Dietary Nitrate Metabolism in Porcine Ocular Tissues Determined Using 15N-Labeled Sodium Nitrate Supplementation.

Nitrate (NO3-) obtained from the diet is converted to nitrite (NO2-) and subsequently to nitric oxide (NO) within the body. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite that are similar to those in blood. Notably, cornea and sclera exhibited the capability to reduce nitrate to nitrite. To gain deeper insights into nitrate metabolism in porcine eyes, our current study involved feeding pigs either NaCl or Na15NO3 and assessing the levels of total and 15N-labeled NO3-/NO2- in various ocular tissues. Three hours after Na15NO3 ingestion, a marked increase in 15NO3- and 15NO2- was observed in all parts of the eye; in particular, the aqueous and vitreous humor showed a high 15NO3- enrichment (77.5 and 74.5%, respectively), similar to that of plasma (77.1%) and showed an even higher 15NO2- enrichment (39.9 and 35.3%, respectively) than that of plasma (19.8%). The total amounts of NO3- and NO2- exhibited patterns consistent with those observed in 15N analysis. Next, to investigate whether nitrate or nitrite accumulate proportionally after multiple nitrate treatments, we measured nitrate and nitrite contents after supplementing pigs with Na15NO3 for five consecutive days. In both 15N-labeled and total nitrate and nitrite analysis, we did not observe further accumulation of these ions after multiple treatments, compared to a single treatment. These findings suggest that dietary nitrate supplementation exerts a significant influence on nitrate and nitrite levels and potentially NO levels in the eye and opens up the possibility for the therapeutic use of dietary nitrate/nitrite to enhance or restore NO levels in ocular tissues.

Structure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1.

Ubiquitination, catalyzed usually by a three-enzyme cascade (E1, E2, E3), regulates various eukaryotic cellular processes. E3 ligases are the most critical components of this catalytic cascade, determining both substrate specificity and polyubiquitination linkage specificity. Here, we reveal the mechanism of a naturally occurring E3-independent ubiquitination reaction of a unique human E2 enzyme UBE2E1 by solving the structure of UBE2E1 in complex with substrate SETDB1-derived peptide. Guided by this peptide sequence-dependent ubiquitination mechanism, we developed an E3-free enzymatic strategy SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized ubiquitinated sites, ubiquitin chain linkages and lengths. Notably, this strategy can also be used to generate site-specific branched ubiquitin chains or even NEDD8-modified proteins. Our work not only deepens the understanding of how an E3-free substrate ubiquitination reaction occurs in human cells, but also provides a practical approach for obtaining ubiquitinated proteins to dissect the biochemical functions of ubiquitination.

Sequential release of interacting proteins and Ub-modifying enzymes by disulfide heterotypic ubiquitin reagents.

Heterotypic ubiquitin (Ub) chains have emerged as fundamental components in a wide range of cellular processes. The integrative identification of Ub-interacting proteins (readers) and Ub-modifying enzymes (writers and erasers) that selectively recognize and regulate heterotypic ubiquitination may provide crucial insights into these processes. In this study, we employed the bifunctional molecule-assisted (CAET) strategy to develop a type of disulfide bond-activated heterotypic Ub reagents, which allowed to enrich heterotypic Ub-interacting proteins and modifying enzymes simultaneously. The sequential release of readers which are non-covalently bound and writers or erasers which are covalently conjugated by using urea and reductant, respectively, combined with label-free quantitative (LFQ) MS indicated that these heterotypic Ub reagents would facilitate future investigations into functional roles played by heterotypic Ub chains.

Glycemia and Gluconeogenesis With Metformin and Liraglutide: A Randomized Trial in Youth-onset Type 2 Diabetes.

OBJECTIVE: Elevated rates of gluconeogenesis are an early pathogenic feature of youth-onset type 2 diabetes (Y-T2D), but targeted first-line therapies are suboptimal, especially in African American (AA) youth. We evaluated glucose-lowering mechanisms of metformin and liraglutide by measuring rates of gluconeogenesis and ß-cell function after therapy in AA Y-T2D. METHODS: In this parallel randomized clinical trial, 22 youth with Y-T2D-age 15.3 ± 2.1 years (mean ± SD), 68% female, body mass index (BMI) 40.1 ± 7.9 kg/m2, duration of diagnosis 1.8 ± 1.3 years-were randomized to metformin alone (Met) or metformin + liraglutide (Lira) (Met + Lira) and evaluated before and after 12 weeks. Stable isotope tracers were used to measure gluconeogenesis [2H2O] and glucose production [6,6-2H2]glucose after an overnight fast and during a continuous meal. ß-cell function (sigma) and whole-body insulin sensitivity (mSI) were assessed during a frequently sampled 2-hour oral glucose tolerance test. RESULTS: At baseline, gluconeogenesis, glucose production, and fasting and 2-hour glucose were comparable in both groups, though Met + Lira had higher hemoglobin A1C. Met + Lira had a greater decrease from baseline in fasting glucose (-2.0 ± 1.3 vs -0.6 ± 0.9 mmol/L, P = .008) and a greater increase in sigma (0.72 ± 0.68 vs -0.05 ± 0.71, P = .03). The change in fractional gluconeogenesis was similar between groups (Met + Lira: -0.36 ± 9.4 vs Met: 0.04 ± 12.3%, P = .9), and there were no changes in prandial gluconeogenesis or mSI. Increased glucose clearance in both groups was related to sigma (r = 0.63, P = .003) but not gluconeogenesis or mSI. CONCLUSION: Among Y-T2D, metformin with or without liraglutide improved glycemia but did not suppress high rates of gluconeogenesis. Novel therapies that will enhance ß-cell function and target the elevated rates of gluconeogenesis in Y-T2D are needed.

Consumption of sucralose- and acesulfame-potassium-containing diet soda alters the relative abundance of microbial taxa at the species level: findings of two pilot studies.

Sucralose and acesulfame-potassium consumption alters gut microbiota in rodents, with unclear effects in humans. We examined effects of three-times daily sucralose- and acesulfame-potassium-containing diet soda consumption for 1 (n = 17) or 8 (n = 8) weeks on gut microbiota composition in young adults. After 8 weeks of diet soda consumption, the relative abundance of Proteobacteria, specifically Enterobacteriaceae, increased; and, increased abundance of two Proteobacteria taxa was also observed after 1 week of diet soda consumption compared with sparkling water. In addition, three taxa in the Bacteroides genus increased following 1 week of diet soda consumption compared with sparkling water. The clinical relevance of these findings and effects of sucralose and acesulfame-potassium consumption on human gut microbiota warrant further investigation in larger studies. Clinical trial registration: NCT02877186 and NCT03125356.

Effects of Low-Calorie Sweetener Restriction on Glycemic Variability and Cardiometabolic Health in Children with Type 1 Diabetes: Findings of a Pilot and Feasibility Study.

Low-calorie sweeteners (LCS) are commonly consumed by children with type 1 diabetes (T1D), yet their role in cardiometabolic health is unclear. This study examined the feasibility, acceptability, and preliminary effects of 12 weeks of LCS restriction among children with T1D. Children (n = 31) with T1D completed a two-week run-in (n = 28) and were randomly assigned to avoid LCS (LCS restriction, n = 15) or continue their usual LCS intake (n = 13). Feasibility was assessed using recruitment, retention, and adherence rates percentages. Acceptability was assessed through parents completing a qualitative interview (subset, n = 15) and a satisfaction survey at follow-up. Preliminary outcomes were between-group differences in change in average daily time-in-range (TIR) over 12 weeks (primary), and other measures of glycemic variability, lipids, inflammatory biomarkers, visceral adiposity, and dietary intake (secondary). Linear regression, unadjusted and adjusted for age, sex, race, and change in BMI, was used to compare mean changes in all outcomes between groups. LCS restriction was feasible and acceptable. No between-group differences in change in TIR or other measures of glycemic variability were observed. However, significant decreases in TNF-alpha (-0.23 ± 0.08 pg/mL) and improvements in cholesterol (-0.31 ± 0.18 mmol/L) and LDL (-0.60 ± 0.39 mmol/L) were observed with usual LCS intake, compared with LCS restriction. Those randomized to LCS restriction did not report increases in total or added sugar intake, and lower energy intake was reported in both groups (-190.8 ± 106.40 kcal LCS restriction, -245.3 ± 112.90 kcal usual LCS intake group). Decreases in percent energy from carbohydrates (-8.5 ± 2.61) and increases in percent energy from protein (3.2 ± 1.16) and fat (5.2 ± 2.02) were reported with usual LCS intake compared with LCS restriction. Twelve weeks of LCS restriction did not compromise glycemic variability or cardiometabolic outcomes in this small sample of youth with T1D. Further examination of LCS restriction among children with T1D is warranted.

Distribution of dietary nitrate and its metabolites in rat tissues after 15N-labeled nitrate administration.

The reduction pathway of nitrate (NO3-) and nitrite (NO2-) to nitric oxide (NO) contributes to regulating many physiological processes. To examine the rate and extent of dietary nitrate absorption and its reduction to nitrite, we supplemented rat diets with Na15NO3-containing water (1 g/L) and collected plasma, urine and several tissue samples. We found that plasma and urine showed 8.8- and 11.7-fold increases respectively in total nitrate concentrations in 1-day supplementation group compared to control. In tissue samples-gluteus, liver and eyes-we found 1.7-, 2.4- and 4.2-fold increases respectively in 1-day supplementation group. These increases remained similar in 3-day supplementation group. LC-MS/MS analysis showed that the augmented nitrate concentrations were primarily from the exogenously provided 15N-nitrate. Overall nitrite concentrations and percent of 15N-nitrite were also greatly increased in all samples after nitrate supplementation; eye homogenates showed larger increases compared to gluteus and liver. Moreover, genes related to nitrate transport and reduction (Sialin, CLC and XOR) were upregulated after nitrate supplementation for 3 days in muscle (Sialin 2.3-, CLC1 1.3-, CLC3 2.1-, XOR 2.4-fold) and eye (XOR 1.7-fold) homogenates. These results demonstrate that dietary nitrate is quickly absorbed into circulation and tissues, and it can be reduced to nitrite in tissues (and likely to NO) suggesting that nitrate-enriched diets can be an efficient intervention to enhance nitrite and NO bioavailability.

Correlation between patient-specific quality assurance in volumetric modulated arc therapy and 2D dose image features.

In radiotherapy, air-filled ion chamber detectors are ubiquitously used in routine dose measurements for treatment planning. However, its use has been restricted by intrinsic low spatial resolution barriers. We developed one procedure for patient-specific quality assurance (QA) in arc radiotherapy by coalescing two adjacent measurement images into a single image to improve spatial resolution and sampling frequency, and investigated how different spatial resolutions affect the QA results. PTW 729 and 1500 ion chamber detectors were used for dosimetric verification via coalescing two measurements with 5 mm-couch shift and the isocenter, and only isocenter measurement, which we call coalescence and standard acquisition (SA). Statistical process control (SPC), process capability analysis (PCA), and receiver operating characteristic (ROC) curve were used to compare the performance of the two procedures in determining tolerance levels and identifying clinically relevant errors. By analyzing 1256 γ values calculated on interpolated data points, our results indicated that detector 1500 showed higher averages in coalescence cohorts at different tolerance criteria and the dispersion degrees were spread out smaller. Detector 729 yielded a slightly lower process capability of 0.79, 0.76, 1.10, and 1.34, but detector 1500 exhibited somewhat different results of 0.94, 1.42, 1.19, and 1.60 in magnitude. The results of SPC individual control chart showed that cases in coalescence cohorts with γ values lowering its lower control limit (LCL) were greater than those in SA cohorts for detector 1500. A combination of the width of multi-leaf collimator (MLC) leaf, the cross-sectional area of the single detector, and the spacing between adjacent detectors might lead to discrepancies in percent γ values across diverse spatial resolution scenarios. The accuracy of reconstructed volume dose is mainly determined by the interpolation algorithm used in dosimetric systems. The magnitude of filling factor in the ion chamber detectors determined its ability to detect dose deviations. SPC and PCA results indicated that coalescence procedure could detect more potential failure QA results than SA while enhancing action thresholds.

Radiotherapy for primary thyroid adenoid cystic carcinoma.

Primary thyroid adenoid cystic carcinoma (PTACC) is an extremely rare type of mucin-secreting adenocarcinoma. Currently, it is difficult to diagnose, and it lacks standard treatment protocols. We report the case of a 53-year-old female patient with PTACC who underwent additional intensity-modulated radiotherapy 1 month after surgical treatment with an uneventful course. No invasion or distant metastasis was detected at the 7-month follow-up after radiotherapy, and the prognosis was favorable. In this case, herein, we have summarized the diagnostic features of the disease and proposed that postoperative adjuvant radiotherapy can significantly improve the patient's prognosis. Finally, we further confirmed the important role of radiotherapy in PTACC by reviewing relevant literature, which may provide clinicians with valuable treatment experience.

15 N-labeled dietary nitrate supplementation increases human skeletal muscle nitrate concentration and improves muscle torque production.

AIM: Dietary nitrate (NO3 - ) supplementation increases nitric oxide bioavailability and can enhance exercise performance. We investigated the distribution and metabolic fate of ingested NO3 - at rest and during exercise with a focus on skeletal muscle. METHODS: In a randomized, crossover study, 10 healthy volunteers consumed 12.8 mmol 15 N-labeled potassium nitrate (K15 NO3 ; NIT) or potassium chloride placebo (PLA). Muscle biopsies were taken at baseline, at 1- and 3-h post-supplement ingestion, and immediately following the completion of 60 maximal intermittent contractions of the knee extensors. Muscle, plasma, saliva, and urine samples were analyzed using chemiluminescence to determine absolute [NO3 - ] and [NO2 - ], and by mass spectrometry to determine the proportion of NO3 - and NO2 - that was 15 N-labeled. RESULTS: Neither muscle [NO3 - ] nor [NO2 - ] were altered by PLA. Following NIT, muscle [NO3 - ] (but not [NO2 - ]) was elevated at 1-h (from ~35 to 147 nmol/g, p < 0.001) and 3-h, with almost all of the increase being 15 N-labeled. There was a significant reduction in 15 N-labeled muscle [NO3 - ] from pre- to post-exercise. Relative to PLA, mean muscle torque production was ~7% greater during the first 18 contractions following NIT. This improvement in torque was correlated with the pre-exercise 15 N-labeled muscle [NO3 - ] and the magnitude of decline in 15 N-labeled muscle [NO3 - ] during exercise (r = 0.66 and r = 0.62, respectively; p < 0.01). CONCLUSION: This study shows, for the first time, that skeletal muscle rapidly takes up dietary NO3 - , the elevated muscle [NO3 - ] following NO3 - ingestion declines during exercise, and muscle NO3 - dynamics are associated with enhanced torque production during maximal intermittent muscle contractions.

Longitudinal multi-omics analyses of the gut-liver axis reveals metabolic dysregulation in hepatitis C infection and cirrhosis.

The gut and liver are connected via the portal vein, and this relationship, which includes the gut microbiome, is described as the gut-liver axis. Hepatitis C virus (HCV) can infect the liver and cause fibrosis with chronic infection. HCV has been associated with an altered gut microbiome; however, how these changes impact metabolism across the gut-liver axis and how this varies with disease severity and time is unclear. Here we used multi-omics analysis of portal and peripheral blood, faeces and liver tissue to characterize the gut-liver axis of patients with HCV across a fibrosis severity gradient before (n = 29) and 6 months after (n = 23) sustained virologic response, that is, no detection of the virus. Fatty acids were the major metabolites perturbed across the liver, portal vein and gut microbiome in HCV, especially in patients with cirrhosis. Decreased fatty acid degradation by hepatic peroxisomes and mitochondria was coupled with increased free fatty acid (FFA) influx to the liver via the portal vein. Metatranscriptomics indicated that Anaerostipes hadrus-mediated fatty acid synthesis influences portal FFAs. Both microbial fatty acid synthesis and portal FFAs were associated with enhanced hepatic fibrosis. Bacteroides vulgatus-mediated intestinal glycan breakdown was linked to portal glycan products, which in turn correlated with enhanced portal inflammation in HCV. Paired comparison of patient samples at both timepoints showed that hepatic metabolism, especially in peroxisomes, is persistently dysregulated in cirrhosis independently of the virus. Sustained virologic response was associated with a potential beneficial role for Methanobrevibacter smithii, which correlated with liver disease severity markers. These results develop our understanding of the gut-liver axis in HCV and non-HCV liver disease aetiologies and provide a foundation for future therapies.

Non-Nutritive Sweeteners in Human Amniotic Fluid and Cord Blood: Evidence of Transplacental Fetal Exposure.

OBJECTIVE: This study aimed to investigate human fetal exposure to non-nutritive sweeteners (NNS) by analyzing amniotic fluid and umbilical cord blood. STUDY DESIGN: Concentrations of four NNS (acesulfame-potassium [ace-K], saccharin, steviol glucuronide, and sucralose) were measured in amniotic fluid (n = 13) and cord blood samples (n = 15) using liquid chromatography-mass spectrometry. Amniotic fluid samples were obtained for research purposes at the time of term elective cesarean birth or clinically indicated third trimester amnioreduction at Mercy Hospital for Women (Melbourne, Australia). All except four women were in the fasting state. Cord blood samples were obtained from an independent cohort of newborns whose mothers were enrolled in a separate clinical trial at the National Institutes of Health. RESULTS: Ten of 13 amniotic fluid samples contained at least one NNS (ace-K, saccharin, steviol glucuronide, and/or sucralose). Maximum amniotic fluid NNS concentrations of ace-K, saccharin, steviol glucuronide, and sucralose were 78.9, 55.9, 93.5, and 30.6 ng/mL, respectively. Ace-K and saccharin were present in 100% and 80% of the cord blood samples, with maximal concentrations of 6.5 and 2.7 ng/mL, respectively. Sucralose was not detected and steviol glucuronide was not measurable in any of the cord blood samples. CONCLUSION: Our results provide evidence of human transplacental transmission of NNS. Based on results predominantly obtained from rodent models, we speculate that NNS exposure may adversely influence the offsprings' metabolic health. Well-designed, prospective clinical trials are necessary to understand the impact of NNS intake during pregnancy on human development and long-term health. KEY POINTS: · NNS consumption during pregnancy has increased in recent years.. · Maternal NNS intake during pregnancy is associated with preterm birth and higher infant weight gain in epidemiologic studies.. · In rodents, in utero NNS exposure induces metabolic abnormalities in mothers and their offspring, alters offspring gut microbiota composition, and promotes sweet taste preference in adulthood.. · It is presently unknown whether and to what degree maternal NNS ingestion in humans leads to direct in utero exposure.. · This study provides the first evidence of in utero NNS exposure in humans and highlights the urgent need to investigate clinical consequences of early life NNS exposure on metabolism, weight, taste preference, and general health..

Highly Efficient Separation of C8 Aromatic Isomers by Rationally Designed Nonaromatic Metal-Organic Frameworks.

The separation of C8 aromatics (xylenes and ethylbenzene) remains one of the most challenging industrial separations due to their similar structures and properties. Suitable adsorbents that can distinguish the small differences among isomers are urgently demanded. Herein, we demonstrate a strategy to realize the precise discrimination of C8 aromatics by constructing a nonaromatic confined pore environment with mixed polycycloalkane-type ligands. The nonaromatic low-polar pore environment avoids strong convergent interactions between the framework and the common phenyl rings while creating possibilities to amplify the difference between host-guest/guest-guest interactions regarding the different methyl (ethyl) group positions of isomers. The resultant metal-organic framework, ZUL-C3, with either tetragonal or monoclinic lattice, exhibits outstanding separation performance for C8 aromatics, not only realizing the simultaneous separation of four isomers from each other but also setting a benchmark for the dynamic separation performance of OX/PX and OX/MX.

The role transition of radiotherapy for the treatment of liver cancer in the COVID-19 era.

The uncontrollable COVID-19 crises in the SARS-CoV-2 high-prevalence areas have greatly disrupted the routine treatment of liver cancer and triggered a role transformation of radiotherapy for liver cancer. The weight of radiotherapy in the treatment algorithm for liver cancer has been enlarged by the COVID-19 pandemic, which is helpful for the optimal risk-benefit profile.

Tanshinone IIA Accomplished Protection against Radiation-Induced Cardiomyocyte Injury by Regulating the p38/p53 Pathway.

Background: Radiotherapy is one of the major strategies for treating tumors, and it inevitably causes damage to relevant tissues and organs during treatment. Radiation-induced heart disease (RIHD) refers to radiation-induced cardiovascular adverse effects caused by thoracic radiotherapy. Currently, there is no uniform standard in the treatment of RIHD. Methods: In our group study, by administering a dose of 4 Gy radiation, we established a radiation injured cardiomyocyte model and explored the regulatory relationship between tanshinone IIA and p38 MAPK in cardiomyocyte injury. We assessed cell damage and proliferation using clonogenic assay and lactate dehydrogenase (LDH) release assay. The measures of antioxidant activity and oxidative stress were conducted using superoxide dismutase (SOD) and reactive oxygen species (ROS). The apoptosis rate and the relative expression of apoptotic proteins were conducted using flow cytometry and western blot. To assess p38 and p53 expressions and phosphorylation levels, western blot was performed. Results: Experimental results suggested that tanshinone IIA restored cell proliferation in radiation-induced cardiomyocyte injury (∗∗P < 0.01), and the level of LDH release decreased (∗P < 0.05). Meanwhile, tanshinone IIA could decrease the ROS generation induced by radiation (∗∗P < 0.01) and upregulate the SOD level (∗∗P < 0.01). Again, tanshinone IIA reduced radiation-induced cardiomyocyte apoptosis (∗∗P < 0.01). Finally, tanshinone IIA downregulated radiation-induced p38/p53 overexpression (∗∗∗P < 0.001). Conclusions: The treatment effects of tanshinone IIA against radiation-induced myocardial injury may be through the regulation of the p38/p53 pathway.

Highly stretchable ionically crosslinked acrylate elastomers inspired by polyelectrolyte complexes.

Dynamic bonds are a powerful approach to tailor the mechanical properties of elastomers and introduce shape-memory, self-healing, and recyclability. Among the library of dynamic crosslinks, electrostatic interactions among oppositely charged ions have been shown to enable tough and resilient elastomers and hydrogels. In this work, we investigate the mechanical properties of ionically crosslinked ethyl acrylate-based elastomers assembled from oppositely charged copolymers. Using both infrared and Raman spectroscopy, we confirm that ionic interactions are established among polymer chains. We find that the glass transition temperature of the complex is in between the two individual copolymers, while the complex demonstrates higher stiffness and more recovery, indicating that ionic bonds can strengthen and enhance recovery of these elastomers. We compare cycles to increasing strain levels at different strain rates, and hypothesize that at fast strain rates ionic bonds dynamically break and reform while entanglements do not have time to slip, and at slow strain rates ionic interactions are disrupted and these entanglements slip significantly. Further, we show that a higher ionic to neutral monomer ratio can increase the stiffness, but its effect on recovery is minimal. Finally, taking advantage of the versatility of acrylates, ethyl acrylate is replaced with the more hydrophilic 2-hydroxyethyl acrylate, and the latter is shown to exhibit better recovery and self-healing at a cost of stiffness and strength. The design principles uncovered for these easy-to-manufacture polyelectrolyte complex-inspired bulk materials can be broadly applied to tailor elastomer stiffness, strength, inelastic recovery, and self-healing for various applications.

Environmental footprints of Chinese foods and beverages: Literature-based construction of a LCA database.

To accurately estimate and model the impact of food consumption and potential dietary changes on environment and climate change, the need for country specific data is evident. This study developed a Chinese Food Life Cycle Assessment Database (CFLCAD) in which Greenhouse Gas Emissions (GHGE) for 80 food items, Water Use (WU) for 93 food items and Land Use (LU) for 50 food items were collected through a literature review. To estimate the environmental footprints of food from production to consumption, the study applied conversion factors for the edible portion of food, food loss ratio and processing, storage, packaging, transportation, and food preparation stages. In addition, when no LCA data of a certain food was available, data from food groups with similar nutritional composition or cultivation condition were used as proxies. The database covered 17 food groups and each food item was referenced to the Chinese Food Composition Table and has a unique food code. The CFLCAD can be used to link individual-level food consumption data with nutrition survey in China, to allow for a more accurate estimation of the environmental footprints of Chinese diets.

Photocaging of Activity-Based Ubiquitin Probes via a C-Terminal Backbone Modification Strategy.

Photocaged, activity-based ubiquitin (Ub) probes (Ub-ABPs) have been developed for the time-resolved probing of deubiquitinating enzyme (DUB) activities, but many Ub-ABPs are still challenging to photocage because their warheads (e.g. propargylamide (PA) or dehydroalanine (Dha)) are difficult to temporally block and activate. Here, we describe a new C-terminal backbone modification strategy for the construction of photocaged Ub-ABPs in which a light-sensitive group is placed at the backbone amide bond of the Ub Gly75. This strategy enabled the facile generation of cell-permeable photocaged Ub-PA and Dha probes that could be activated to capture DUBs after photo-irradiation, and were used to profile DUBs in cells under specially designed conditions (e.g. in cells experiencing oxidative stress) or DUBs with isopeptide linkage selectivity. This backbone modification strategy is anticipated to provide a general solution for the development of photocaged Ub ABPs bearing any warheads for DUB profiling.

Postprandial Plasma Lipidomics Reveal Specific Alteration of Hepatic-derived Diacylglycerols in Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Hepatic energy metabolism is a dynamic process modulated by multiple stimuli. In nonalcoholic fatty liver disease (NAFLD), human studies typically focus on the static fasting state. We hypothesized that unique postprandial alterations in hepatic lipid metabolism are present in NAFLD. METHODS: In a prospective clinical study, 37 patients with NAFLD and 10 healthy control subjects ingested a standardized liquid meal with pre- and postprandial blood sampling. Postprandial plasma lipid kinetics were characterized at the molecular lipid species level by untargeted lipidomics, cluster analysis, and lipid particle isolation, then confirmed in a mouse model. RESULTS: There was a specific increase of multiple plasma diacylglycerol (DAG) species at 4 hours postprandially in patients with NAFLD but not in controls. This was replicated in a nonalcoholic steatohepatitis mouse model, where postprandial DAGs increased in plasma and concomitantly decreased in the liver. The increase in plasma DAGs appears early in the disease course, is dissociated from NAFLD severity and obesity, and correlates with postprandial insulin levels. Immunocapture isolation of very low density lipoprotein in human samples and stable isotope tracer studies in mice revealed that elevated postprandial plasma DAGs reflect hepatic secretion of endogenous, rather than meal-derived lipids. CONCLUSIONS: We identified a selective insulin-related increase in hepatic secretion of endogenously derived DAGs after a mixed meal as a unique feature of NAFLD. DAGs are known to be lipotoxic and associated with atherosclerosis. Although it is still unknown whether the increased exposure to hepatic DAGs contributes to extrahepatic manifestations and cardiovascular risk in NAFLD, our study highlights the importance of extending NAFLD research beyond the fasting state.