Cytidine deaminases APOBEC3C and APOBEC3D promote DNA replication stress resistance in pancreatic cancer cells.

Gemcitabine is a potent inhibitor of DNA replication and is a mainstay therapeutic for diverse cancers, particularly pancreatic ductal adenocarcinoma (PDAC). However, most tumors remain refractory to gemcitabine therapies. Here, to define the cancer cell response to gemcitabine, we performed genome-scale CRISPR-Cas9 chemical-genetic screens in PDAC cells and found selective loss of cell fitness upon disruption of the cytidine deaminases APOBEC3C and APOBEC3D. Following gemcitabine treatment, APOBEC3C and APOBEC3D promote DNA replication stress resistance and cell survival by deaminating cytidines in the nuclear genome to ensure DNA replication fork restart and repair in PDAC cells. We provide evidence that the chemical-genetic interaction between APOBEC3C or APOBEC3D and gemcitabine is absent in nontransformed cells but is recapitulated across different PDAC cell lines, in PDAC organoids and in PDAC xenografts. Thus, we uncover roles for APOBEC3C and APOBEC3D in DNA replication stress resistance and offer plausible targets for improving gemcitabine-based therapies for PDAC.

Sex differences in kidney metabolism may reflect sex-dependent outcomes in human diabetic kidney disease.

Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) and progresses faster in males than in females. We identify sex-based differences in kidney metabolism and in the blood metabolome of male and female individuals with diabetes. Primary human proximal tubular epithelial cells (PTECs) from healthy males displayed increased mitochondrial respiration, oxidative stress, apoptosis, and greater injury when exposed to high glucose compared with PTECs from healthy females. Male human PTECs showed increased glucose and glutamine fluxes to the TCA cycle, whereas female human PTECs showed increased pyruvate content. The male human PTEC phenotype was enhanced by dihydrotestosterone and mediated by the transcription factor HNF4A and histone demethylase KDM6A. In mice where sex chromosomes either matched or did not match gonadal sex, male gonadal sex contributed to the kidney metabolism differences between males and females. A blood metabolomics analysis in a cohort of adolescents with or without diabetes showed increased TCA cycle metabolites in males. In a second cohort of adults with diabetes, females without DKD had higher serum pyruvate concentrations than did males with or without DKD. Serum pyruvate concentrations positively correlated with the estimated glomerular filtration rate, a measure of kidney function, and negatively correlated with all-cause mortality in this cohort. In a third cohort of adults with CKD, male sex and diabetes were associated with increased plasma TCA cycle metabolites, which correlated with all-cause mortality. These findings suggest that differences in male and female kidney metabolism may contribute to sex-dependent outcomes in DKD.

Computational Expansion of High-Resolution-MSn Spectral Libraries.

Commonly, in MS-based untargeted metabolomics, some metabolites cannot be confidently identified due to ambiguities in resolving isobars and structurally similar species. To address this, analytical techniques beyond traditional MS2 analysis, such as MSn fragmentation, can be applied to probe metabolites for additional structural information. In MSn fragmentation, recursive cycles of activation are applied to fragment ions originating from the same precursor ion detected on an MS1 spectrum. This resonant-type collision-activated dissociation (CAD) can yield information that cannot be ascertained from MS2 spectra alone, which helps improve the performance of metabolite identification workflows. However, most approaches for metabolite identification require mass-to-charge (m/z) values measured with high resolution, as this enables the determination of accurate mass values. Unfortunately, high-resolution-MSn spectra are relatively rare in spectral libraries. Here, we describe a computational approach to generate a database of high-resolution-MSn spectra by converting existing low-resolution-MSn spectra using complementary high-resolution-MS2 spectra generated by beam-type CAD. Using this method, we have generated a database, derived from the NIST20 MS/MS database, of MSn spectral trees representing 9637 compounds and 19386 precursor ions where at least 90% of signal intensity was converted from low-to-high resolution.

Protocol for mapping the metabolome and lipidome of medulloblastoma cells using liquid chromatography-mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics and lipidomics have recently been used to show that MYC-amplified group 3 medulloblastoma tumors are driven by metabolic reprogramming. Here, we present a protocol to extract metabolites and lipids from human medulloblastoma brain tumor-initiating cells and normal neural stem cells. We describe untargeted LC-MS methods that can be used to achieve extensive coverage of the polar metabolome and lipidome. Finally, we detail strategies for metabolite identification and data analysis. For complete details on the use and execution of this protocol, please refer to Gwynne et al.1.

Cancer-selective metabolic vulnerabilities in MYC-amplified medulloblastoma.

MYC-driven medulloblastoma (MB) is an aggressive pediatric brain tumor characterized by therapy resistance and disease recurrence. Here, we integrated data from unbiased genetic screening and metabolomic profiling to identify multiple cancer-selective metabolic vulnerabilities in MYC-driven MB tumor cells, which are amenable to therapeutic targeting. Among these targets, dihydroorotate dehydrogenase (DHODH), an enzyme that catalyzes de novo pyrimidine biosynthesis, emerged as a favorable candidate for therapeutic targeting. Mechanistically, DHODH inhibition acts on target, leading to uridine metabolite scarcity and hyperlipidemia, accompanied by reduced protein O-GlcNAcylation and c-Myc degradation. Pyrimidine starvation evokes a metabolic stress response that leads to cell-cycle arrest and apoptosis. We further show that an orally available small-molecule DHODH inhibitor demonstrates potent mono-therapeutic efficacy against patient-derived MB xenografts in vivo. The reprogramming of pyrimidine metabolism in MYC-driven medulloblastoma represents an unappreciated therapeutic strategy and a potential new class of treatments with stronger cancer selectivity and fewer neurotoxic sequelae.

Cauda equina syndrome-the questions.

Cauda equina syndrome is a devastating condition often following an innocent pathology in the form of a disc prolapse. The effect on sufferers, however, can be lifelong. It is necessary to make a diagnosis as expeditiously as possible via adequate history, clinical examination and appropriate imaging to offer treatment, in the form of decompressive surgery within 48 hours. It is extremely important to communicate adequately with the patient and their family recording all the relevant details including those of expected outcome. National guidelines are likely to be of value to clinicians and patients.

Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli.

The pathogenic strategy of Escherichia coli and many other gram-negative pathogens relies on the translocation of a specific set of proteins, called effectors, into the eukaryotic host cell during infection. These effectors act in concert to modulate host cell processes in favor of the invading pathogen. Injected by the type III secretion system (T3SS), the effector arsenal of enterohemorrhagic E. coli (EHEC) O157:H7 features at least eight individual NleG effectors, which are also found across diverse attaching and effacing pathogens. NleG effectors share a conserved C-terminal U-box E3 ubiquitin ligase domain that engages with host ubiquitination machinery. However, their specific functions and ubiquitination targets have remained uncharacterized. Here, we identify host proteins targeted for ubiquitination-mediated degradation by two EHEC NleG family members, NleG5-1 and NleG2-3. NleG5-1 localizes to the host cell nucleus and targets the MED15 subunit of the Mediator complex, while NleG2-3 resides in the host cytosol and triggers degradation of Hexokinase-2 and SNAP29. Our structural studies of NleG5-1 reveal a distinct N-terminal α/ß domain that is responsible for interacting with host protein targets. The core of this domain is conserved across the NleG family, suggesting this domain is present in functionally distinct NleG effectors, which evolved diversified surface residues to interact with specific host proteins. This is a demonstration of the functional diversification and the range of host proteins targeted by the most expanded effector family in the pathogenic arsenal of E. coli.

Galectin-9 binds IgM-BCR to regulate B cell signaling.

The galectin family of secreted lectins have emerged as important regulators of immune cell function; however, their role in B-cell responses is poorly understood. Here we identify IgM-BCR as a ligand for galectin-9. Furthermore, we show enhanced BCR microcluster formation and signaling in galectin-9-deficient B cells. Notably, treatment with exogenous recombinant galectin-9 nearly completely abolishes BCR signaling. We investigated the molecular mechanism for galectin-9-mediated inhibition of BCR signaling using super-resolution imaging and single-particle tracking. We show that galectin-9 merges pre-existing nanoclusters of IgM-BCR, immobilizes IgM-BCR, and relocalizes IgM-BCR together with the inhibitory molecules CD45 and CD22. In resting naive cells, we use dual-color super-resolution imaging to demonstrate that galectin-9 mediates the close association of IgM and CD22, and propose that the loss of this association provides a mechanism for enhanced activation of galectin-9-deficient B cells.