The Michael addition of thiols to 13-oxo-octadecadienoate (13-oxo-ODE) with implications for LC-MS analysis of glutathione conjugation.

Unsaturated fatty acid ketones with αß,γδ conjugation are susceptible to Michael addition of thiols, with unresolved issues on the site of adduction and precise structures of the conjugates. Herein we reacted 13-keto-octadecadienoic acid (13-oxo-ODE or 13-KODE) with glutathione (GSH), N-acetyl-cysteine, and ß-mercaptoethanol and identified the adducts. HPLC-UV analyses indicated none of the products exhibit a conjugated enone UV chromophore, a result that conflicts with the literature and is relevant to the mass spectral interpretation of 1,4 versus 1,6 thiol adduction. Aided by the development of an HPLC solvent system that separates the GSH diastereomers and thus avoids overlap of signals in proton NMR experiments, we established the two major conjugates are formed by 1,6 addition of GSH at the 9-carbon of 13-oxo-ODE with the remaining double bond α to the thiol in the 10,11 position. N-acetyl cysteine reacts similarly, while ß-mercaptoethanol gives equal amounts of 1,4 and 1,6 addition products. Equine glutathione transferase catalyzed 1,6 addition of GSH to the two major diastereomers in 44:56 proportions. LC-MS in positive ion mode gives a product ion interpreted before as evidence of 1,4-thiol adduction, whereas here we find this ion using the authentic 1,6 adduct. LC-MS with negative ion APCI gave a fragment selective for 1,4 adduction. These results clarify the structures of thiol conjugates of a prototypical unsaturated keto-fatty acid and have relevance to the application of LC-MS for the structural analysis of keto-fatty acid glutathione conjugation.

The consumption of animal products is associated with plasma levels of alpha-aminoadipic acid (2-AAA).

BACKGROUND AND AIMS: The cardiometabolic disease-associated metabolite, alpha-aminoadipic acid (2-AAA) is formed from the breakdown of the essential dietary amino acid lysine. However, it was not known whether elevated plasma levels of 2-AAA are related to dietary nutrient intake. We aimed to determine whether diet is a determinant of circulating 2-AAA in healthy individuals, and whether 2-AAA is altered in response to dietary modification. METHODS AND RESULTS: We investigated the association between 2-AAA and dietary nutrient intake in a cross-sectional study of healthy individuals (N = 254). We then performed a randomized cross-over dietary intervention trial to investigate the effect of lysine supplementation (1 week) on 2-AAA in healthy individuals (N = 40). We further assessed the effect of a vegetarian diet on 2-AAA in a short-term (4-day) dietary intervention trial in healthy omnivorous women (N = 35). We found that self-reported dietary intake of animal products, including meat, poultry, and seafood, was associated with higher plasma 2-AAA cross-sectionally (P < 0.0001). Supplementary dietary lysine (5g/day) caused no significant increase in plasma 2-AAA; however, plasma 2-AAA was altered by general dietary modification. Further, plasma 2-AAA was significantly reduced by a short-term vegetarian diet (P = 0.003). CONCLUSION: We identified associations between plasma 2-AAA and consumption of animal products, which were validated in a vegetarian dietary intervention trial, but not in a trial designed to specifically increase the 2-AAA amino acid precursor lysine. Further studies are warranted to investigate whether implementation of a vegetarian diet improves cardiometabolic risk in individuals with elevated 2-AAA.

Tumor therapy by targeting extracellular hydroxyapatite using novel drugs: A paradigm shift.

BACKGROUND: It has been shown that tumor microenvironment (TME) hydroxyapatite (HAP) is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME-HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue. We provide strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME-HAP) using a novel therapeutic can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects. METHODS: We developed an injectable cation exchange nano particulate sulfonated polystyrene solution (NSPS) that we engineered to dissolve TME-HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism. This was evaluated in cell culture using 4T1, MDA-MB-231 triple negative breast cancer cells, MCF10 normal breast cells, and H292 lung cancer cells, and in vivo using orthotopic mouse models of cancer that contained detectable microenvironment HAP including breast (MMTV-Neu, 4T1, and MDA-MB-231), prostate (PC3) and colon (HCA7) cancer using 18 F-NaF for HAP and 18 F-FDG for glucose metabolism with PET imaging. On the other hand, H292 lung tumor cells that lacked detectable microenvironment HAP and MCF10a normal breast cells that do not produce HAP served as negative controls. Tumor microenvironment pH levels following injection of NSPS were evaluated via Chemical Exchange Saturation (CEST) MRI and via ex vivo methods. RESULTS: Within 24 h of adding the small concentration of 1X of NSPS (~7 µM), we observed significant tumor cell death (~ 10%, p < 0.05) in 4T1 and MDA-MB-231 cell cultures that contain HAP but ⟨2% in H292 and MCF10a cells that lack detectable HAP and in controls. Using CEST MRI, we found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration. in the tumors that contained TME-HAP in mouse models, MMTV-Neu, 4T1, and MDA-MB-231, PC3, and HCA7, there was a significant reduction (p<0.05) in 18 F-Na Fuptake post NSPS treatment as expected; 18 F- uptake in the tumor = 3.8 ± 0.5 %ID/g (percent of the injected dose per gram) at baseline compared to 1.8 ±0.5 %ID/g following one-time treatment with 100 mg/kg NSPS. Of similar importance, is that 18 F-FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 h of treatment with one-time NSPS which persisted for at least one week. Additionally, tumor growth was significantly slower (p < 0.05) in the mice treated with one-time NSPS. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone. CONCLUSION: Dissolution of TME-HAP using our novel NSPS has the potential to provide a new treatment paradigm to enhance the management of cancer patients with poor prognosis.

An early-life microbiota metabolite protects against obesity by regulating intestinal lipid metabolism.

The mechanisms by which the early-life microbiota protects against environmental factors that promote childhood obesity remain largely unknown. Using a mouse model in which young mice are simultaneously exposed to antibiotics and a high-fat (HF) diet, we show that Lactobacillus species, predominant members of the small intestine (SI) microbiota, regulate intestinal epithelial cells (IECs) to limit diet-induced obesity during early life. A Lactobacillus-derived metabolite, phenyllactic acid (PLA), protects against metabolic dysfunction caused by early-life exposure to antibiotics and a HF diet by increasing the abundance of peroxisome proliferator-activated receptor γ (PPAR-γ) in SI IECs. Therefore, PLA is a microbiota-derived metabolite that activates protective pathways in the small intestinal epithelium to regulate intestinal lipid metabolism and prevent antibiotic-associated obesity during early life.

Association of alpha-aminoadipic acid with cardiometabolic risk factors in healthy and high-risk individuals.

Introduction: Plasma levels of the metabolite alpha-aminoadipic acid (2-AAA) have been associated with risk of type 2 diabetes (T2D) and atherosclerosis. However, little is known about the relationship of 2-AAA to other cardiometabolic risk markers in pre-disease states, or in the setting of comorbid disease. Methods: We measured circulating 2-AAA using two methods in 1) a sample of 261 healthy individuals (2-AAA Study), and 2) in a sample of 134 persons comprising 110 individuals with treated HIV, with or without T2D, a population at high risk of metabolic disease and cardiovascular events despite suppression of circulating virus, and 24 individuals with T2D without HIV (HATIM Study). We examined associations between plasma 2-AAA and markers of cardiometabolic health within each cohort. Results and discussion: We observed differences in 2-AAA by sex and race in both cohorts, with higher levels observed in men compared with women, and in Asian compared with Black or white individuals (P<0.05). There was no significant difference in 2-AAA by HIV status within individuals with T2D in the HATIM Study. We confirmed associations between 2-AAA and dyslipidemia in both cohorts, where high 2-AAA associated with low HDL cholesterol (P<0.001) and high triglycerides (P<0.05). As expected, within the cohort of people with HIV, 2-AAA was higher in the setting of T2D compared to pre-diabetes or normoglycemia (P<0.001). 2-AAA was positively associated with body mass index (BMI) in the 2-AAA Study, and with waist circumference and measures of visceral fat volume in HATIM (all P<0.05). Further, 2-AAA associated with increased liver fat in persons with HIV (P<0.001). Our study confirms 2-AAA as a marker of cardiometabolic risk in both healthy individuals and those at high cardiometabolic risk, reveals relationships with adiposity and hepatic steatosis, and highlights important differences by sex and race. Further studies are warranted to establish molecular mechanisms linking 2-AAA to disease in other high-risk populations.

Association of alpha-aminoadipic acid (2-AAA) with cardiometabolic risk factors in healthy and high-risk individuals.

Plasma levels of the metabolite alpha-aminoadipic acid (2-AAA) have been associated with risk of type 2 diabetes (T2D) and atherosclerosis. However, little is known about the relationship of 2-AAA to other cardiometabolic risk markers in pre-disease states, or in the setting of comorbid disease. We measured circulating 2-AAA using two methods in 1) a sample of 261 healthy individuals (2-AAA Study), and 2) in a sample of 134 persons comprising 110 individuals with treated HIV, with or without T2D, a population at high risk of metabolic disease and cardiovascular events despite suppression of circulating virus, and 24 individuals with T2D without HIV (HATIM Study). We examined associations between plasma 2-AAA and markers of cardiometabolic health within each cohort. We observed differences in 2-AAA by sex and race in both cohorts, with higher levels observed in men compared with women, and in Asian compared with Black or white individuals (P<0.05). There was no significant difference in 2-AAA by HIV status within individuals with T2D in the HATIM Study. We confirmed associations between 2-AAA and dyslipidemia in both cohorts where high 2-AAA associated with low HDL cholesterol (P<0.001) and high triglycerides (P<0.05). As expected, within the cohort of people with HIV, 2-AAA was higher in the setting of T2D compared to pre-diabetes or normoglycemia (P<0.001). 2-AAA was positively associated with body mass index (BMI) in the 2-AAA Study, and with waist circumference and measures of visceral fat volume in HATIM (all P<0.05). Further, 2-AAA associated with increased liver fat in persons with HIV (P<0.001). Our study confirms 2-AAA as a marker of cardiometabolic risk in both healthy individuals and those at high cardiometabolic risk, reveals relationships with adiposity and hepatic steatosis, and highlights important differences by sex and race. Further studies are warranted to establish molecular mechanisms linking 2-AAA to disease in other high-risk populations.

Evaluation of ω-alkynyl-labeled linoleic and arachidonic acids as substrates for recombinant lipoxygenase pathway enzymes.

ω-Alkynyl-fatty acids can be used as probes for covalent binding to intracellular macromolecules. To inform future in vivo studies, we determined the rates of reaction of ω-alkynyl-labeled linoleate with recombinant enzymes of the skin 12R-lipoxygenase (12R-LOX) pathway involved in epidermal barrier formation (12R-LOX, epidermal lipoxygenase-3 (eLOX3), and SDR9C7). We also examined the reactivity of ω-alkynyl-arachidonic acid with representative lipoxygenase enzymes employing either "carboxyl end-first" substrate binding (5S-LOX) or "tail-first" (platelet-type 12S-LOX). ω-Alkynyl-linoleic acid was oxygenated by 12R-LOX at 62 ± 9 % of the rate compared to linoleic acid, the alkynyl-9R-HPODE product was isomerized by eLOX3 at only 43 ± 1 % of the natural substrate, whereas its epoxy alcohol product was converted to epoxy ketone linoleic by an NADH-dependent dehydrogenase (SDR9C7) with 91 ± 1 % efficiency. The results suggest the optimal approach will be application of the 12R-LOX/eLOX3-derived epoxyalcohol, which should be most efficiently incorporated into the pathway and allow subsequent analysis of covalent binding to epidermal proteins. Regarding the orientation of substrate binding in LOX catalysis, our results and previous reports suggest the ω-alkynyl group has a stronger inhibitory effect on tail-first binding, as might be expected. Beyond slowing the reaction, however, we found that the tail-first binding and transformation of ω-alkynyl-arachidonic acid by platelet-type 12S-LOX results in almost complete enzyme inactivation, possibly due to reactive intermediates blocking the enzyme active site. Overall, the results reinforce the conclusion that ω-alkynyl-fatty acids are suitable for selected applications after appropriate reactivity is established.

Two C18 hydroxy-cyclohexenone fatty acids from mammalian epidermis: Potential relation to 12R-lipoxygenase and covalent binding of ceramides.

A key requirement in forming the water permeability barrier in the mammalian epidermis is the oxidation of linoleate esterified in a skin-specific acylceramide by the sequential actions of 12R-lipoxygenase, epidermal lipoxygenase-3, and the epoxyalcohol dehydrogenase SDR9C7 (short-chain dehydrogenase-reductase family 7 member 9). By mechanisms that remain unclear, this oxidation pathway promotes the covalent binding of ceramides to protein, forming a critical structure of the epidermal barrier, the corneocyte lipid envelope. Here, we detected, in porcine, mouse, and human epidermis, two novel fatty acid derivatives formed by KOH treatment from precursors covalently bound to protein: a "polar" lipid chromatographing on normal-phase HPLC just before omega-hydroxy ceramide and a "less polar" lipid nearer the solvent front. Approximately 100 µg of the novel lipids were isolated from porcine epidermis, and the structures were established by UV-spectroscopy, LC-MS, GC-MS, and NMR. Each is a C18 fatty acid and hydroxy-cyclohexenone with the ring on carbons C9-C14 in the polar lipid and C8-C13 in the less polar lipid. Overnight culture of [14C]linoleic acid with whole mouse skin ex vivo led to recovery of the 14C-labeled hydroxy-cyclohexenones. We deduce they are formed from covalently bound precursors during the KOH treatment used to release esterified lipids. KOH-induced intramolecular aldol reactions from a common precursor can account for their formation. Discovery of these hydroxy-cyclohexenones presents an opportunity for a reverse pathway analysis, namely to work back from these structures to identify their covalently bound precursors and relationship to the linoleate oxidation pathway.

Ornithine Decarboxylase in Gastric Epithelial Cells Promotes the Immunopathogenesis of Helicobacter pylori Infection.

Colonization by Helicobacter pylori is associated with gastric diseases, ranging from superficial gastritis to more severe pathologies, including intestinal metaplasia and adenocarcinoma. The interplay of the host response and the pathogen affect the outcome of disease. One major component of the mucosal response to H. pylori is the activation of a strong but inefficient immune response that fails to control the infection and frequently causes tissue damage. We have shown that polyamines can regulate H. pylori-induced inflammation. Chemical inhibition of ornithine decarboxylase (ODC), which generates the polyamine putrescine from l-ornithine, reduces gastritis in mice and adenocarcinoma incidence in gerbils infected with H. pylori However, we have also demonstrated that Odc deletion in myeloid cells enhances M1 macrophage activation and gastritis. Here we used a genetic approach to assess the specific role of gastric epithelial ODC during H. pylori infection. Specific deletion of the gene encoding for ODC in gastric epithelial cells reduces gastritis, attenuates epithelial proliferation, alters the metabolome, and downregulates the expression of immune mediators induced by H. pylori Inhibition of ODC activity or ODC knockdown in human gastric epithelial cells dampens H. pylori-induced NF-κB activation, CXCL8 mRNA expression, and IL-8 production. Chronic inflammation is a major risk factor for the progression to more severe pathologies associated with H. pylori infection, and we now show that epithelial ODC plays an important role in mediating this inflammatory response.

Cystathionine γ-lyase exacerbates Helicobacter pylori immunopathogenesis by promoting macrophage metabolic remodeling and activation.

Macrophages play a crucial role in the inflammatory response to the human stomach pathogen Helicobacter pylori, which infects half of the world's population and causes gastric cancer. Recent studies have highlighted the importance of macrophage immunometabolism in their activation state and function. We have demonstrated that the cysteine-producing enzyme cystathionine γ-lyase (CTH) is upregulated in humans and mice with H. pylori infection. Here, we show that induction of CTH in macrophages by H. pylori promoted persistent inflammation. Cth-/- mice had reduced macrophage and T cell activation in H. pylori-infected tissues, an altered metabolome, and decreased enrichment of immune-associated gene networks, culminating in decreased H. pylori-induced gastritis. CTH is downstream of the proposed antiinflammatory molecule, S-adenosylmethionine (SAM). Whereas Cth-/- mice exhibited gastric SAM accumulation, WT mice treated with SAM did not display protection against H. pylori-induced inflammation. Instead, we demonstrated that Cth-deficient macrophages exhibited alterations in the proteome, decreased NF-κB activation, diminished expression of macrophage activation markers, and impaired oxidative phosphorylation and glycolysis. Thus, through altering cellular respiration, CTH is a key enhancer of macrophage activation, contributing to a pathogenic inflammatory response that is the universal precursor for the development of H. pylori-induced gastric disease.

VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites.

RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCSs) as platforms for the generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and in vivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes luminal filling of multivesicular bodies (MVBs), CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives the biogenesis of a select RNA-containing EV population.

Iron deficiency linked to altered bile acid metabolism promotes Helicobacter pylori-induced inflammation-driven gastric carcinogenesis.

Gastric carcinogenesis is mediated by complex interactions among Helicobacter pylori, host, and environmental factors. Here, we demonstrate that H. pylori augmented gastric injury in INS-GAS mice under iron-deficient conditions. Mechanistically, these phenotypes were not driven by alterations in the gastric microbiota; however, discovery-based and targeted metabolomics revealed that bile acids were significantly altered in H. pylori-infected mice with iron deficiency, with significant upregulation of deoxycholic acid (DCA), a carcinogenic bile acid. The severity of gastric injury was further augmented when H. pylori-infected mice were treated with DCA, and, in vitro, DCA increased translocation of the H. pylori oncoprotein CagA into host cells. Conversely, bile acid sequestration attenuated H. pylori-induced injury under conditions of iron deficiency. To translate these findings to human populations, we evaluated the association between bile acid sequestrant use and gastric cancer risk in a large human cohort. Among 416,885 individuals, a significant dose-dependent reduction in risk was associated with cumulative bile acid sequestrant use. Further, expression of the bile acid receptor transmembrane G protein-coupled bile acid receptor 5 (TGR5) paralleled the severity of carcinogenic lesions in humans. These data demonstrate that increased H. pylori-induced injury within the context of iron deficiency is tightly linked to altered bile acid metabolism, which may promote gastric carcinogenesis.

Salmonella enterica serovar Typhimurium uses anaerobic respiration to overcome propionate-mediated colonization resistance.

The gut microbiota benefits the host by limiting enteric pathogen expansion (colonization resistance), partially via the production of inhibitory metabolites. Propionate, a short-chain fatty acid produced by microbiota members, is proposed to mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm). Here, we show that S. Tm overcomes the inhibitory effects of propionate by using it as a carbon source for anaerobic respiration. We determine that propionate metabolism provides an inflammation-dependent colonization advantage to S. Tm during infection. Such benefit is abolished in the intestinal lumen of Salmonella-infected germ-free mice. Interestingly, S. Tm propionate-mediated intestinal expansion is restored when germ-free mice are monocolonized with Bacteroides thetaiotaomicron (B. theta), a prominent propionate producer in the gut, but not when mice are monocolonized with a propionate-production-deficient B. theta strain. Taken together, our results reveal a strategy used by S. Tm to mitigate colonization resistance by metabolizing microbiota-derived propionate.

Monolayer autoxidation of arachidonic acid to epoxyeicosatrienoic acids as a model of their potential formation in cell membranes.

In light of the importance of epoxyeicosatrienoic acids (EETs) in mammalian pathophysiology, a nonenzymatic route that might form these monoepoxides in cells is of significant interest. In the late 1970s, a simple system of arranging linoleic acid molecules on a monolayer on silica was devised and shown to yield monoepoxides as the main autoxidation products. Here, we investigated this system with arachidonic acid and characterized the primary products. By the early stages of autoxidation (∼10% conversion of arachidonic acid), the major products detected by LC-MS and HPLC-UV were the 14,15-, 11,12-, and 8,9-EETs, with the 5,6-EET mainly represented as the 5-δ-lactone-6-hydroxyeicosatrienoate as established by 1H-NMR. The EETs were mainly the cis epoxides as expected, with minor trans configuration EETs among the products. 1H-NMR analysis in four deuterated solvents helped clarify the epoxide configurations. EET formation in monolayers involves intermolecular reaction with a fatty acid peroxyl radical, producing the EET and leaving an incipient and more reactive alkoxyl radical, which in turn gives rise to epoxy-hydro(pero)xides and other polar products. The monolayer alignment of fatty acid molecules resembles the arrangements of fatty acids in cell membranes and, under conditions of lipid peroxidation, this intermolecular mechanism might contribute to EET formation in biological membranes.

Plasma Arginine and Citrulline are Elevated in Diabetic Retinopathy.

PURPOSE: To determine if plasma levels of six arginine-related and citrulline-related metabolites (arginine, citrulline, asymmetric dimethylarginine [ADMA], ornithine, proline, and argininosuccinate) differ between patients with type 2 diabetes and diabetic retinopathy (DR) and type 2 diabetic controls or between patients with proliferative DR (PDR) and non-proliferative DR (NPDR). DESIGN: Cross-sectional study. METHODS: Adults with type 2 diabetes were recruited from the Vanderbilt Eye Institute. Exclusion criteria included non-diabetic retinal disease. Plasma metabolite levels were quantified in 159 diabetic controls and 156 DR patients (92 NPDR, 64 PDR) using isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Metabolite levels were compared using Wilcoxon Rank Sum test and logistic regressions adjusting for age, sex, hemoglobin A1c, diabetes duration, statin use, and angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker use. A secondary analysis that included creatinine in the regression model was performed for the subset of patients with available creatinine values (135 diabetic controls, 100 DR patients [58 NPDR, 42 PDR]). RESULTS: Multivariable logistic regression analyses determined that arginine (OR = 1.20, [1.06-1.38], P = .0067) and citrulline (OR = 1.53, [1.20-1.98], P = .0025) were significantly elevated in DR patients compared to diabetic controls. While ADMA differed between NPDR and PDR patients in the primary analysis (OR = 1.56, [1.15-2.16], P = .0051), it was not significantly different when adjusting for creatinine (OR = 1.30, [0.90-1.91], P = .15). CONCLUSIONS: Plasma arginine and citrulline were significantly elevated in type 2 diabetic patients with DR compared to diabetic controls. None of the tested metabolites significantly differed between NPDR and PDR patients in the adjusted analysis.

Intravitreal HDAC Inhibitor Belinostat Effectively Eradicates Vitreous Seeds Without Retinal Toxicity In Vivo in a Rabbit Retinoblastoma Model.

Purpose: Current melphalan-based regimens for intravitreal chemotherapy for retinoblastoma vitreous seeds are effective but toxic to the retina. Thus, alternative agents are needed. Based on the known biology of histone deacetylases (HDACs) in the retinoblastoma pathway, we systematically studied whether the HDAC inhibitor belinostat is a viable, molecularly targeted alternative agent for intravitreal delivery that might provide comparable efficacy, without toxicity. Methods: In vivo pharmacokinetic experiments in rabbits and in vitro cytotoxicity experiments were performed to determine the 90% inhibitory concentration (IC90). Functional toxicity by electroretinography and structural toxicity by optical coherence tomography (OCT), OCT angiography, and histopathology were evaluated in rabbits following three injections of belinostat 350 µg (2× IC90) or 700 µg (4× IC90), compared with melphalan 12.5 µg (rabbit equivalent of the human dose). The relative efficacy of intravitreal belinostat versus melphalan to treat WERI-Rb1 human cell xenografts in rabbit eyes was directly quantified. RNA sequencing was used to assess belinostat-induced changes in RB cell gene expression. Results: The maximum nontoxic dose of belinostat was 350 µg, which caused no reductions in electroretinography parameters, retinal microvascular loss on OCT angiography, or retinal degeneration. Melphalan caused severe retinal structural and functional toxicity. Belinostat 350 µg (equivalent to 700 µg in the larger human eye) was equally effective at eradicating vitreous seeds in the rabbit xenograft model compared with melphalan (95.5% reduction for belinostat, P < 0.001; 89.4% reduction for melphalan, P < 0.001; belinostat vs. melphalan, P = 0.10). Even 700 µg belinostat (equivalent to 1400 µg in humans) caused only minimal toxicity. Widespread changes in gene expression resulted. Conclusions: Molecularly targeted inhibition of HDACs with intravitreal belinostat was equally effective as standard-of-care melphalan but without retinal toxicity. Belinostat may therefore be an attractive agent to pursue clinically for intravitreal treatment of retinoblastoma.

Knock-Out of DHTKD1 Alters Mitochondrial Respiration and Function, and May Represent a Novel Pathway in Cardiometabolic Disease Risk.

Cardiometabolic disease affects the majority of individuals worldwide. The metabolite α-aminoadipic acid (2-AAA) was identified as a biomarker of Type 2 Diabetes (T2D). However, the mechanisms underlying this association remain unknown. DHTKD1, a central gene in the 2-AAA pathway, has been linked to 2-AAA levels and metabolic phenotypes. However, relatively little is known about its function. Here we report that DHTKD1 knock-out (KO) in HAP-1 cells leads to impaired mitochondrial structure and function. Despite impaired mitochondrial respiration and less ATP production, normal cell proliferation rate is maintained, potentially through a series of compensatory mechanisms, including increased mitochondrial content and Akt activation, p38, and ERK signaling. Common variants in DHTKD1 associate with Type 2 Diabetes and cardiometabolic traits in large genome-wide associations studies. These findings highlight the vital role of DHTKD1 in cellular metabolism and establish DHTKD1-mediated mitochondrial dysfunction as a potential novel pathway in cardiometabolic disease.

Hypoxic preconditioning protects against ischemic kidney injury through the IDO1/kynurenine pathway.

Prolonged cellular hypoxia leads to energetic failure and death. However, sublethal hypoxia can trigger an adaptive response called hypoxic preconditioning. While prolyl-hydroxylase (PHD) enzymes and hypoxia-inducible factors (HIFs) have been identified as key elements of oxygen-sensing machinery, the mechanisms by which hypoxic preconditioning protects against insults remain unclear. Here, we perform serum metabolomic profiling to assess alterations induced by two potent cytoprotective approaches, hypoxic preconditioning and pharmacologic PHD inhibition. We discover that both approaches increase serum kynurenine levels and enhance kynurenine biotransformation, leading to preservation of NAD+ in the post-ischemic kidney. Furthermore, we show that indoleamine 2,3-dioxygenase 1 (Ido1) deficiency abolishes the systemic increase of kynurenine and the subsequent renoprotection generated by hypoxic preconditioning and PHD inhibition. Importantly, exogenous administration of kynurenine restores the hypoxic preconditioning in the context of Ido1 deficiency. Collectively, our findings demonstrate a critical role of the IDO1-kynurenine axis in mediating hypoxic preconditioning.

Lipocalin Blc is a potential heme-binding protein.

Lipocalins are a superfamily of functionally diverse proteins defined by a well-conserved tertiary structure despite variation in sequence. Lipocalins bind and transport small hydrophobic molecules in organisms of all kingdoms. However, there is still uncertainty regarding the function of some members of the family, including bacterial lipocalin Blc from Escherichia coli. Here, we present evidence that lipocalin Blc may be involved in heme binding, trans-periplasmic transport, or heme storage. This conclusion is supported by a cocrystal structure, mass-spectrometric data, absorption titration, and in silico analysis. Binding of heme is observed at low micromolar range with one-to-one ligand-to-protein stoichiometry. However, the absence of classical coordination to the iron atom leaves the possibility that the primary ligand of Blc is another tetrapyrrole.