Acute Fetal Metabolomic Changes in Twins Undergoing Fetoscopic Surgery for Twin-Twin Transfusion Syndrome.

Fetuses undergo major surgical stress as well as fluid shifts secondary to both twin-twin transfusion (TTTS) as well as the fetoscopic surgery for treatment of TTTS. While the pathophysiology of TTTS is understood, the acute metabolic changes that fetuses experience from fetoscopic surgery are not. We sought to evaluate the changes in recipient metabolomic profile secondary to TTTS surgery. Amniotic fluid was collected at the beginning and end of four TTTS surgical cases performed from 12/2022-2/2023. Samples were immediately processed and evaluated via NMR-based Metabolomics Facility protocol. In univariate analysis, 12 metabolites (glucose, lactate, and 10 key amino acids) showed statistically significant changes between the beginning and end of the surgery. Among these, 11 metabolites decreased at the end, while only lactate increased. Supervised oPLS-DA modeling revealed pyruvate and lactate as the two metabolites most impact on the variance between cases, and that 40% of metabolomic changes could be attributed directly to the timing that the sample was taken (i.e., if pre- or postoperatively). These results indicate significant metabolic changes in the recipient twin during fetoscopic surgery for TTTS. These findings of decreased glucose, increased lactate, and decreased amnio acids would indicate increased catabolism during surgery. This study raises questions regarding optimal maternal and fetal nutrition during surgery and if nutritional status could be optimized to further improve twin survival during fetoscopic surgery.

A randomized phase 2 trial of oral vitamin A for graft-versus-host disease in children and young adults.

ABSTRACT: Vitamin A plays a key role in the maintenance of gastrointestinal homeostasis and promotes a tolerogenic phenotype in tissue resident macrophages. We conducted a prospective randomized double-blinded placebo-controlled clinical trial in which 80 recipients of hematopoietic stem cell transplantation (HSCT) were randomized 1:1 to receive pretransplant high-dose vitamin A or placebo. A single oral dose of vitamin A of 4000 IU/kg, maximum 250 000 IU was given before conditioning. The primary end point was incidence of acute graft-versus-host disease (GVHD) at day +100. In an intent-to-treat analysis, incidence of acute GVHD was 12.5% in the vitamin A arm and 20% in the placebo arm (P = .5). Incidence of acute gastrointestinal (GI) GVHD was 2.5% in the vitamin A arm (P = .09) and 12.5% in the placebo arm at day +180. Incidence of chronic GVHD was 5% in the vitamin A arm and 15% in the placebo arm (P = .02) at 1 year. In an "as treated" analysis, cumulative incidence of acute GI GVHD at day +180 was 0% and 12.5% in recipients of vitamin A and placebo, respectively (P = .02), and cumulative incidence of chronic GVHD was 2.7% and 15% in recipients of vitamin A and placebo, respectively (P = .01). The only possibly attributable toxicity was asymptomatic grade 3 hyperbilirubinemia in 1 recipient of vitamin A at day +30, which self-resolved. Absolute CCR9+ CD8+ effector memory T cells, reflecting gut T-cell trafficking, were lower in the vitamin A arm at day +30 after HSCT (P = .01). Levels of serum amyloid A-1, a vitamin A transport protein with proinflammatory effects, were lower in the vitamin A arm. The vitamin A arm had lower interleukin-6 (IL-6), IL-8, and suppressor of tumorigenicity 2 levels and likely a more favorable gut microbiome and short chain fatty acids. Pre-HSCT oral vitamin A is inexpensive, has low toxicity, and reduces GVHD. This trial was registered at www.ClinicalTrials.gov as NCT03202849.

Fetal lung hypoxia and energetic cell failure in the nitrofen-induced congenital diaphragmatic hernia rat model.

PURPOSE: Congenital diaphragmatic hernia (CDH) pathogenesis is poorly understood. We hypothesize that fetal CDH lungs are chronically hypoxic because of lung hypoplasia and tissue compression, affecting the cell bioenergetics as a possible explanation for abnormal lung development. METHODS: To investigate this theory, we conducted a study using the rat nitrofen model of CDH. We evaluated the bioenergetics status using H1 Nuclear magnetic resonance and studied the expression of enzymes involved in energy production, the hypoxia-inducible factor 1α, and the glucose transporter 1. RESULTS: The nitrofen-exposed lungs have increased levels of hypoxia-inducible factor 1α and the main fetal glucose transporter, more evident in the CDH lungs. We also found imbalanced AMP:ATP and ADP:ATP ratios, and a depleted energy cellular charge. Subsequent transcription levels and protein expression of the enzymes involved in bioenergetics confirm the attempt to prevent the energy collapse with the increase in lactate dehydrogenase C, pyruvate dehydrogenase kinase 1 and 2, adenosine monophosphate deaminase, AMP-activated protein kinase, calcium/calmodulin-dependent protein kinase 2, and liver kinase B1, while decreasing ATP synthase. CONCLUSION: Our study suggests that changes in energy production could play a role in CDH pathogenesis. If confirmed in other animal models and humans, this could lead to the development of novel therapies targeting the mitochondria to improve outcomes.

Drug resistance mechanisms create targetable proteostatic vulnerabilities in Her2+ breast cancers.

Oncogenic kinase inhibitors show short-lived responses in the clinic due to high rate of acquired resistance. We previously showed that pharmacologically exploiting oncogene-induced proteotoxic stress can be a viable alternative to oncogene-targeted therapy. Here, we performed extensive analyses of the transcriptomic, metabolomic and proteostatic perturbations during the course of treatment of Her2+ breast cancer cells with a Her2 inhibitor covering the drug response, resistance, relapse and drug withdrawal phases. We found that acute Her2 inhibition, in addition to blocking mitogenic signaling, leads to significant decline in the glucose uptake, and shutdown of glycolysis and of global protein synthesis. During prolonged therapy, compensatory overexpression of Her3 allows for the reactivation of mitogenic signaling pathways, but fails to re-engage the glucose uptake and glycolysis, resulting in proteotoxic ER stress, which maintains the protein synthesis block and growth inhibition. Her3-mediated cell proliferation under ER stress during prolonged Her2 inhibition is enabled due to the overexpression of the eIF2 phosphatase GADD34, which uncouples protein synthesis block from the ER stress response to allow for active cell growth. We show that this imbalance in the mitogenic and proteostatic signaling created during the acquired resistance to anti-Her2 therapy imposes a specific vulnerability to the inhibition of the endoplasmic reticulum quality control machinery. The latter is more pronounced in the drug withdrawal phase, where the de-inhibition of Her2 creates an acute surge in the downstream signaling pathways and exacerbates the proteostatic imbalance. Therefore, the acquired resistance mechanisms to oncogenic kinase inhibitors may create secondary vulnerabilities that could be exploited in the clinic.

Maternal regulation of biliary disease in neonates via gut microbial metabolites.

Maternal seeding of the microbiome in neonates promotes a long-lasting biological footprint, but how it impacts disease susceptibility in early life remains unknown. We hypothesized that feeding butyrate to pregnant mice influences the newborn's susceptibility to biliary atresia, a severe cholangiopathy of neonates. Here, we show that butyrate administration to mothers renders newborn mice resistant to inflammation and injury of bile ducts and improves survival. The prevention of hepatic immune cell activation and survival trait is linked to fecal signatures of Bacteroidetes and Clostridia and increases glutamate/glutamine and hypoxanthine in stool metabolites of newborn mice. In human neonates with biliary atresia, the fecal microbiome signature of these bacteria is under-represented, with suppression of glutamate/glutamine and increased hypoxanthine pathways. The direct administration of butyrate or glutamine to newborn mice attenuates the disease phenotype, but only glutamine renders bile duct epithelial cells resistant to cytotoxicity by natural killer cells. Thus, maternal intake of butyrate influences the fecal microbial population and metabolites in newborn mice and the phenotypic expression of experimental biliary atresia, with glutamine promoting survival of bile duct epithelial cells.

Role of GM-CSF in regulating metabolism and mitochondrial functions critical to macrophage proliferation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts pleiotropic effects on macrophages and is required for self-renewal but the mechanisms responsible are unknown. Using mouse models with disrupted GM-CSF signaling, we show GM-CSF is critical for mitochondrial turnover, functions, and integrity. GM-CSF signaling is essential for fatty acid ß-oxidation and markedly increased tricarboxylic acid cycle activity, oxidative phosphorylation, and ATP production. GM-CSF also regulated cytosolic pathways including glycolysis, pentose phosphate pathway, and amino acid synthesis. We conclude that GM-CSF regulates macrophages in part through a critical role in maintaining mitochondria, which are necessary for cellular metabolism as well as proliferation and self-renewal.

Fibrinogen activates focal adhesion kinase (FAK) promoting colorectal adenocarcinoma growth.

BACKGROUND: We previously showed that fibrinogen is a major determinant of the growth of a murine model of colorectal cancer (CRC). OBJECTIVE: Our aim was to define the mechanisms coupling fibrin(ogen) to CRC growth. RESULTS: CRC tumors transplanted into the dorsal subcutis of Fib- mice were less proliferative and demonstrated increased senescence relative to those grown in Fib+ mice. RNA-seq analyses of Fib+ and Fib- tumors revealed 213 differentially regulated genes. One gene highly upregulated in tumors from Fib- mice was stratifin, encoding 14-3-3σ, a master regulator of proliferation/senescence. In a separate cohort, we observed significantly increased protein levels of 14-3-3σ and its upstream and downstream targets (i.e., p53 and p21) in tumors from Fib- mice. In vitro analyses demonstrated increased tumor cell proliferation in a fibrin printed three-dimensional environment compared with controls, suggesting that fibrin(ogen) in the tumor microenvironment promotes tumor growth in this context via a tumor cell intrinsic mechanism. In vivo analyses showed diminished activation of focal adhesion kinase (FAK), a key negative regulator of p53, in Fib- tumors. Furthermore, nuclear magnetic resonance-based metabolomics demonstrated significantly reduced metabolic activity in tumors from Fib- relative to Fib+ mice. Together, these findings suggest that fibrin(ogen)-mediated engagement of colon cancer cells activates FAK, which inhibits p53 and its downstream targets including 14-3-3σ and p21, thereby promoting cellular proliferation and preventing senescence. CONCLUSIONS: These studies suggest that fibrin(ogen) is an important component of the colon cancer microenvironment and may be exploited as a potential therapeutic target.

Lung Metabolomics Profiling of Congenital Diaphragmatic Hernia in Fetal Rats.

Congenital diaphragmatic hernia (CDH) is characterized by the herniation of abdominal contents into the thoracic cavity during the fetal period. This competition for fetal thoracic space results in lung hypoplasia and vascular maldevelopment that can generate severe pulmonary hypertension (PH). The detailed mechanisms of CDH pathogenesis are yet to be understood. Acknowledgment of the lung metabolism during the in-utero CDH development can help to discern the CDH pathophysiology changes. Timed-pregnant dams received nitrofen or vehicle (olive oil) on E9.5 day of gestation. All fetal lungs exposed to nitrofen or vehicle control were harvested at day E21.5 by C-section and processed for metabolomics analysis using nuclear magnetic resonance (NMR) spectroscopy. The three groups analyzed were nitrofen-CDH (NCDH), nitrofen-control (NC), and vehicle control (VC). A total of 64 metabolites were quantified and subjected to statistical analysis. The multivariate analysis identified forty-four metabolites that were statistically different between the three groups. The highest Variable importance in projection (VIP) score (>2) metabolites were lactate, glutamate, and adenosine 5'-triphosphate (ATP). Fetal CDH lungs have changes related to oxidative stress, nucleotide synthesis, amino acid metabolism, glycerophospholipid metabolism, and glucose metabolism. This work provides new insights into the molecular mechanisms behind the CDH pathophysiology and can explore potential novel treatment targets for CDH patients.

Reduced Gut Microbiome Diversity and Metabolome Differences in Rhinoceros Species at Risk for Iron Overload Disorder.

Iron overload disorder (IOD) affects many wildlife species cared for ex situ. Two of the four rhinoceros species in human care, Sumatran rhinoceros (Dicerorhinus sumatrensis) and black rhinoceros (Diceros bicornis), are susceptible, whereas the other two, white rhinoceros (Ceratotherium simum) and greater one-horned (GOH) rhinoceros (Rhinoceros unicornis), are relatively resistant to IOD. Complex interrelationships exist between mammalian hosts, their indigenous gut microbiota, metabolome, physical condition, and iron availability. The goal of this study was to gain insight into these relationships within the family Rhinocerotidae. Specific objectives were to (1) characterize the gut microbiome and metabolome of four rhinoceros species; (2) compare the microbiome and metabolome of IOD-susceptible and IOD-resistant rhinoceros species; and (3) identify variation in the microbiome and metabolome associated with compromised health or disease in IOD-susceptible rhinoceroses. Fecal samples were collected from 31 rhinoceroses (Sumatran rhinoceros, n = 3; black rhinoceros, n = 6; GOH rhinoceros, n = 9; white rhinoceros, n = 13) located at five facilities, and matched fecal aliquots were processed for microbiome and metabolome analyses using 16S rRNA gene sequencing and nuclear magnetic resonance spectroscopy, respectively. Despite the phylogenetic disparity and dissimilar zoo diets of the hosts, the structure of the fecal microbiota of the two IOD-susceptible rhinoceros species were more closely related to each other than to those of the two IOD-resistant species (Bray-Curtis dissimilarity; IOD-susceptible vs. IOD-resistant p-value < 0.001). In addition, IOD-susceptible rhinoceroses exhibited less microbial diversity than their IOD-resistant relatives (Shannon diversity; p-value < 0.001) which could have health implications. Of note, the black rhinoceros was distinct among the four rhinoceros species with the most divergent fecal metabolome; interestingly, it contained higher concentrations of short chain fatty acids. Neither age nor sex were associated with differences in microbial community composition (p = 0.253 and 0.488, respectively) or fecal metabolomic profile (p = 0.634 and 0.332, respectively). Differences in the distal gut microbiomes between IOD-resistant and IOD-susceptible rhinoceroses support hypotheses that gut microbes play a role in host iron acquisition, and further studies and experiments to test these hypotheses are warranted.