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This review summarizes the recent advances in the development of photoreactive bioorthogonal lipid probes, and the use of these lipid probes in mapping diverse biological pathways in mammalian cells using emerging chemoproteomic approaches.

Discovery of novel antibiotics targeting multidrug-resistant (MDR) pathogens such as A. baumannii is an urgent, unmet medical need. Our work has highlighted the potential of this unique scaffold to annihilate MDR A. baumannii alone and in combination with amikacin both in vitro and in animals, that too without inducing resistance. Further in depth analysis identified central metabolism to be a putative target. Taken together, these experiments lay down the foundation for effective management of infections caused due to highly MDR pathogens.

Type III polyketide synthases (PKSs) found across

Phagocytosis is an important evolutionary conserved process, essential for clearing pathogens and cellular debris in higher organisms, including humans. This well-orchestrated innate immunological response is intricately regulated by numerous cellular factors, important amongst which are the immunomodulatory lysophosphatidylserines (lyso-PSs) and the pro-apoptotic oxidized phosphatidylserines (PSs) signalling lipids. Interestingly, in mammals, both these signalling lipids are physiologically regulated by the lipase ABHD12, mutations of which cause the human neurological disorder PHARC. Despite the biomedical significance of this lipase, detailed mechanistic studies and the specific contribution of ABHD12 to innate processes like phagocytosis remain poorly understood. Here, by immunohistochemical and immunofluorescence approaches, using the murine model of PHARC, we show, that upon an inflammatory stimulus, activated microglial cells in the cerebellum of mice deficient in ABHD12 have an amoeboid morphology, increased soma size and display heightened phagocytosis activity. We also report that upon an inflammatory stimulus, cerebellar levels of ABHD12 increase to possibly metabolize the heightened oxidized PS levels, temper phagocytosis and, in turn, control neuroinflammation during oxidative stress. Next, to complement these findings, with the use of biochemical approaches in cultured microglial cells, we show that the pharmacological inhibition and/or genetic deletion of ABHD12 results in increased phagocytic uptake in a fluorescent bead uptake assay. Together, our studies provide compelling evidence that ABHD12 plays an important role in regulating phagocytosis in cerebellar microglial cells and provides a possible explanation, as to why human PHARC subjects display neuroinflammation and atrophy in the cerebellum.

Persulfides and polysulfides, collectively known as the sulfane sulfur pool along with hydrogen sulfide (H2S), play a central role in cellular physiology and disease. Exogenously enhancing these species in cells is an emerging therapeutic paradigm for mitigating oxidative stress and inflammation that are associated with several diseases. In this study, we present a unique approach of using the cell's own enzyme machinery coupled with an array of artificial substrates to enhance the cellular sulfane sulfur pool. We report the synthesis and validation of artificial/unnatural substrates specific for 3-mercaptopyruvate sulfurtransferase (3-MST), an important enzyme that contributes to sulfur trafficking in cells. We demonstrate that these artificial substrates generate persulfides in vitro as well as mediate sulfur transfer to low molecular weight thiols and to cysteine-containing proteins. A nearly 100-fold difference in the rates of H2S production for the various substrates is observed supporting the tunability of persulfide generation by the 3-MST enzyme/artificial substrate system. Next, we show that the substrate 1a permeates cells and is selectively turned over by 3-MST to generate 3-MST-persulfide, which protects against reactive oxygen species-induced lethality. Lastly, in a mouse model, 1a is found to significantly mitigate neuroinflammation in the brain tissue. Together, the approach that we have developed allows for the on-demand generation of persulfides in vitro and in vivo using a range of shelf-stable, artificial substrates of 3-MST, while opening up possibilities of harnessing these molecules for therapeutic applications., A persulfide/hydrogen sulfide generation strategy through artificial substrates for 3-mercaptopyruvate sulfurtransferase (3-MST) is reported, which enhances cellular persulfides, attenuates reactive oxygen species (ROS), and alleviates inflammation.

The serine hydrolase (SH) superfamily is, perhaps, one of the largest functional enzyme classes in all forms of life and consists of proteases, peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytic-type reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of chemical proteomics techniques, activity-based protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlases for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, we initially report a bioinformatics analysis toward the identification and categorization of nonredundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the full complement of SH activities during various developmental stages and in different adult tissues of Drosophila. Finally, as a proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult D. melanogaster, and we propose new research directions, resources, and tools that this study can provide to the fly community.

Inefficient physiological transitions are known to cause metabolic disorders. Therefore, investigating mechanisms that constitute molecular switches in a central metabolic organ like the liver becomes crucial. Specifically, upstream mechanisms that control temporal engagement of transcription factors, which are essential to mediate physiological fed–fast–refed transitions are less understood. SIRT1, a NAD(+)-dependent deacetylase, is pivotal in regulating hepatic gene expression and has emerged as a key therapeutic target. Despite this, if/how nutrient inputs regulate SIRT1 interactions, stability, and therefore downstream functions are still unknown. Here, we establish nutrient-dependent O-GlcNAcylation of SIRT1, within its N-terminal domain, as a crucial determinant of hepatic functions. Our findings demonstrate that during a fasted-to-refed transition, glycosylation of SIRT1 modulates its interactions with various transcription factors and a nodal cytosolic kinase involved in insulin signaling. Moreover, sustained glycosylation in the fed state causes nuclear exclusion and cytosolic ubiquitin-mediated degradation of SIRT1. This mechanism exerts spatiotemporal control over SIRT1 functions by constituting a previously unknown molecular relay. Of note, loss of SIRT1 glycosylation discomposed these interactions resulting in aberrant gene expression, mitochondrial dysfunctions, and enhanced hepatic gluconeogenesis. Expression of nonglycosylatable SIRT1 in the liver abrogated metabolic flexibility, resulting in systemic insulin resistance, hyperglycemia, and hepatic inflammation, highlighting the physiological costs associated with its overactivation. Conversely, our study also reveals that hyperglycosylation of SIRT1 is associated with aging and high-fat–induced obesity. Thus, we establish that nutrient-dependent glycosylation of SIRT1 is essential to gate its functions and maintain physiological fitness.

Significance Mycobacterium tuberculosis ( Mtb ) is the etiological agent of human tuberculosis (TB). Mtb can persist inside host macrophages by successfully adapting to intracellular conditions. Acquisition of balanced amounts of essential micronutrients is one such important process. Our studies have identified a metallophore produced on demand to restore Mtb zinc metabolic imbalance. These diacyl-diisonitrile lipopeptides, named kupyaphores, are specifically induced during infection and move in and out of cells to protect bacteria from host-mediated nutritional deprivation and intoxication. Furthermore, we identify an Mtb isonitrile hydratase homolog , expressed in low-zinc conditions, which probably facilitates zinc release from kupyaphores. Identification of this zinc acquisition strategy could provide opportunities in future to understand systemic zinc dysbiosis and associated manifestations in TB patients.

Chain-length-specific subsets of diacylglycerol (DAG) lipids are proposed to regulate differential physiological responses ranging from signal transduction to modulation of the membrane properties. However, the mechanism or molecular players regulating the subsets of DAG species remain unknown. Here, we uncover the role of a conserved eukaryotic protein family, DISCO-interacting protein 2 (DIP2) as a homeostatic regulator of a chemically distinct subset of DAGs using yeast, fly, and mouse models. Genetic and chemical screens along with lipidomics analysis in yeast reveal that DIP2 prevents the toxic accumulation of specific DAGs in the logarithmic growth phase, which otherwise leads to endoplasmic reticulum stress. We also show that the fatty acyl-AMP ligase-like domains of DIP2 are essential for the redirection of the flux of DAG subspecies to storage lipid, triacylglycerols. DIP2 is associated with vacuoles through mitochondria-vacuole contact sites and such modulation of selective DAG abundance by DIP2 is found to be crucial for optimal vacuole membrane fusion and consequently osmoadaptation in yeast. Thus, the study illuminates an unprecedented DAG metabolism route and provides new insights on how cell fine-tunes DAG subspecies for cellular homeostasis and environmental adaptation.Lipids, such as fats and hormones, constitute one of the main building blocks of cells. There are thousands of different lipids each with distinctive chemical properties that allow them to carry out specific roles. For example, a group of lipids called diacylglycerols help cells perform a myriad of tasks, like sensing external signals, making membranes, and storing energy. The production and breakdown of diacylglycerols is therefore tightly regulated. However, very little is known about the molecules involved in this metabolic process. One possible candidate is the enzyme DIP2 which is comprised of a protein module known as FAAL (short for fatty acyl-AMP ligase). FAAL belongs to a family of enzymes that synthesize lipid-like molecules in bacteria. In 2021, a group of researchers tracked the evolutionary trajectory of these bacterial proteins and found that most of them were lost in eukaryotes, such as animals and fungi. FAAL-like proteins, however, had been retained through evolution and incorporated in to DIP2. Here, Mondal, Kinatukara et al. – including some of the researchers involved in the 2021 study – have used a combination of genetic and biochemical experiments to investigate whether and how DIP2 contributes to lipid metabolism in eukaryotes. They found that yeast cells without the gene for DIP2 had higher levels of diacylglycerols which hampered the shape and function of certain cellular compartments. The mutant cells were also unable to convert diacylglycerols in to another group of lipids which are involved in energy storage. This effect was observed in fruit flies and mice lacking DIP2, suggesting that this role for DIP2 is conserved across most eukaryotes. Further experiments in yeast cells revealed that unlike other enzymes that metabolize diacylglycerols, DIP2 only acted on a sub-population of diacylglycerols at specific locations and times. Furthermore, yeast cells lacking DIP2 could still grow under ideal conditions, but could not cope with high or low salt concentrations in their surroundings, suggesting that the enzyme helps cells deal with environmental stresses. Since DIP2 is found in most eukaryotes, understanding how it works could be useful for multiple branches of biology. For example, some pathogenic fungi that cause diseases in crop plants and humans also rely on DIP2. Further studies are needed to better understand the role that DIP2 plays in other eukaryotic species which may shed light on other processes the enzyme is involved in

Phagocytosis is an important physiological process, which, in higher organisms, is a means of fighting infections and clearing cellular debris. During phagocytosis, detrimental foreign particles (e.g. pathogens and apoptotic cells) are engulfed by phagocytes (e.g. macrophages), enclosed in membrane-bound vesicles called phagosomes, and transported to the lysosome for eventual detoxification. During this well-choreographed process, the nascent phagosome (also called early phagosome, EP) undergoes a series of spatiotemporally regulated changes in its protein and lipid composition and matures into a late phagosome (LP), which subsequently fuses with the lysosomal membrane to form the phagolysosome. While several elegant proteomic studies have identified the role of unique proteins during phagosomal maturation, the corresponding lipidomic studies are sparse. Recently, we reported a comparative lipidomic analysis between EPs and LPs and showed that ceramides are enriched on the LPs. Further, we found that this ceramide accumulation on LPs was orchestrated by ceramide synthase 2, inhibition of which hampers phagosomal maturation. Following up on this study, here, using biochemical assays, we first show that the increased ceramidase activity on EPs also significantly contributes to the accumulation of ceramides on LPs. Next, leveraging lipidomics, we show that de novo ceramide synthesis does not significantly contribute to the ceramide accumulation on LPs, while concomitant to increased ceramides, glucosylceramides are substantially elevated on LPs. We validate this interesting finding using biochemical assays and show that LPs indeed have heightened glucosylceramide synthase activity. Taken together, our studies provide interesting insights and possible new roles of sphingolipid metabolism during phagosomal maturation.

Kumar et al. describe a detailed pathway for channeling fat from the liver into blood across fed/fasted cycles. Insulin, phosphatidic acid, and kinesin collaborate in hepatocytes to deliver lipid droplets to the smooth ER, where they are catabolized to supply fat for lipoprotein production and secretion., Triglyceride-rich lipid droplets (LDs) are catabolized with high efficiency in hepatocytes to supply fatty acids for producing lipoprotein particles. Fasting causes a massive influx of adipose-derived fatty acids into the liver. The liver in the fasted state is therefore bloated with LDs but, remarkably, still continues to secrete triglycerides at a constant rate. Here we show that insulin signaling elevates phosphatidic acid (PA) dramatically on LDs in the fed state. PA then signals to recruit kinesin-1 motors, which transport LDs to the peripherally located smooth ER inside hepatocytes, where LDs are catabolized to produce lipoproteins. This pathway is down-regulated homeostatically when fasting causes insulin levels to drop, thus preventing dangerous elevation of triglycerides in the blood. Further, we show that a specific peptide against kinesin-1 blocks triglyceride secretion without any apparent deleterious effects on cells. Our work therefore reveals fundamental mechanisms that maintain lipid homeostasis across metabolic states and leverages this knowledge to propose a molecular target against hyperlipidemia.

Publisher's version (útgefin grein), The alarming global rise in fatalities from multidrug-resistant Staphylococcus aureus (S. aureus) infections has underscored a need to develop new therapies to address this epidemic. Chemoproteomics is valuable in identifying targets for new drugs in different human diseases including bacterial infections. Targeting functional cysteines is particularly attractive, as they serve critical catalytic functions that enable bacterial survival. Here, we report an indole-based quinone epoxide scaffold with a unique boat-like conformation that allows steric control in modulating thiol reactivity. We extensively characterize a lead compound (4a), which potently inhibits clinically derived vancomycin-resistant S. aureus. Leveraging diverse chemoproteomic platforms, we identify and biochemically validate important transcriptional factors as potent targets of 4a. Interestingly, each identified transcriptional factor has a conserved catalytic cysteine residue that confers antibiotic tolerance to these bacteria. Thus, the chemical tools and biological targets that we describe here prospect new therapeutic paradigms in combatting S. aureus infections., The authors thank the Department of Biotechnology (DBT), Government of India (BT/PR15848/MED/29/1025/2016 to H.C. and S.C.), a Wellcome Trust DBT India Alliance Intermediate Fellowship (IA/I/15/2/502058 to S.S.K.) and a DST-FIST Infrastructure Development Grant (to IISER Pune Biology) for the financial support for our research. The Council for Scientific and Industrial Research (CSIR) and the Department of Science and Technology—Innovation in Science Pursuit for Inspired Research (DST-INSPIRE) for graduate student fellowships.

Reactive oxygen species (ROS) are transient, highly reactive intermediates or byproducts produced during oxygen metabolism. However, when innate mechanisms are unable to cope with sequestration of surplus ROS, oxidative stress results, in which excess ROS damage biomolecules. Oxidized phosphatidylserine (PS), a proapoptotic 'eat me' signal, is produced in response to elevated ROS, yet little is known regarding its chemical composition and metabolism. Here, we report a small molecule that generates ROS in different mammalian cells. We used this molecule to detect, characterize and study oxidized PS in mammalian cells. We developed a chemical-genetic screen to identify enzymes that regulate oxidized PS in mammalian cells and found that the lipase ABHD12 hydrolyzes oxidized PS. We validated these findings in different physiological settings including primary peritoneal macrophages and brains from Abhd12-/- mice under inflammatory stress, and in the process, we functionally annotated an enzyme regulating oxidized PS in vivo.

Background: This study addresses an important gap, as it is the first US nationwide, epidemiologic study of ADPKD incidence and prevalence. Summary: This 3-year, observational study utilized data from Truven Health MarketScan® administrative claims, as well as cross-sectional data from the National Ambulatory Medical Care Survey (NAMCS). We estimated the annual incidence and diagnosed prevalence using population-based data on over 170 million de-identified patients to provide the most current epidemiologic estimates available. The ADPKD-diagnosed prevalence was 4.3 per 10,000 in the NAMCS, which closely corresponded with age-adjusted rates from patients with either commercial insurance or employer-sponsored Medicare supplemental insurance. The annual incidence was 0.62 per 10,000. Both nationwide data sets indicate that approximately 140,000 patients are currently diagnosed in the USA. We also found significant differences by gender and age. Females are nearly twice as likely as males to be diagnosed in early adulthood, while the incidence in males was highest in those aged 65 years or older. ADPKD appears more likely to be diagnosed in men after disease progression or the development of chronic kidney disease. Key Messages: Our results revealed striking age and gender differences in the incidence of ADPKD. Young women are diagnosed with ADPKD at nearly twice the rate of young men, perhaps due to the use of ultrasound in women during child-bearing years. This points to a need for increased recognition of ADPKD, with an emphasis on younger men in particular. ADPKD has been inaccurately perceived as a common condition based on misinterpretation of early epidemiologic data (1957) confirmed by our data and recent European data. ADPKD affects approximately 140,000 patients in the USA and meets the criterion for a rare disease. Our results indicate a need for further study of gender and ADPKD diagnosis, progression, management, and outcomes.

The sirtuins and histone deacetylases are the best characterized members of the lysine deacetylase (KDAC) enzyme family. Recently, we annotated the "orphan" enzyme ABHD14B (α/β-hydrolase domain containing protein # 14B) as a novel KDAC and showed this enzyme's ability to transfer an acetyl-group from protein lysine residue(s) to coenzyme-A to yield acetyl-coenzyme-A, thereby, expanding the repertoire of this enzyme family. However, the role of ABHD14B in metabolic processes is not fully elucidated. Here, we investigated the role of this enzyme using mammalian cell knockdowns in a combined transcriptomics and metabolomics analysis. We found from these complementary experiments in vivo that the loss of ABHD14B results in significantly altered glucose metabolism, specifically the decreased flux of glucose through glycolysis and the citric acid cycle. Further, we show that depleting hepatic ABHD14B in mice also results in defective systemic glucose metabolism, particularly during fasting. Taken together, our findings illuminate the important metabolic functions that the KDAC ABHD14B plays in mammalian physiology and poses new questions regarding the role of this hitherto cryptic metabolism-regulating enzyme.

The serine hydrolase (SH) superfamily is perhaps, one of the largest functional enzyme classes in all forms of life, and consists of proteases, peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytic-type reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of the chemical proteomics technique, activity-based protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlas’ for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, here, we initially report a bioinformatics analysis towards the identification and categorization of non-redundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the full complement of SH activities during various developmental stages and in different adult tissues of Drosophila. Finally, as proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult Drosophila melanogaster, and together, we prospect new research directions, resources and tools that this study can provide to the fly community.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tuberculosis (TB) patients suffer from progressive and debilitating loss of muscle mass and function, referred to as cachexia. Though a multifactorial condition, cachexia in cancer is promoted by systemic zinc redistribution and accumulation in muscles. Clinical studies with TB patients indeed show zinc dyshomeostasis. We therefore set out to understand mechanisms by which Mycobacterium tuberculosis (Mtb) govern zinc metallostasis at the host-pathogen interface. Here, we report a novel zinc metallophore from Mtb that restores zinc metabolic imbalance. These diisonitrile lipopeptides, named kupyaphores are transiently induced early-on during macrophage infection and also in infected mice lungs. Kupyaphores protects bacteria from host-mediated nutritional deprivation and intoxication. Kupyaphore Mtb mutant strain cannot mobilize zinc and shows reduced fitness in mice. Further, we characterize Mtb encoded isonitrile hydratase that could mediate intracellular zinc release through covalent modification of kupyaphores. Our studies could provide a molecular link between TB-induced altered zinc homeostasis and associated cachexia.

Lysophospholipids are potent hormone-like signalling biological lipids that regulate many important biological processes in mammals (including humans). Lysophosphatidic acid and sphingosine-1-phosphate represent the best studied examples for this lipid class, and their metabolic enzymes and/or cognate receptors are currently under clinical investigation for treatment of various neurological and autoimmune diseases in humans. Over the past two decades, the lysophsophatidylserines (lyso-PSs) have emerged as yet another biologically important lysophospholipid, and deregulation in its metabolism has been linked to various human pathophysiological conditions. Despite its recent emergence, an exhaustive review summarizing recent advances on lyso-PSs and the biological pathways that this bioactive lysophospholipid regulates has been lacking. To address this, here, we summarize studies that led to the discovery of lyso-PS as a potent signalling biomolecule, and discuss the structure, its detection in biological systems, and the biodistribution of this lysophospholipid in various mammalian systems. Further, we describe in detail the enzymatic pathways that are involved in the biosynthesis and degradation of this lipid and the putative lyso-PS receptors reported in the literature. Finally, we discuss the various biological pathways directly regulated by lyso-PSs in mammals and prospect new questions for this still emerging biomedically important signalling lysophospholipid.

Vitamin D, a secosteroid, plays an important role in several physiological processes, and its deficiency can lead to numerous pathophysiological conditions in humans. The primary objective of this study was to develop and validate the robustness of a mass spectrometry-based method capable of quantifying 25(OH)D3 for an upcoming epidemiological survey in India and to pilot test it on healthy volunteers. We first describe the development and validation of various experimental parameters that ascertain the robustness and reliability of 25-hydroxy-vitamin D3 (25(OH)D3) extractions and quantitative measurements from Dried Blood Spot (DBS) samples, where we used eight disks of 3 mm each, punched from the circular spot covering the entire circumference of the spot. Next, we conducted a pilot study, comparing 25(OH)D3 levels from serum and DBS samples from 45 participants using a protocol developed for specifically this purpose. We found that the mean 25(OH)D3 concentrations in DBS samples were comparable to the serum levels (P > 0.05). In summary, our extraction and LC–MS protocol for quantitative 25(OH)D3 measurements are robust and reproducible, and will serve as an invaluable tool for upcoming epidemiological surveys in India and perhaps around the world.

Disco-interacting protein 2 is a highly conserved three-domain protein with two tandem Adenylate-forming domains. It is proposed to influence the processes involved in neuronal development by influencing lipid metabolism and remains to be characterized. In this study, we show that Disco-interacting protein 2a null mice do not exhibit overt phenotype defects. However, the body composition differences were observed in these mice under different dietary regimens. The neutral lipid composition of two different diets was characterized, and it was observed that the new-born mice grow relatively slower than the wild-type mice with delayed appearance of features such as dentition when fed with high-triacylglycerol NIN-formulation diet. The high-diacylglycerol Safe-formulation diet was found to accumulate more fat mass in mice than those fed with high-triacylglycerol NIN-formulation diet beyond 10 months. These findings point to a proposed relationship between dietary components (particularly the lipid composition) and body composition along with the growth of neonates in mice lacking the gene Disco-interacting protein 2a.

Lysophosphatidylserine (lyso-PS), a lysophospholipid derived from phosphatidylserine (PS), has emerged as a potent signaling lipid in mammalian physiology. In vivo, the metabolic serine hydrolases ABHD16A and ABHD12 are major lipases that biosynthesize and degrade lyso-PS, respectively. Of biomedical relevance, deleterious mutations to ABHD12 cause accumulation of lyso-PS in the brain, and this deregulated lyso-PS metabolism leads to the human genetic neurological disorder PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract). While the roles of ABHD16A and ABHD12 in lyso-PS metabolism in the mammalian brain are well established, the anatomical and (sub)cellular localizations of both lipases and the functional cross-talk between them with respect to regulating lyso-PS lipids remain under investigated. Here, using subcellular organelle fractionation, biochemical assays, and immunofluorescence-based high-resolution microscopy, we show that the PS lipase ABHD16A is an endoplasmic reticulum-localized enzyme, an organelle intricately regulating cellular PS levels. In addition, leveraging immunohistochemical analysis using genetic ABHD16A and ABHD12 knockout mice as important controls, we map the anatomical distribution of both of these lipases in tandem in the murine brain and show for the first time the distinct localization of these lipases to different regions and cells of the cerebellum. We complement the aforementioned immunohistochemical studies by quantitatively measuring lyso-PS concentrations in various brain regions using mass spectrometry and find that the cerebellar lyso-PS levels are most affected by deletion of ABHD16A (decreased) or ABHD12 (increased). Taken together, our studies provide new insights into lyso-PS signaling in the cerebellum, the most atrophic brain region in human PHARC subjects.

Treatment options for patients with acute myeloid leukemia (AML), who are unfit for induction chemotherapy are unsatisfactory. Overall survival (OS) superiority has not been demonstrated in randomized controlled trials (RCT) in this population, challenging the value of available therapies. We sought to assess the relative value of approved therapies using value-assessment tools. Clinical, safety, quality-of-life (QOL), supportive care, and resource utilization outcomes data were abstracted from RCTs and examined using value-assessment frameworks. Three RCTs, one each of azacitidine, decitabine, and low-dose cytarabine were identified. OS was not statistically significant and secondary outcomes including response rates, rates of transfusion independence, the frequency of hospitalizations and changes in QOL were reported differently across trials. Value-assessment tools considered OS as the primary efficacy endpoint without consideration to response rates. The NCCN Evidence BlocksTM were most successful in considering secondary endpoints. With the move toward value-based care, understanding how these value tools apply to AML patients is critical.

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a rare genetic human neurological disorder caused by null mutations to the Abhd12 gene, which encodes the integral membrane serine hydrolase enzyme ABHD12. Although the role that ABHD12 plays in PHARC is understood, the thorough biochemical characterization of ABHD12 is lacking. Here, we report the facile synthesis of mono-1-(fatty)acyl-glycerol lipids of varying chain lengths and unsaturation and use this lipid substrate library to biochemically characterize recombinant mammalian ABHD12. The substrate profiling study for ABHD12 suggested that this enzyme requires glycosylation for optimal activity and that it has a strong preference for very-long-chain lipid substrates. We further validated this substrate profile against brain membrane lysates generated from WT and ABHD12 knockout mice. Finally, using cellular organelle fractionation and immunofluorescence assays, we show that mammalian ABHD12 is enriched on the endoplasmic reticulum membrane, where most of the very-long-chain fatty acids are biosynthesized in cells. Taken together, our findings provide a biochemical explanation for why very-long-chain lipids (such as lysophosphatidylserine lipids) accumulate in the brains of ABHD12 knockout mice, which is a murine model of PHARC.

Phagocytosis is an evolutionarily conserved biological process where pathogens or cellular debris are cleared by engulfing them in a membrane-enclosed cellular compartment called the phagosome. The formation, maturation, and subsequent degradation of a phagosome is an important immune response essential for protection against many pathogens. Yet, the global lipid profile of phagosomes remains unknown, especially as a function of their maturation in immune cells. Here, we show using mass spectrometry based quantitative lipidomics that the ceramide class of lipids, especially very long chain ceramides, are enriched on maturing phagosomes with a concomitant decrease in the biosynthetic precursors of ceramides. We thus posit a new function for the enzyme ceramide synthase during phagocytosis in mammalian macrophages. Biochemical assays, cellular lipid feeding experiments, and pharmacological blockade of ceramide synthase together show that this enzyme indeed controls the flux of ceramides on maturing phagosomes. We also find similar results in the primitive eukaryote Dictyostelium discoideum, suggesting that ceramide enrichment may be evolutionarily conserved and likely an indispensible step in phagosome maturation.

In humans, lysophosphatidylserines (lyso-PSs) are potent lipid regulators of important immunological processes. Given their structural diversity and commercial paucity, here, we report the synthesis of methyl-esters of lyso-PS (Me-lyso-PS), containing medium to very-long chain (VLC) lipid tails. We show that Me-lyso-PSs are excellent substrates for the lyso-PS lipase ABHD12, and that these synthetic lipids are acted upon by cellular carboxylesterases to produce lyso-PSs. Next, in macrophages, we demonstrate that VLC lyso-PSs orchestrate pro-inflammatory responses and in turn neuroinflammation via a Toll-like receptor 2 (TLR2) dependent pathway. We also show that long chain (LC) lyso-PSs robustly induce intracellular cyclic AMP production, cytosolic calcium influx, and phosphorylation of the nodal extracellular signal-regulated kinase (ERK) to regulate macrophage activation via a TLR2-independent pathway. Finally, we report that LC lyso-PSs potently elicit histamine release during the mast cell degranulation process, and that ABHD12 is the major lyso-PS lipase in these immune cells.

Significance The liver secretes lipids in a controlled manner despite vast changes in its internal lipid content. This buffering function of the liver is essential for lipid/energy homeostasis, but its molecular and cellular mechanism is unknown. We show that motor protein kinesin transports lipid droplets (LDs) to the endoplasmic reticulum (ER) in liver cells, engineering ER−droplet contacts and supplying lipids to the ER for secretion as lipoprotein. However, when fasting induces massive lipid accumulation in liver, kinesin is removed from LDs, inhibiting lipid supply to the ER and homeostatically tempering lipid secretion from liver in a fasted state. Interestingly, reducing kinesin also blocks propagation of hepatitis-C virus inside liver cells, possibly because viral proteins cannot transfer from the ER to LDs., Despite massive fluctuations in its internal triglyceride content, the liver secretes triglyceride under tight homeostatic control. This buffering function is most visible after fasting, when liver triglyceride increases manyfold but circulating serum triglyceride barely fluctuates. How the liver controls triglyceride secretion is unknown, but is fundamentally important for lipid and energy homeostasis in animals. Here we find an unexpected cellular and molecular mechanism behind such control. We show that kinesin motors are recruited to triglyceride-rich lipid droplets (LDs) in the liver by the GTPase ARF1, which is a key activator of lipolysis. This recruitment is activated by an insulin-dependent pathway and therefore responds to fed/fasted states of the animal. In fed state, ARF1 and kinesin appear on LDs, consequently transporting LDs to the periphery of hepatocytes where the smooth endoplasmic reticulum (sER) is present. Because the lipases that catabolize LDs in hepatocytes reside on the sER, LDs can now be catabolized efficiently to provide triglyceride for lipoprotein assembly and secretion from the sER. Upon fasting, insulin is lowered to remove ARF1 and kinesin from LDs, thus down-regulating LD transport and sER–LD contacts. This tempers triglyceride availabiity for very low density lipoprotein assembly and allows homeostatic control of serum triglyceride in a fasted state. We further show that kinesin knockdown inhibits hepatitis-C virus replication in hepatocytes, likely because translated viral proteins are unable to transfer from the ER to LDs.

The metabolic serine hydrolase family is, arguably, one of the largest functional enzyme classes in mammals, including humans, comprising 1-2% of the total proteome. This enzyme family uses a conserved nucleophilic serine residue in the active site to perform diverse hydrolytic reactions and consists of proteases, lipases, esterases, amidases, and transacylases, which are prototypical members of this family. In humans, this enzyme family consists of >250, of which approximately 40% members remain unannotated, in terms of both their endogenous substrates and the biological pathways that they regulate. The enzyme ABHD14B, an outlying member of this family, is also known as CCG1/TAFII250-interacting factor B, as it was found to be associated with transcription initiation factor TFIID. The crystal structure of human ABHD14B was determined more than a decade ago; however, its endogenous substrates remain elusive. In this paper, we annotate ABHD14B as a lysine deacetylase (KDAC), showing this enzyme's ability to transfer an acetyl group from a post-translationally acetylated lysine to coenzyme A (CoA), to yield acetyl-CoA, while regenerating the free amine of protein lysine residues. We validate these findings by in vitro biochemical assays using recombinantly purified human ABHD14B in conjunction with cellular studies in a mammalian cell line by knocking down ABHD14B and by identification of a putative substrate binding site. Finally, we report the development and characterization of a much-needed, exquisitely selective ABHD14B antibody, and using it, we map the cellular and tissue distribution of ABHD14B and prospective metabolic pathways that this enzyme might biologically regulate.

Purpose Costs associated with unplanned readmissions among patients with heart failure with and without hyponatremia were studied. Methods This study estimated the costs of patients hospitalized for heart failure (HF) discharged with or without corrected sodium. A model was developed to monetize the 30-day readmission risk based on hyponatremia correction. Costs of discharging patient with corrected versus uncorrected hyponatremia were estimated using readmission rates from a previously published study and hospitalization costs from the Healthcare Costs and Utilization Cost Project and the Premier Healthcare Database. Results Discharging patients with HF and hyponatremia increased costs from $488-$569 per discharge compared to patients with corrected hyponatremia. This range reflected differences in readmission rates and sources of hospitalization costs. Sensitivity analyses showed hospitalization costs and readmission rates had the largest impact on model results. Conclusion A retrospective study supports the value of upfront monitoring and correction of low serum sodium levels before discharge among patients with HF and hyponatremia by presenting an economic argument in addition to the clinical rational for reducing risk of readmission.

Insulin resistance (IR) is known to precede onset of type 2 diabetes and increased oxidative stress appears to be a deleterious factor leading to IR. In this study, we evaluated ability of pterostilbene (PTS), a methoxylated analogue of resveratrol and a known antioxidant, to reverse palmitic acid (PA)-mediated IR in HepG2 cells. PTS prevented reactive oxygen species (ROS) formation and subsequent oxidative lipid damage by reducing the expression of NADPH oxidase 3 (NOX3) in PA treated HepG2 cells. Hepatic glucose production was used as a measure of IR and PTS reversed PA-mediated increase in hepatic glucose production by reducing expression of genes coding for gluconeogenic enzymes namely glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate carboxylase (PC); and their transcription factors cAMP response element binding protein (CREB) and fork head class Box O (FOXO1) along with its coactivator peroxisome proliferator-activated receptor gamma co-activator-1 α (PGC1α). PTS reversed PA-mediated activation of c-Jun N-terminal kinase (JNK), which in turn altered insulin signalling pathway by phosphorylating IRS-1 at Ser 307, leading to inhibition of phosphorylation of Akt and GSK-3β. PTS also reduced PA-mediated lipid accumulation by reducing expression of transcription factors SREBP1c and PPARα. SREBP1c activates genes involved in fatty acid and triglyceride synthesis while PPARα activates CPT1, a rate limiting enzyme for controlling entry and oxidation of fatty acids into mitochondria. PTS, however, did not influence PA uptake confirmed by using BODIPY-labelled fluorescent C16 fatty acid analogue. Thus, our data provides a possible mechanistic explanation for reversal of PA-mediated IR in HepG2 cells.

Familial amyotrophic lateral sclerosis (ALS) is an incurable, late-onset motor neuron disease, linked strongly to various causative genetic loci. ALS8 codes for a missense mutation, P56S, in VAMP-associated protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase our understanding of the disease and lead to potential therapeutics. We developed a quantitative high-throughput Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy to score for genes involved in the modulation of aggregates of the fly orthologue, VAP(P58S), fused with GFP. A targeted RNA interference screen against 900 genes identified 150 hits that modify aggregation, including the ALS loci Sod1 and TDP43 (also known as TBPH), as well as genes belonging to the mTOR pathway. Further, a system to measure the extent of VAP(P58S) aggregation in the Drosophila larval brain was developed in order to validate the hits from the cell-based screen. In the larval brain, we find that reduction of SOD1 levels or decreased mTOR signalling reduces aggregation, presumably by increasing the levels of cellular reactive oxygen species (ROS). The mechanism of aggregate clearance is, primarily, proteasomal degradation, which appears to be triggered by an increase in ROS. We have thus uncovered an interesting interplay between SOD1, ROS and mTOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead to better understanding of the initiation and progression of ALS. This article has an associated First Person interview with the first author of the paper.

Familial amyotrophic lateral sclerosis (ALS) is an incurable, late-onset motor neuron disease, linked strongly to various causative genetic loci. ALS8 codes for a missense mutation, P56S, in VAMP-associated protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase our understanding of the disease and lead to potential therapeutics. We developed a quantitative high-throughput Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy to score for genes involved in the modulation of aggregates of the fly orthologue, VAP(P58S), fused with GFP. A targeted RNA interference screen against 900 genes identified 150 hits that modify aggregation, including the ALS loci Sod1 and TDP43 (also known as TBPH), as well as genes belonging to the mTOR pathway. Further, a system to measure the extent of VAP(P58S) aggregation in the Drosophila larval brain was developed in order to validate the hits from the cell-based screen. In the larval brain, we find that reduction of SOD1 levels or decreased mTOR signalling reduces aggregation, presumably by increasing the levels of cellular reactive oxygen species (ROS). The mechanism of aggregate clearance is, primarily, proteasomal degradation, which appears to be triggered by an increase in ROS. We have thus uncovered an interesting interplay between SOD1, ROS and mTOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead to better understanding of the initiation and progression of ALS.This article has an associated First Person interview with the first author of the paper.

This study analyzed hospital readmission rates of patients with schizophrenia who were treated with long-acting injectable antipsychotics (LAIs) or with oral antipsychotics after being discharged from a hospitalization.Medical claims of patients with schizophrenia who were ages 18-64 and had a first hospitalization for a serious mental illness (index hospitalization, October 2007 through September 2012) and at least one prescription for a first- or second-generation antipsychotic were analyzed from the Truven Health MarketScan Multi-State Medicaid Database. Analyses were conducted for patients with a sole diagnosis of schizophrenia (N=1,450) and for all patients with schizophrenia (N=15,556), which added patients with a codiagnosis of bipolar disorder or major depressive disorder. Probability of rehospitalization for any cause at 30 and 60 days after the initial hospitalization was assessed with multivariate logistic regression and propensity score matching (PSM) methods. The PSM model matched age, preindex use of LAIs or short-acting injectables, and select comorbidities between the LAI and the oral antipsychotics groups.LAIs were associated with significantly lower probability of rehospitalization compared with oral antipsychotics at 60 days for schizophrenia-only patients (adjusted odds ratio [AOR]=.60, 95% confidence interval [CI]=.41-.90) and for all patients (AOR=.70, CI=.52-.95). The absolute difference in probability of rehospitalization for all patients was significantly lower by 5.0% at 60 days in the LAI group compared with the oral antipsychotics group.Compared with use of oral antipsychotics, use of LAIs was associated with fewer readmissions of Medicaid patients with schizophrenia within 60 days after an index hospitalization.

Schizophrenia presents a substantial clinical and economic burden to the health-care system. In QUAlity of LIfe with AbiliFY Maintena (QUALIFY), a randomized head-to-head study of aripiprazole once-monthly 400 mg (AOM 400) compared with paliperidone palmitate (PP; 78–234 mg/mo), AOM 400 demonstrated greater improvement in health-related quality of life and functioning in patients with stable schizophrenia. The present analysis used health economics assessment data collected during the QUALIFY study to determine the direct medical and pharmacy costs and the cost-effectiveness associated with each treatment over 6 months. Compared with those receiving PP, patients receiving AOM 400 incurred significantly lower direct total costs ($8908±186 vs $9675±190, p=0.005) and treatment costs ($7967±113 vs $8706±116, p

Objective The objective of this study was to estimate the US societal economic burden of schizophrenia and update the 2002 reported costs of $62.7 billion given the disease management and health care structural changes of the last decade. Methods A prevalence-based approach was used to assess direct health care costs, direct non-health care costs, and indirect costs associated with schizophrenia (ICD-9 codes 295.xx) for 2013, with cost adjustments where necessary. Direct health care costs were estimated using a retrospective matched cohort design using the Truven Health Analytics MarketScan Commercial Claims and Encounters, Medicare Supplemental, and Medicaid Multistate databases. Direct non-health care costs were estimated for law enforcement, homeless shelters, and research and training. Indirect costs were estimated for productivity loss from unemployment, reduced work productivity among the employed, premature mortality (ie, suicide), and caregiving. Results The economic burden of schizophrenia was estimated at $155.7 billion ($134.4 billion-$174.3 billion based on sensitivity analyses) for 2013 and included excess direct health care costs of $37.7 billion (24%), direct non-health care costs of $9.3 billion (6%), and indirect costs of $117.3 billion (76%) compared to individuals without schizophrenia. The largest components were excess costs associated with unemployment (38%), productivity loss due to caregiving (34%), and direct health care costs (24%). Conclusions Schizophrenia is associated with a significant economic burden where, in addition to direct health care costs, indirect and non-health care costs are strong contributors, suggesting that therapies should aim at improving not only symptom control but also cognition and functional performance, which are associated with substantial non-health care and indirect costs.

Drosophilamethyltransferase (Mt2) has been implicated in methylation of both DNA and tRNA. In this study, we demonstrate that loss ofMt2activity leads to an age dependent decline of immune function in the adult fly. A newly eclosed adult has mild immune defects that exacerbate in a fifteen-day oldMt2−/−fly. The age dependent effects appear to be systemic, including disturbances in lipid metabolism, changes in cell shape of hemocytes and significant fold changes in levels of transcripts related to host defense. Lipid imbalance, as measured by quantitative lipidomics, correlates with immune dysfunction with high levels of immunomodulatory lipids, sphingosine-1phosphate (S1P) and ceramides, along with low levels of storage lipids. Activity assays on fly lysates confirm the age dependent increase in S1P and concomitant reduction of S1P lyase activity. We hypothesize thatMt2functions to regulate genetic loci such as S1P lyase and this regulation is essential for robust host defense as the animal ages. Our study uncovers novel links between age dependentMt2function, innate immune response and lipid homeostasis.

Myrlene S Aigbogun,1 Sizhu Liu,2 Siddhesh A Kamat,1 Christophe Sapin,3 Amy M Duhig,2 Leslie Citrome4 1Health Economics and Outcomes Research, Otsuka Pharmaceutical Development and Commercialization, Inc, Princeton, NJ, USA; 2Global Health Economics and Outcomes Research, Xcenda, Palm Harbor, FL, USA; 3Global Analytics, Lundbeck, Paris, France; 4Psychiatry and Behavioral Sciences, New York Medical College, Valhalla, NY, USA Objective: This study used a decision-analytic framework to assess the cost-effectiveness of brexpiprazole vs comparator branded therapies for reducing relapses and hospitalizations among adults with schizophrenia from a US payer perspective.Methods: An economic model was developed to assess patients with stable schizophrenia initiating treatment with brexpiprazole (1–4 mg), cariprazine (1–6 mg), or lurasidone (40–80 mg) over a 1-year period. After 6 months, patients remained on treatment or discontinued due to relapse, adverse events, or other reasons. Patients who discontinued due to relapse or adverse events were assumed to have switched to other therapy, and those who discontinued due to other reasons were assumed to have received no therapy. Primary outcomes were incremental cost per relapse avoided and hospitalization avoided, and the secondary outcome was cost per quality-adjusted life-year (QALY) gained. Sensitivity and scenario analyses were also conducted.Results: Brexpiprazole was associated with the highest per-patient clinical effectiveness (avoided relapses 0.637, avoided hospitalizations 0.719, QALYs 0.707) among comparators, followed by cariprazine (avoided relapses 0.590, avoided hospitalizations 0.683, QALYs 0.683) and lurasidone (avoided relapses 0.400, avoided hospitalizations 0.536, QALYs 0.623). Annual per-patient health-care costs were lowest for brexpiprazole ($20,510), followed by cariprazine ($22,282) and lurasidone ($25,510). Brexpiprazole was the least costly and most effective treatment strategy for all outcomes. Results were sensitive to relapse rates and daily cost of brexpiprazole. Limitations include data principally obtained from drug-specific randomized withdrawal studies and lack of direct-comparison trials.Conclusion: This analysis evaluated brexpiprazole treatment for the reduction of schizophrenia relapses and hospitalizations over a 1-year period compared to other recently available branded antipsychotics, and excluded generic antipsychotic treatments. Brexpiprazole treatment may lead to clinical benefits and medical cost savings, and provides a cost-effective treatment option for patients relatively to other branded second-generation antipsychotics. Keywords: schizophrenia, cost-effectiveness, relapse prevention, cost-benefit, indirect analysis, event avoided, hospitalization avoided, brexpiprazole