We have demonstrated a new workflow, highlighting the benefits of both techniques to obtain metabolomic and lipidomic data, which has realized for the first time the combination of these two complementary and powerful technologies. NMR spectroscopy is frequently used to obtain quantitative metabolite information from cells and tissues.

Unlabeled aspirin as an activatable theranostic MRI agent for breast cancer.

Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is emerging as an alternative to gadolinium-based contrast MRI. We have evaluated the possibility of CEST MRI of orthotopic breast tumor xenografts with unlabeled aspirin's conversion to salicylic acid (SA) through various enzymatic activities, most notably inhibition of cyclooxygenase (COX)-1/-2 enzymes. : We measured the COX-1/-2 expression in four breast cancer cell lines by Western Blot analysis and selected the highest and lowest expressing cell lines.

Factorial Design to Optimize Matrix Spraying Parameters for MALDI Mass Spectrometry Imaging.

Matrix deposition is a critical step in obtaining reproducible and spatially representative matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging data. To date, few limited studies have examined the optimization of matrix spraying parameters for maximizing analyte extraction while minimizing analyte delocalization. Herein, we present a study using automated pneumatic spraying with a heated sample-holder tray to determine an optimized model for mouse whole kidney lipid imaging using a 2,5-dihydroxybenzoic acid matrix in which the solvent flow rate, nozzle velocity, and sample heating were optimized using a two-level factorial experimental design.

Pneumatically Sprayed Gold Nanoparticles for Mass Spectrometry Imaging of Neurotransmitters.

Using citrate-capped gold nanoparticles (AuNPs) for laser desorption ionization mass spectrometry (LDI-MS) is an approach that has demonstrated broad applicability to ionization of different classes of molecules. Here, we show a simple AuNP-based approach for the ionization of neurotransmitters. Specifically, the detection of acetylcholine, dopamine, epinephrine, glutamine, 4-aminobutyric acid, norepinephrine, octopamine, and serotonin was achieved at physiologically relevant concentrations in serum and homogenized tissue.

Perfluoro--Butyl Hydroxyprolines as Sensitive, Conformationally Responsive Molecular Probes: Detection of Protein Kinase Activity by F NMR.

F NMR spectroscopy provides the ability to quantitatively analyze single species in complex solutions but is often limited by the modest sensitivity inherent to NMR. 4- and 4-Perfluoro--buyl hydroxyproline contain 9 equivalent fluorines, in amino acids with strong conformational preferences. In order to test the ability to use these amino acids as sensitive probes of protein modifications, the perfluoro--buyl hydroxyprolines were incorporated into substrate peptides of the protein kinases PKA and Akt.

Cellular resolution in clinical MALDI mass spectrometry imaging: the latest advancements and current challenges.

Mass spectrometry (MS) is the workhorse of metabolomics, proteomics and lipidomics. Mass spectrometry imaging (MSI), its extension to spatially resolved analysis of tissues, is a powerful tool for visualizing molecular information within the histological context of tissue. This review summarizes recent developments in MSI and highlights current challenges that remain to achieve molecular imaging at the cellular level of clinical specimens.

Focus on the glycerophosphocholine pathway in choline phospholipid metabolism of cancer.

Activated choline metabolism is a hallmark of carcinogenesis and tumor progression, which leads to elevated levels of phosphocholine and glycerophosphocholine in all types of cancer tested so far. Magnetic resonance spectroscopy applications have played a key role in detecting these elevated choline phospholipid metabolites. To date, the majority of cancer-related studies have focused on phosphocholine and the Kennedy pathway, which constitutes the biosynthesis pathway for membrane phosphatidylcholine.

Phosphorylation-dependent protein design: design of a minimal protein kinase-inducible domain.

Protein kinases and phosphatases modulate protein structure and function, which in turn regulate cellular activities. The development of novel proteins and protein motifs that are responsive to protein phosphorylation provides new ways to probe the functions of individual protein kinases and the intracellular effects of their activation and downregulation. Herein we develop a minimal motif that is responsive to protein phosphorylation, termed a minimal protein kinase-inducible domain.

Wireless control of cellular function by activation of a novel protein responsive to electromagnetic fields.

The Kryptopterus bicirrhis (glass catfish) is known to respond to electromagnetic fields (EMF). Here we tested its avoidance behavior in response to static and alternating magnetic fields stimulation. Using expression cloning we identified an electromagnetic perceptive gene (EPG) from the K.

Perfluoro-tert-butyl Homoserine Is a Helix-Promoting, Highly Fluorinated, NMR-Sensitive Aliphatic Amino Acid: Detection of the Estrogen Receptor·Coactivator Protein-Protein Interaction by F NMR.

Highly fluorinated amino acids can stabilize proteins and complexes with proteins, via enhanced hydrophobicity, and provide novel methods for identification of specific molecular events in complex solutions, via selective detection by F NMR and the absence of native F signals in biological contexts. However, the potential applications of F NMR in probing biological processes are limited both by the strong propensities of most highly fluorinated amino acids for the extended conformation and by the relatively modest sensitivity of NMR spectroscopy, which typically constrains measurements to mid-micromolar concentrations. Herein, we demonstrate that perfluoro-tert-butyl homoserine exhibits a propensity for compact conformations, including α-helix and polyproline helix (PPII), that is similar to that of methionine.

Synthesis of Perfluoro-tert-butyl Tyrosine, for Application in F NMR, via a Diazonium-Coupling Reaction.

A practical synthesis of the novel highly fluorinated amino acid Fmoc-perfluoro-tert-butyl tyrosine was developed. The sequence proceeds in two steps from commercially available Fmoc-4-NH-phenylalanine via diazotization followed by diazonium coupling reaction with perfluoro-tert-butanol. In peptides, perfluoro-tert-butyl tyrosine was detected in 30 s by NMR spectroscopy at 500 nM peptide concentration due to nine chemically equivalent fluorines that are a sharp singlet by F NMR.

(2S,4R)- and (2S,4S)-perfluoro-tert-butyl 4-hydroxyproline: two conformationally distinct proline amino acids for sensitive application in 19F NMR.

(2S,4R)- and (2S,4S)-perfluoro-tert-butyl 4-hydroxyproline were synthesized (as Fmoc-, Boc-, and free amino acids) in 2-5 steps. The key step of each synthesis was a Mitsunobu reaction with perfluoro-tert-butanol, which incorporated a perfluoro-tert-butyl group, with nine chemically equivalent fluorines. Both amino acids were incorporated in model α-helical and polyproline helix peptides.