Faculty Bibliography

The Integrated Stress Response (ISR) mediates cellular adaptation to endoplasmic reticulum (ER) stress, amino acid deprivation, and mitochondrial dysfunction. The ISR regulates gene expression in part by preferentially translating the transcription factor ATF4, a process regulated by upstream open reading frames (uORFs) in its 5' leader. In Drosophila, Xrp1 is another transcription factor induced during the ISR, but the precise underlying mechanism remains unclear. Here, we report that Xrp1 induction in response to ER stress is regulated by both its uORFs and the main ORF sequence. Xrp1 has seven splice isoforms, and the two predominant transcripts expressed in eye imaginal discs contain uORFs. Expressing the ER stress-imposing ninaEG69D transgene in this tissue induced Xrp1 expression without significantly changing the Xrp1 splice isoform composition. The uORF-containing 5' leaders, particularly the AUG codon of the second uORF, inhibited DsRed expression when placed upstream of the reporter. Unlike ATF4, the uORF-containing 5' leader alone was insufficient to mediate the main ORF induction, but Xrp1 induction occurred in ninaEG69D-expressing discs when Xrp1's 5' leader and the main ORF sequence were both present. Functionally, Xrp1 was required to maintain the integrity of Drosophila photoreceptors exposed to constant light. In a different disease model, parkin mutants activated Xrp1 target gene expression in specific tissues and Xrp1 loss enhanced the viability of parkin mutant flies during adult eclosion. These results provide molecular and pathological insights into Xrp1 regulation and function in disease models.

BACKGROUND:Transmural healing (TMH) indicates resolution of inflammation in all bowel wall layers and is an emerging therapeutic target in Crohn's disease (CD). Standardized sonographic criteria for TMH and early improvement, termed Transmural Response (TMR), have not been established. This systematic review synthesizes published definitions to provide an up-to-date overview of the current evidence base for intestinal ultrasound (IUS)-based assessment in CD. METHODS:This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Comprehensive searches of databases identified full-text articles that pre-specified TMH, TMR or normal/abnormal bowel on trans-abdominal IUS in pediatric or adult participants with CD. Definitions were summarized descriptively. RESULTS:Eighty-three full-text studies (8033 patients) met eligibility criteria; 39 (47%) defined TMH and 22 (27%) defined TMR. TMH definitions most often included bowel-wall thickness (BWT) ≤ 3mm (31/39, 79%), absent or minimal Doppler flow (25/39, 64%), and preserved bowel wall stratification (10/39, 26%). All TMR definitions required BWT reduction, but thresholds varied (absolute ≥ 1 mm or relative ≥ 25% in 16/22, 73%). Nine studies (9/22, 41%) also required Doppler flow improvement and 4/22 (18%) included additional criteria. Pediatric-specific criteria were reported in 2 TMH and one TMR studies, extrapolating from adult BWT values. Heterogeneity precluded quantitative pooling. CONCLUSIONS:Standardized IUS definitions of TMH and TMR in CD are lacking. Consistent, validated criteria are essential to enable reproducible ultrasound endpoints, support treat-to-target strategies, and facilitate incorporation of IUS into CD clinical trials and routine care.

BACKGROUND/UNASSIGNED:-deficiency in epicardium-derived cells (EPDCs) contributes to fibro-inflammatory signaling and ACM pathogenesis. METHODS/UNASSIGNED:in cardiomyocytes (Pkp2-cKO), in EPDC (Pkp2-eKO), or in both cardiomyocyte and EPDC (Pkp2-ceKO) via the tissue-specific expression of tamoxifen-inducible Cre recombinase. Nonmyocyte populations were isolated 21 days posttamoxifen injection for single-cell RNA-sequencing. Immunohistochemistry, flow cytometry, quantitative reverse transcription polymerase chain reaction, and echocardiography were used to interrogate cardiac physiology and cellular composition. RESULTS/UNASSIGNED:deletion in cardiomyocytes induced a moderate fibro-inflammatory EPDC phenotype, while deletion in EPDC did not elicit a pathological phenotype, suggesting cardiomyocyte involvement is necessary for ACM pathogenesis. Proinflammatory fibroblasts acquired the senescence-associated secretory phenotype, correlating with elevated senescence associated-βgal staining in the right ventricle. Gene expression, flow cytometry, and histological data also revealed an exaggerated inflammatory response in Pkp2-ceKO mice, which progresses from right to left ventricular predominance. Importantly, macrophages and B cells accumulate in both Pkp2-cKO and Pkp2-ceKO mice compared with controls. Although B-cell depletion delays the early inflammatory and fibrosis response, it did not alter end-stage cardiac physiology. CONCLUSIONS/UNASSIGNED:

Among the ways by which oncogenic KRAS upregulates glycolysis in cancer is direct interaction of KRAS4A with hexokinase 1 (HK1), but the mechanism is unknown. HK1 associates with the outer mitochondrial membrane (OMM) where its allosteric regulation depends on homodimerization. Using affinity capture, FRET, and blue native gels, we show that KRAS4A enhances oligomerization of HK1 on the OMM. Modeling the HK1/KRAS4A complex with AlphaFold3 predicts that the membrane association sequences of both HK1 and KRAS4A are oriented toward the OMM. Super-resolution microscopy showed colocalization of HK1 and KRAS4A on the OMM with HK1 enriched at discrete locations. Single-molecule tracking reveals HK1 diffusing freely along the OMM and dwelling at discrete regions where two molecules can be seen to colocalize transiently. KRAS4A expression decreased the diffusion coefficient of HK1 on the organelle. Thus, KRAS4A alters the dynamics of HK1 on the OMM and promotes oligomerization.

De novo protein synthesis is required for long-lasting synaptic plasticity and memory, but it comes with a great metabolic cost. In the mammalian brain, it remains unclear which cell types and biological mechanisms are critical for sensing and responding to increased metabolic demand. Here, we demonstrate that microglia, the resident macrophages of the brain, are required for metabolic coupling between endothelial cells, astrocytes, and neurons, which fuels protein synthesis in active neurons. Increasing metabolic demand via a motor task stimulates microglia to secrete the hypoxia-responsive protein CYR61, which increases glucose transporter expression in brain vasculature. Depleting microglia reduces training-induced metabolic fluxes and neuronal protein synthesis, which can be reproduced by blocking CYR61 signaling. Thus, we define a neuroimmune metabolic circuit that is required for on-demand protein synthesis in mouse motor cortex.

A deficit in dietary protein elicits a nutrient-specific appetite, yet the underlying mechanisms remain poorly understood. In this work, we identify coordinated neuronal and systemic mechanisms in Drosophila that drive an essential amino acid (EAA)-specific appetite. EAA deprivation increases neuropeptide CNMamide (CNMa) expression in gut enterocytes, activating enteric neurons and ellipsoid body neurons in the brain to promote EAA intake through two complementary pathways: a rapid neuronal gut-brain axis and a slower hormonal route. CNMa suppresses the activity of sugar-sensing diuretic hormone 44 (DH44) neurons, thereby reducing carbohydrate intake and biasing feeding toward EAAs. Similarly, protein deprivation in mice promotes an EAA-specific appetite independently of fibroblast growth factor 21 (FGF21). Together, these findings reveal multilayered gut-brain mechanisms that regulate nutrient-specific feeding and maintain EAA homeostasis across species.

Lysosomal phospholipid degradation produces two types of metabolites, either 2-lysophospholipids with saturated fatty acids in sn-1 position or 1-lysophospholipids with unsaturated fatty acids in sn-2 position. They may either be degraded further or re-used for phospholipid synthesis. We found that LPLAT7 (LPGAT1), an acyltransferase of the endoplasmic reticulum, re-acylates specifically lysosome-derived 1-lysophospholipids that carry an unsaturated chain. The enzymatic activity of LPLAT7 was specific for stearoyl-CoA and 1-lyso-2-acyl positional isomers of unsaturated lysophospholipids. In Huh7 cells, Lplat7 knockout prevented the reacylation of 1-lysophospholipids generated by the lysosomal degradation of exogenous ²H-phosphatidylcholine. Inhibition of lysosomal phospholipid degradation reduced the abundance of 1-stearoyl-2-unsaturated PC in Huh7 cells. Lplat7 knockout blunted the loss of unsaturated lysophosphatidylcholine (LPC) in response to lysosomal inhibition, suggesting that LPLAT7 consumes unsaturated LPC formed by lysosomes. In mice, Lplat7 knockout increased the concentration of unsaturated lysophospholipids, reduced the abundance of 1-stearoyl-2-unsaturated species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, and inhibited the regeneration of cellular membranes. It also triggered the accumulation of triglycerides, confirming earlier reports that unsaturated lysophospholipids induce lipid droplet formation. Thus, by re-acylating unsaturated 1-lysophospholipids, LPLAT7 shifts lipid metabolism from the biogenesis of lipid droplets to the biogenesis of membranes.

BACKGROUND:Exercise links with improved cancer outcomes following a diagnosis of primary breast cancer but experimental evidence and molecular mechanistic interrogation from preclinical studies are limited. The purpose of this study was to evaluate the effects, and dose‒response, of exercise in mouse models of breast cancer, as well as elucidate cancer cell extrinsic and intrinsic responses. METHODS:Independent in vivo modeling was used to investigate the effects of exercise across distinct breast cancer models. Unbiased transcriptomic and metabolomic analyses, alongside cellular and proteomic interrogation, were used to determine tumor microenvironment (TME)- and cancer cell-specific effects. RESULTS:Exercise inhibited breast cancer growth and metastasis across multiple syngeneic mouse models compared to sham control. Tumor growth inhibition was independent of estrogen receptor status, and in the 4T1 model, exercise exerted non-dose-dependent effects. In the Met1 model, exercise decreased TME immune cell content, particularly tumor-associated macrophages, while promoting an activated anticancer innate immune cell gene signature. Concurrent in vivo cancer cell-intrinsic effects were characterized by broad transcriptomic reprogramming including downregulation of metabolic pathways and upregulation of pathways regulating proliferation and apoptosis. Whole tumor metabolomic analyses unveiled broad shifts including decreased nicotinamide adenine dinucleotide (NAD+) and lactate, as well as availability of biosynthetic precursors. Finally, in silico analyses identified TME ligands, such as High Mobility Group Box 2 (HMGB2) and Cardiotrophin-1 (CTF1) as candidate drivers of downstream gene expression changes in cancer cells. CONCLUSION/CONCLUSIONS:Exercise suppresses breast cancer progression, which occurs in conjunction with broad reprogramming of immune TME-cancer processes and their interaction.