FooDOxS: a database of oxidized sterols content in foods.

Dietary oxidized sterols (DOxS) are cholesterol-like molecules known to exert pro-inflammatory, pro-oxidant, and pro-apoptotic effects, among others. We present the FooDOxS database, a comprehensive compilation of DOxS content in over 1680 food items from 120 publications across 25 countries, augmented by data generated by our group. This database reports DOxS content in foods classified under the NOVA and What We Eat in America (WWEIA) systems, allowing a comprehensive and statistically robust summary of DOxS content in foods. Notably, we evaluated the efficacy of using NOVA and WWEIA classifications in capturing DOxS variations across food categories. Our findings provide insights into the strengths and limitations of these classification systems, enhancing their utility for assessing dietary components. This research contributes to the understanding of DOxS in food processing and suggests refinements for classification systems, holding promise for improved food safety and public health assessments.

AMPK regulates phagophore-to-autophagosome maturation.

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example, by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK-mediated inhibition of phagophore tethering to donor membrane. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

Oxidative Status of Ultra-Processed Foods in the Western Diet.

Ultra-processed foods (UPFs) have gained substantial attention in the scientific community due to their surging consumption and potential health repercussions. In addition to their well-established poor nutritional profile, UPFs have been implicated in containing various dietary oxidized sterols (DOxSs). These DOxSs are associated with a spectrum of chronic diseases, including cardiometabolic conditions, cancer, diabetes, Parkinson's, and Alzheimer's disease. In this study, we present a comprehensive database documenting the presence of DOxSs and other dietary metabolites in >60 UPFs commonly consumed as part of the Western diet. Significant differences were found in DOxS and phytosterol content between ready-to-eat (RTE) and fast foods (FFs). Biomarker analysis revealed that DOxS accumulation, particularly 25-OH and triol, can potentially discriminate between RTEs and FFs. This work underscores the potential utility of dietary biomarkers in early disease detection and prevention. However, an essential next step is conducting exposure assessments to better comprehend the levels of DOxS exposure and their association with chronic diseases.

Cholesterol crystals induce mechanical trauma, inflammation, and neo-vascularization in solid cancers as in atherosclerosis.

Background and aims: Cancer and atherosclerosis share common risk factors and inflammatory pathways that promote their proliferation via vascular endothelial growth factor (VEGF). Because CCs cause mechanical injury and inflammation in atherosclerosis, we investigated their presence in solid cancers and their activation of IL-1ß, VEGF, CD44, and Ubiquityl-Histone H2B (Ub-H2B), that promote cancer growth. Methods: Tumor specimens from eleven different types of human cancers and atherosclerotic plaques were assessed for CCs, free cholesterol content and IL1-ß by microscopy, immunohistochemistry, and biochemical analysis. Breast and colon cancer cell lines were cultured with and without CCs to select for expression of VEGF, CD44, and Ub-H2B. Western blot and immunofluorescence were performed on cells to assess the effect of CCs on signaling pathways. Results: Cancers displayed higher CC content (+2.29 ± 0.74 vs +1.46 ± 0.84, p < 0.0001), distribution (5.06 ± 3.13 vs 2.86 ± 2.18, p < 0.001) and free cholesterol (3.63 ± 4.02 vs 1.52 ± 0.56 µg/mg, p < 0.01) than cancer free marginal tissues and similarly for atherosclerotic plaques and margins (+2.31 ± 0.51 vs +1.44 ± 0.79, p < 0.02; 14.0 ± 5.74 vs 8.14 ± 5.52, p < 0.03; 0.19 ± 0.14 vs 0.09 ± 0.04 µg/mg, p < 0.02) respectively. Cancers displayed significantly increased expression of IL1-ß compared to marginal tissues. CCs treated cancer cells had increased expression of VEGF, CD44, and Ub-H2B compared to control. By microscopy, CCs were found perforating cancer tumors similar to plaque rupture. Conclusions: These findings suggest that CCs can induce trauma and activate cytokines that enhance cancer growth as in atherosclerosis.

AMPK Regulates Phagophore-to-Autophagosome Maturation.

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK mediated inhibition of phagophores tethering to donor membranes. Our results clarify AMPK's regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

Tracking the transition from an ATG9A vesicle to an autophagosome.

ABBREVIATIONS: ATG: autophagy-related proteins; ULK1/2: Unc-51-Like activating Kinases; PI3Ks: Phosphoinositide 3-Kinases; ATG2A: autophagy-related protein 2A; ATG5: autophagy-related protein 5; ATG16: autophagy-related protein 16; ATG8: autophagy-related protein 8; U2OS: human bone osteosarcoma epithelial cell; LC3B: microtubule-associated protein 1A/1B Light Chain 3B; GABARAPL1: GABA type A Receptor-Associated Protein Like 1; ATG9A: autophagy-related protein 9A; ATG13: autophagy-related protein 13; SQSTM1: Sequestosome-1/p62; WIPI2: WD repeat domain, Phosphoinositide Interacting 2; PI3P: Phosphoinositide-3-phosphate.

Quantitative analysis of autophagy reveals the role of ATG9 and ATG2 in autophagosome formation.

Autophagy is a catabolic pathway required for the recycling of cytoplasmic materials. To define the mechanisms underlying autophagy it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of cell lines expressing HaloTagged autophagy factors from their endogenous loci, we analyzed the abundance, single-molecule dynamics, and autophagosome association kinetics of autophagy proteins involved in autophagosome biogenesis. We demonstrate that autophagosome formation is inefficient and ATG2-mediated tethering to donor membranes is a key commitment step in autophagosome formation. Furthermore, our observations support the model that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles, and that the ULK1 complex and PI3-kinase form a positive feedback loop required for autophagosome formation. Finally, we demonstrate that the duration of autophagosome biogenesis is ∼110 s. In total, our work provides quantitative insight into autophagosome biogenesis and establishes an experimental framework to analyze autophagy in human cells.

Dietary exposure assessment of infant formula and baby foods' oxidized lipids in the US population.

Baby Foods (BFs) and Infant formulas (IFs) are the main sources of nutrition for an infant throughout the 1st year of life. Various enriched products are commercially available for parents seeking to fulfill their baby's nutritional needs. Consequently, different bioactive lipids are present in BFs and IFs, including dietary oxysterols (DOxS), whose known toxicity has been associated with mutagenicity, cancer, and other chronic diseases. In this work, we performed an exposure assessment of 25 bioactive lipids on IFs (n = 30) and BFs (n = 13) commercially available in the US. To determine dietary exposure, we used EPA's SHEDS-HT probabilistic model. Even though ß-Sitosterol was the most exposed bioactive lipid with 75,410 µg/day, cholesterol was the most absorbed compound during the entire first year (19.3 mg/day). Additionally, we found 7α-hydroxycholesterol (7α-OH) as a potential DOxS biomarker of the BFs manufacturing process. This is the first time an infant's exposure assessment (including DOxS) after BFs and IFs consumption is performed, enabling much-needed information regarding these hazardous compounds and their potential effects on infants' health.

Evaluation of the nutritional quality of ultra-processed foods (ready to eat + fast food): Fatty acids, sugar, and sodium.

The average American consumes more than 50% of their total dietary energy from ultra-processed foods (UPFs). From a nutritional standpoint, as UPFs intake increases, fiber, vitamin, and mineral intake decrease. High consumption of UPFs, mainly from fast foods (FF) and ready-to-eat (RTE) food items, emerges as a critical public health concern linking nutritional quality and food safety. In the present work, a systematic database of the fatty acid composition of the most consumed UPFs in the Midwest is reported. Saturated and monounsaturated fatty acids were predominant in RTE (42.5%) and FF (43.2%), respectively. In addition, the fatty acid profile in UPFs is reported according to six food categories: meat and poultry, eggs and derivatives, dairy products, seafood, baby foods, and others. Meat and poultry, and dairy products were the dominant food categories among UPFs. Meanwhile, polyunsaturated fatty acids were abundant in the eggs and seafood groups UPFs (61.8% and 46.4%, respectively) regardless of the food group. Furthermore, no significant differences were found in sugar content in UPFs. Caloric content was positively correlated with sodium (ρ = 0.748) and price (ρ = 0.534). The significance of this study relies on providing new quantitative data on the fat, sodium, and sugar contents of the most consumed UPFs in the Midwestern area of the United States. This information suggests paying more attention to these nutritional attributes, aiming to reduce their incorporation in UPF preparations. Additionally, more quantitative data are needed regarding other nutritional parameters such as protein and lipid degradation in UPFs. PRACTICAL APPLICATION: This study provides a profile of the fatty acid composition of the most consumed UPFs in the Midwestern region of the United States, as well as correlations with fat, sodium, and sugar contents in UPFs. The information offered a new perspective on the nutrition quality of UPFs, suggesting the reduction of the incorporation of these attributes in UPFs. Additionally, it will help define priority interventions for more advanced precision nutrition, especially for vulnerable populations, for example, children and older people. The overall decrease in added sugar and sodium and the service size in UPFs will significantly improve the nutritional quality of the Western diet.

Lipid profiling and dietary assessment of infant formulas reveal high intakes of major cholesterol oxidative product (7-ketocholesterol).

Approximately two-thirds of US infants receive infant formula (IF) as a primary or sole nutritional source during the first six months of life. IF is available in a variety of commercial presentations; from a manufacturing standpoint, they can be categorized as powder- (PIF) or liquid- (LIF) based formulations. Thirty commercial IFs were analyzed in their oxidative and non-oxidative lipid profiles. We identified 7-ketocholesterol - a major end-product of cholesterol oxidation - as a potential biomarker of IF manufacturing. The statistical analysis allowed a re-classification of IF based on their metabolomic fingerprint, resulting in three groups assigned with low-to-high oxidative status. Finally, we modeled the dietary intake of cholesterol, sterols, and 7-ketocholesterol in the first year of life. The database provided in this study will be instrumental for scientists interested in infant nutrition, to establish bases for epidemiological studies aimed to find connections between nutrition and diet-associated diseases, such as sitosterolemia.

Efficacy and Safety of Non-Anesthesiologist Administration of Propofol Sedation in Endoscopic Ultrasound: A Propensity Score Analysis.

In spite of promising preliminary results, evidence supporting the use of non-anesthesiologist-administered propofol sedation (NAAP) in endoscopic ultrasound (EUS) procedures is still limited. The aim of this manuscript was to examine the safety and efficacy of NAAP as compared to anesthesiologist-administered propofol sedation in EUS procedures performed in a referral center. Out of 832 patients referred to our center between 2016 and 2019, after propensity score matching two groups were compared: 305 treated with NAAP and 305 controls who underwent anesthesiologist-administered propofol sedation. The primary outcome was the rate of major complications. The median age was 67 years and the proportion of patients with comorbidities was 31.8% in both groups. One patient in each group (0.3%) experienced a major complication, whereas minor complications were observed in 13 patients in the NAAP group (4.2%) and 10 patients in the control group (3.2%; p = 0.52). Overall pain during the procedure was 2.3 ± 1 in group 1 and 1.8 ± 1 in group 2 (p = 0.67), whereas pain/discomfort upon awakening was rated as 1 ± 0.5 in both groups (p = 0.72). NAAP is safe and effective even in advanced EUS procedures. Further randomized-controlled trials (RCTs) are warranted to confirm these findings.

Flavonoids Ability to Disrupt Inflammation Mediated by Lipid and Cholesterol Oxidation.

Flavonoids are plant secondary metabolites that act as protectants against harmful effects of UV-B radiation inasmuch as biotic stress, conferring at the same time pigmentation of fruits and leaves [67]. The term "flavonoid" refers to phenolics having a basic skeleton of diphenylpropane (C6-C3-C6), which consists of two aromatic rings linked through three carbons that usually form an oxygenated heterocycle [25, 52]. Flavonoids are broken down into several different sub-categories based on their chemical structure. The main subclasses commonly found in food items are: flavonols, flavones, flavanones, flavan-3-ols, proanthocyanidins, and anthocyanins [44, 67]. Figure 19.1 depicts the major classification of flavonoids according to their chemical structure. Their occurrence in food matrices has been extensively reviewed [39, 44], and has been subject of extensive research in the last decades. Table 19.1 contains a few examples of compounds from each of the subcategory, with the fruit (berry) in which they are commonly found. The monomeric unit of flavonoids can dimerize and polymerize to form other important high molecular weight molecules; this is the case of proanthocyanidins, that are polymers of flavan-3-ols or flavanols. Not only do these compounds act as plant protectants, but they can also be very beneficial to human health. Cohorts studies performed in the early '90 have shown that dietary consumption of flavonoids was inversely associated with morbidity and mortality from coronary heart disease [31, 32]. These findings have opened an intensive field of research on the effects of flavonoids and flavonoids-rich food extracts in cardiovascular diseases (CVD) progression, particularly in the modulating CVD-associated oxidative stress and inflammation. In this short review, we will summarize the current findings in flavonoids beneficial effects in preventing CVD through inhibition of initial stages of CVD progression. Given the magnitude of scientific literature in the field, we will focus on two strictly mechanistic aspects: inhibition of chemical-induced LDL oxidation, and the effect of flavonoids in the monocyte/macrophages activation pathways.

Picturing the Membrane-assisted Choreography of Cytochrome P450 with Lipid Nanodiscs.

Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, and requires enzymatic partners as well as environmental factors that tune its complex dynamic activity. P450 and its reducing counterparts are membrane-bound proteins which are believed to dynamically interact to form functional complexes. Increasing experimental evidence signifies the role (s) of protein-lipid interactions in P450's catalytic function and efficiency. The challenges posed by the membrane have severely limited high-resolution understanding of the molecular interfaces of these interactions. Nevertheless, recent NMR studies have provided piercing insights into the dynamic structural interactions that enable the function of P450. In this review, we will discuss different biomimetic approaches relevant to unveil molecular interplays at the membrane, focusing on our recent work on lipid-nanodiscs. We also highlight the need to expand the use of nanodiscs, and the power of a combination of cutting-edge solution and solid-state NMR techniques, to study the dynamic structures of P450 as well as other membrane-proteins.

The role of cholesterol oxidation products in food toxicity.

Food consumption can lead to the accumulation of certain chemical compounds able to exert toxic activities against humans. Of mayor interests are those molecules generated during food processing and handling, since their occurrence and distribution depend of many intrinsic and extrinsic factors. Cholesterol - a lipid constituent of mammalian cells - is the precursor of several toxic molecules known as cholesterol oxidation products (COPs). In the last decades, it has been demonstrated that food processing can dramatically trigger COPs accumulation in meats, eggs, dairy products, fish and poultry. On the other hand, countless scientific evidences have pointed out the highly toxic and pathogenic activities of COPs, from cancer stimulation to neurodegenerative disorders, via molecular mechanisms that are largely unexplored. The aim of this review is to merge the evidence on COPs accumulation in foods and their toxic activities through dietary intake, as from in vivo and in vitro studies. We consider that it is imperative to systematically monitor the formation of COPs to bridge these quantitative efforts with a risk exposure assessment on sensitive populations.

Lipid-exchange in nanodiscs discloses membrane boundaries of cytochrome-P450 reductase.

Lipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the sole electron transferase for microsomal oxygenases, possesses a conformational dynamics entwined with its topology. Here, we use peptide-nanodiscs to unveil cytochrome-P450-reductase's lipid boundaries, demonstrating a protein-driven enrichment of ethanolamine lipids (by 25%) which ameliorates by 3-fold CPR's electron-transfer ability.

Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs.

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.

Membrane environment drives cytochrome P450's spin transition and its interaction with cytochrome b5.

Heme's spin-multiplicity is key in determining the enzymatic function of cytochrome P450 (cytP450). The origin of the low-spin state in ferric P450 is still under debate. Here, we report the first experimental demonstration of P450's membrane interaction altering its spin equilibrium which is accompanied by a stronger affinity for cytochrome b5. These results highlight the importance of lipid membrane for the function of P450.

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study.

Cytochrome P450-reductase (CPR) is a versatile NADPH-dependent electron donor located in the cytoplasmic side of the endoplasmic reticulum. It is an electron transferase that is able to deliver electrons to a variety of membrane-bound oxidative partners, including the drug-metabolizing enzymes of the cytochrome P450s (P450). CPR is also stoichiometrically limited compared to its oxidative counterparts, and hypotheses have arisen about possible models that can overcome the stoichiometric imbalance, including quaternary organization of P450 and diffusion-limited models. Described here are results from a single-protein tracking study of fluorescently labeled CPR and cytochrome P450 2C9 (CYP2C9) molecules in which stochastic analysis was used to determine the dissociation constants of CPR/CYP2C9 complexes in a lipid bilayer membrane for the first time. Single-protein trajectories demonstrate the transient nature of these CPR-CYP2C9 interactions, and the measured Kd values are highly dependent on the redox state of CPR. It is shown that CPRox/CYP2C9 complexes have a much higher dissociation constant than CPR2-/CYP2C9 or CPR4-/CYP2C9 complexes, and a model is presented to account for these results. An Arrhenius analysis of diffusion constants was also carried out, demonstrating that the reduced forms of CPR and CYP2C9 interact differently with the biomimetic ER and may, in addition to protein conformational changes, contribute to the observed NADPH-dependent shift in Kd. Finally, it is also shown that the CPRox/CYP2C9 affinity depends on the nature of the ligand, being higher when a substrate is bound, compared to an inhibitor.

Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c.

Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb 5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb 5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb 5-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb 5 in the presence and absence of membrane. Notably, in the presence of membrane, cytb 5 only engaged cyt c at its lower and upper clefts while the membrane-free cytb 5 also uses a distal region. Using restraints generated from both cytb 5 and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. These results demonstrate the importance of studying protein-protein complex formation in membrane mimetic systems. Our results also demonstrate the successful preparation of novel peptide-based lipid nanodiscs, which are detergent-free and possesses size flexibility, and their use for NMR structural studies of membrane proteins.

The catalytic function of cytochrome P450 is entwined with its membrane-bound nature.

Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts-cytochrome P450-reductase and cytochrome b 5 -are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s) played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.