Immunometabolic Maladaptations to the Tumor Microenvironment.

Tumors consist of cancer cells and a wide range of tissue resident and infiltrating cell types. Tumor metabolism, however, has largely been studied on whole tumors or cancer cells and the metabolism of infiltrating immune cells remains poorly understood. It is now clear from a range of analyses and metabolite rescue studies that metabolic adaptations to the tumor microenvironment (TME) directly impede T-cell and macrophage effector functions. The drivers of metabolic adaptation to the TME and metabolic immune suppression include depletion of essential nutrients, accumulation of waste products or immune suppression metabolites, and metabolic signaling through altered posttranslational modifications. Each infiltrating immune cell subset differs, however, with specific metabolic requirements and adaptations that can be maladaptive for antitumor immunity. Here, we review T-cell and macrophage adaptation and metabolic immune suppression in solid tumors. Ultimately, understanding and addressing these challenges will improve cancer immunotherapy and adoptive chimeric antigen receptor T-cell therapies.

Lactoylglutathione promotes inflammatory signaling in macrophages through histone lactoylation.

Chronic, systemic inflammation is a pathophysiological manifestation of metabolic disorders. Inflammatory signaling leads to elevated glycolytic flux and a metabolic shift towards aerobic glycolysis and lactate generation. This rise in lactate corresponds with increased generation of lactoylLys modifications on histones, mediating transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGSH). Here, we report a regulatory role for LGSH in mediating histone lactoylation and inflammatory signaling. In the absence of the primary LGSH hydrolase, glyoxalase 2 (GLO2), RAW264.7 macrophages display significant elevations in LGSH and histone lactoylation with a corresponding potentiation of the inflammatory response when exposed to lipopolysaccharides. An analysis of chromatin accessibility shows that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state; upon stimulation, however, regions of the genome associated with lactoylation become markedly more accessible. Lastly, we demonstrate a spontaneous S-to-S acyltransfer of lactate from LGSH to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGSH, and not intracellular lactate, is the primary driving factor facilitating histone lactoylation and a major contributor to inflammatory signaling.

Lactoylglutathione promotes inflammatory signaling in macrophages.

Chronic, systemic inflammation is a pathophysiological manifestation of metabolic disorders. Inflammatory signaling leads to elevated glycolytic flux and a metabolic shift towards aerobic glycolysis and lactate generation. This rise in lactate corresponds with increased generation of lactoylLys modifications on histones, mediating transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGSH). Here, we report a regulatory role for LGSH in inflammatory signaling. In the absence of the primary LGSH hydrolase, glyoxalase 2 (GLO2), RAW264.7 macrophages display significant elevations in LGSH, while demonstrating a potentiated inflammatory response when exposed to lipopolysaccharides, corresponding with a rise in histone lactoylation. Interestingly, our data demonstrate that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state, however, upon stimulation, regions of the genome associated with lactoylation become markedly more accessible. Lastly, we demonstrate a spontaneous S-to-S acyltransfer of lactate from LGSH to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGSH, and not intracellular lactate, is a primary contributing factor facilitating the inflammatory response.

A comparison of primary and secondary eye removal after open globe injury: A multi-centre study.

BACKGROUND/OBJECTIVES: Our goal was to compare the characteristics and surgical outcomes of patients who underwent primary eye removal surgery after open globe injury with those who underwent secondary eye removal surgery after open globe repair. SUBJECTS/METHODS: This was a retrospective review of subjects who underwent evisceration or enucleation within 3 months of an open globe injury, at three Level I trauma centres in three U.S. cities between July 2014 and July 2020. RESULTS: 19 patients underwent primary eye removal and 20 underwent secondary eye removal. The most common mechanism of trauma in patients who underwent primary eye removal was gunshot. Compared to the secondary eye removal group, patients who underwent primary eye removal were significantly more likely to be male; have longer hospital stays; be discharged to another care facility rather than home; have facial fractures; suffer intracranial injury; and be unable to consent themselves for surgery. Both groups had a low surgical complication rate with one case of socket contracture in each group. CONCLUSIONS: The standard of care for an open globe injury is prompt repair, but there are occasions when the globe is so damaged that it is deemed unrepairable. We found that globes that required primary eye removal were more often due to gunshot wounds, and that there was greater morbidity associated with these injuries. The authors' preferred surgical approach was evisceration with placement of a silicone sphere; patient outcomes demonstrate that this method was found to be safe, with a low complication and infection rate.

Biochemical Mechanisms of Sirtuin-Directed Protein Acylation in Hepatic Pathologies of Mitochondrial Dysfunction.

Mitochondrial protein acetylation is associated with a host of diseases including cancer, Alzheimer's, and metabolic syndrome. Deciphering the mechanisms regarding how protein acetylation contributes to disease pathologies remains difficult due to the complex diversity of pathways targeted by lysine acetylation. Specifically, protein acetylation is thought to direct feedback from metabolism, whereby nutritional status influences mitochondrial pathways including beta-oxidation, the citric acid cycle, and the electron transport chain. Acetylation provides a crucial connection between hepatic metabolism and mitochondrial function. Dysregulation of protein acetylation throughout the cell can alter mitochondrial function and is associated with numerous liver diseases, including non-alcoholic and alcoholic fatty liver disease, steatohepatitis, and hepatocellular carcinoma. This review introduces biochemical mechanisms of protein acetylation in the regulation of mitochondrial function and hepatic diseases and offers a viewpoint on the potential for targeted therapies.

Chemical Labeling and Enrichment of Histone Glyoxal Adducts.

Because of their long half-lives and highly nucleophilic tails, histones are particularly susceptible to accumulating nonenzymatic covalent modifications, such as glycation. The resulting modifications can have profound effects on cellular physiology due to the regulatory role histones play in all DNA-templated processes; however, the complexity of Maillard chemistry on proteins makes tracking and enriching for glycated proteins a challenging task. Here, we characterize glyoxal (GO) modifications on histones using quantitative proteomics and an aniline-derived GO-reactive probe. In addition, we leverage this chemistry to demonstrate that the glycation regulatory proteins DJ-1 and GLO1 reduce levels of histone GO adducts. Finally, we employ a two-round pull-down method to enrich histone H3 GO glycation and map these adducts to specific chromatin regions.

Biochemical genesis of enzymatic and non-enzymatic post-translational modifications.

Post-translational modifications (PTMs) alter protein structure, function, and localization and play a pivotal role in physiological and pathophysiological conditions. Many PTMs arise from endogenous metabolic intermediates and serve as sensors for metabolic feedback to maintain cell growth and homeostasis. A key feature to PTMs is their biochemical genesis, which can result from either non-enzymatic adduction (nPTMs) or through enzyme-catalyzed reactions (ePTMs). The abundance and site-specificity of PTMs are determined by dedicated classes of enzymes that add (writers) or remove (erasers) the chemical addition. In this review we will highlight the biochemical genesis and regulation of a few of the 700+ PTMs that have been identified.

Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.

Sirtuin 2 Regulates Protein LactoylLys Modifications.

Post-translational modifications (PTMs) play roles in both physiological and pathophysiological processes through the regulation of enzyme structure and function. We recently identified a novel PTM, lactoylLys, derived through a nonenzymatic mechanism from the glycolytic by-product, lactoylglutathione. Under physiologic scenarios, glyoxalase 2 prevents the accumulation of lactoylglutathione and thus lactoylLys modifications. What dictates the site-specificity and abundance of lactoylLys PTMs, however, remains unknown. Here, we report sirtuin 2 as a lactoylLys eraser. Using chemical biology and CRISPR-Cas9, we show that SIRT2 controls the abundance of this PTM both globally and on chromatin. These results address a major gap in our understanding of how nonenzymatic PTMs are regulated and controlled.

Trisomy 21 impairs PGE2 production in dermal fibroblasts.

The triplication of human chromosome 21 results in Down syndrome (DS), the most common genetic form of intellectual disability. This aneuploid condition also results in an enhanced risk of a spectrum of comorbid conditions, such as leukemia, early onset Alzheimer's disease, and diabetes. Individuals with DS also display an increased incidence of wound healing complications and resistance to solid tumor development. Due to this unique phenotype and the involvement of eicosanoids in key comorbidities like poor healing and tumor development, we hypothesized that cells from DS individuals would display altered eicosanoid production. Using age- and sex-matched dermal fibroblasts we interrogated this hypothesis. Briefly, assessment of over 90 metabolites derived from cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome p450 systems revealed a possible deficiency in the COX system. Basal gene expression and Western blotting experiments showed significantly decreased gene expression of COX1 and 2, and COX2 protein abundance in DS fibroblasts compared to euploid controls. Further, using two different stressors, scratch wound or LPS, we found that DS fibroblasts could not upregulate COX2 abundance and prostaglandin E2 production. Together, these findings show that dermal fibroblasts from DS individuals have a deficient COX2 response, which may contribute to wound healing complications and tumor resistance in DS.

Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling.

Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.

Advancements in the repair of large upper eyelid defects: A 10-year review.

PURPOSE: The reconstruction of large (>50%) upper eyelid margin defects can be technically challenging, with multiple approaches described in the literature. We sought to review the recent literature for new techniques or modifications to existing techniques. METHODS: We conducted a Pubmed search for technique papers on the reconstruction of large upper eyelid defects published within the past ten years with a minimum of four patients. RESULTS: We identified ten articles, and divided them into techniques that use a bridging flap from the lower eyelid and those that do not. The number of upper eyelids repaired in each article ranged from 4 to 17. Most techniques could be considered either a modification of the Cutler-Beard technique or a novel anterior lamella flap laid over a graft for the posterior lamella. Postoperative complications included upper or lower eyelid cicatricial retraction, trichiasis, entropion, and lagophthalmos. CONCLUSIONS: Surgeons continue to innovate for this challenging reconstructive surgery. Overall, the trend was to use a graft, most commonly tarsoconjunctiva from the contralateral upper lid, to replace the posterior lamella, and a skin flap, from the lower eyelid or from the adjacent periorbital area, to replace the anterior lamella. Bridging techniques utilized the skin; the skin, orbicularis, and conjunctiva; or a tarsoconjunctival flap from the lower eyelid. Non-bridging techniques generally used a tarsoconjunctival or substitute graft for the posterior lamella, and a skin flap for the anterior lamella.

Toxic erythema of chemotherapy secondary to gemcitabine and paclitaxel.

Toxic erythema of chemotherapy (TEC) is an infrequently reported cutaneous condition, with diagnosis predominately based on clinical presentation, histologic findings, and known reported associations. Therefore, it is important to both recognize common presentations of TEC and be mindful of chemotherapeutic agents associated with this cutaneous side effect to prevent misdiagnosis and prolonged time to treatment. Herein, we present a patient with TEC occurring in intertriginous skin (malignant intertrigo) with classic clinical and histologic findings. In our patient this was associated with a combination neoadjuvant gemcitabine and paclitaxel therapy, a relationship that, to our knowledge, has yet to be reported in the literature.

The sunless tanning agent dihydroxyacetone induces stress response gene expression and signaling in cultured human keratinocytes and reconstructed epidermis.

Sunless (chemical) tanning is widely regarded as a safe alternative to solar UV-induced skin tanning known to be associated with epidermal genotoxic stress, but the cutaneous biology impacted by chemical tanning remains largely unexplored. Chemical tanning is based on the formation of melanin-mimetic cutaneous pigments ('melanoidins') from spontaneous amino-carbonyl ('glycation') reactions between epidermal amino acid/protein components and reactive sugars including the glycolytic ketose dihydroxyacetone (DHA). Here, we have examined the cutaneous effects of acute DHA-exposure on cultured human HaCaT keratinocytes and epidermal reconstructs, profiled by gene expression array analysis and immunodetection. In keratinocytes, DHA-exposure performed at low millimolar concentrations did not impair viability while causing a pronounced cellular stress response as obvious from rapid activation of phospho-protein signal transduction [p-p38, p-Hsp27(S15/S78), p-eIF2α] and gene expression changes (HSPA6, HMOX1, CRYAB, CCL3), not observable upon exposure to the non-ketose, tanning-inactive DHA-control glycerol. Formation of advanced glycation end products (AGEs) from posttranslational protein-adduction was confirmed by quantitative mass spectrometric detection of N-ε-(carboxyethyl)-l-lysine (CEL) and N7-carboxyethyl-l-arginine, and skin cells with CRISPR-Cas9-based elimination of the carbonyl stress response gene GLO1 (encoding glyoxalase 1) displayed hypersensitivity to DHA-cytotoxicity. In human epidermal reconstructs a topical use-relevant DHA-dose regimen elicited a comparable stress response as revealed by gene expression array (HSPA1A, HSPA6, HSPD1, IL6, DDIT3, EGR1) and immunohistochemical analysis (CEL, HO-1, p-Hsp27-S78). In DHA-treated SKH-1 hairless mouse skin IHC-detection revealed epidermal occurrence of CEL- and p-Hsp27-epitopes. For comparison, stress response gene expression array analysis was performed in epidermis exposed to a supra-erythemal dose of solar simulated UV (2 MEDs), identifying genes equally or differentially sensitive to either one of these cutaneous stimuli [DHA ('sunless tanning') versus solar UV ('sun-induced tanning')]. Given the worldwide use of chemical tanners in consumer products, these prototype data documenting a DHA-induced specific cutaneous stress response deserve further molecular exploration in living human skin.

Prophylactic Intravitreal Bevacizumab After Plaque Radiotherapy for Uveal Melanoma: Analysis of Visual Acuity, Tumor Response, and Radiation Complications in 1131 Eyes Based on Patient Age.

PURPOSE: The aim of this study was to determine the impact of age on radiation complications after plaque radiotherapy and prophylactic intravitreal bevacizumab for uveal melanoma. DESIGN: Retrospective cohort study. METHODS: Retrospective single-center study of plaque-irradiated uveal melanoma with prophylactic intravitreal bevacizumab at 4-month intervals from July 2000 to January 2018. RESULTS: Of 1131 eyes in 1131 patients, age was <50 years (n = 231), 50 to 70 years (n = 657), or >70 years (n = 243). Comparison by age category (<50 vs 50-70 vs >70 years) revealed the oldest group presenting with greatest tumor basal diameter (11.3 vs 11.3 vs 12.1 mm, P = 0.03) and worst visual acuity (20/40 vs 20/40 vs 20/50, P = 0.02). After plaque (mean follow-up 40 vs 42 vs 32 months, P < 0.001), radiation complications were most common in the youngest age group, including maculopathy (48% vs 39% vs 28%, P < 0.001), extramacular retinopathy (30% vs 25% vs 16%, P = 0.002), and papillopathy (21% vs 18% vs 12%, P = 0.03). The youngest age group had the highest Kaplan-Meier estimated 48-month cumulative probability for radiation maculopathy (62% vs 46% vs 47%, P = 0.001), extramacular retinopathy (36% vs 34% vs 29%, P = 0.03), and papillopathy (29% vs 26% vs 22%, P = 0.13). On subanalysis, the youngest age group had increased 48-month risk of developing radiation maculopathy when compared with the middle [hazard ratio (HR) = 1.5, P = 0.001] and older (HR = 1.6, P = 0.005) age groups and increased 48-month risk of developing extramacular radiation retinopathy compared with the older age group (HR = 1.5, P = 0.04). CONCLUSIONS: After plaque radiotherapy for uveal melanoma and prophylactic intravitreal bevacizumab at 4-month intervals, patients younger than 50 years old have an increased 48-month risk of radiation maculopathy.

Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes.

Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys" modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions.

Serious games for immersive cultural training: creating a living world.

Living worlds offer a nonlinear, unscripted process for experiencing and safely learning the cognitive complexity and nuance of culture through emergent high-fidelity simulation. The 3D Asymmetric Domain Analysis and Training model uses visual, auditory, behavioral, and cultural models for immersive cultural training using the living-world construct.