Exposure to microplastics and human reproductive outcomes: A systematic review.

BACKGROUND: Microplastics, produced through degradation of environmental plastic pollution, have been detected in human tissues including placenta and fetal meconium. Cell culture and animal studies have demonstrated potential reproductive toxicity of these particles; however, their association with adverse fertility or pregnancy outcomes in humans is not known. OBJECTIVES: To synthesise evidence for the presence of microplastics in human reproductive tissue and their associations with environmental exposures and reproductive outcomes. SEARCH STRATEGY: MEDLINE, Embase, Emcare, CINAHL, ClinicalTrials.gov and ICTRP were searched from inception to 03/02/2023. SELECTION CRITERIA: Studies of human participants, assessing presence of microplastics in reproductive tissues, environmental exposures to microplastics, and fertility- or pregnancy-related outcomes. DATA COLLECTION AND ANALYSIS: Two independent reviewers selected studies and extracted data on study characteristics, microplastics detected, environmental exposures and reproductive outcomes. Narrative synthesis was performed due to methodological heterogeneity. MAIN RESULTS: Of 1094 citations, seven studies were included, covering 96 participants. Microplastics composed of 16 different polymer types were detected in both placental and meconium samples. Two studies reported associations between lifestyle factors (daily water intake, use of scrub cleanser or toothpaste, bottled water and takeaway food) and placental microplastics. One study reported associations between meconium microplastics and reduced microbiota diversity. One reported placental microplastic levels correlated with reduced birthweights and 1-minute Apgar scores. CONCLUSIONS: There is a need for high-quality observational studies to assess the effects of microplastics on human reproductive health.

ABHD17 regulation of plasma membrane palmitoylation and N-Ras-dependent cancer growth.

Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.

1,6-Dioxo-2-azaspiro[3.4]oct-2-enes and Related Spirocycles: Heterocycles from [3 + 2] Nitrile Oxide Cycloadditions with 2-Methyleneoxetanes, -Thietanes, and -Azetidines.

Isoxazolines and 4-membered heterocycles are significant structural motifs in numerous synthetic intermediates and natural products. [3 + 2] Cycloadditions between enol ethers and nitrile oxides have been well studied; however, nitrile oxide cycloadditions with 4-membered heterocycles to give spiroisoxazolines are unreported. Here, we showcase the regio- and diastereoselective [3 + 2] nitrile oxide cycloadditions of 2-methyleneoxetanes, -azetidines, and -thietanes to give an array of 1,6-dioxo-2-azaspiro[3.4]oct-2-enes and related spirocycles. 2D NMR experiments suggested that most of the observed diastereoselectivities were dictated by steric interactions; however, dipolarophiles with H bonding donors reversed the stereochemical outcome. X-ray crystallography confirmed the structural assignments.

Proanthocyanidins mitigate bile acid-induced changes in GSTT2 levels in a panel of racially diverse patient-derived primary esophageal cell cultures.

Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for esophageal adenocarcinoma (EAC). Despite similar rates of GERD and other risk factors across racial groups, EAC progression disproportionately impacts Caucasians. We recently reported that elevated tissue levels of the detoxification enzyme GSTT2 in the esophagi of Blacks compared to Caucasians may contribute protection. Herein, we extend our research to investigate whether cranberry proanthocyanidins (C-PAC) mitigate bile acid-induced damage and GSTT2 levels utilizing a racially diverse panel of patient-derived primary esophageal cultures. We have shown that C-PACs mitigate reflux-induced DNA damage through GSTT2 upregulation in a rat esophageal reflux model, but whether effects are recapitulated in humans or differentially based on race remains unknown. We isolated normal primary esophageal cells from Black and Caucasian patients and assessed GSTT2 protein levels and cellular viability following exposure to a bile acid cocktail with and without C-PAC treatment. Constitutive GSTT2 levels were significantly elevated in Black (2.9-fold) compared to Caucasian patients, as were GSTT2 levels in Black patients with GERD. C-PAC treatment induced GSTT2 levels 1.6-fold in primary normal esophageal cells. GSTT2 induction by C-PAC was greatest in cells with constitutively low GSTT2 expression. Overall, C-PAC mitigated bile-induced reductions of GSTT2 and subsequent loss of cell viability regardless of basal GSTT2 expression or race. These data support that C-PAC may be a safe efficacious agent to promote epithelial fitness through GSTT2 induction and in turn protect against bile acid-induced esophageal injury.

Unusual Transformations of Strain-Heightened Oxetanes.

Oxetanes are important motifs for drug discovery and are valuable templates in organic synthesis. Much of their use as synthetic intermediates exploits their inherent strain, often resulting in chain extensions at the expense of the heterocycle. Modifications on the carbon alpha to the oxygen of oxetanes, such as the C═O of ß-lactones, extend the modes of reactivity. Nevertheless, the outcomes are still largely predictable. On the other hand, other alpha modifications, such as a ═CH2, a spiro-oxiranyl moiety, or a spiro-cyclopropyl group, increase strain and open pathways not available to simple oxetanes or ß-lactones. Methods in generating 2-methyleneoxetanes, 1,5-dioxaspiro[3.2]hexanes, and 4-oxaspiro[2.3]hexanes have been developed by us and others. To date, reactions of these systems have sometimes been predictable, but often the outcomes have been unexpected. This has provided fertile ground for thinking about what controls reactivity and what other reaction pathways might be accessible to these strain-heightened oxetanes.This Account summarizes the published literature on the most straightforward approaches to 2-methyleneoxetanes, dioxaspirohexanes, and oxaspirohexanes and on their reactivity. In contrast to simple oxetanes, reactions of 2-methyleneoxetanes with nucleophiles at C4 release an enolate rather than an alkoxide. Also, 2-methyleneoxetanes can be converted to homopropargyl alcohols or undergo a silicon accelerated isomerization/electrocyclic ring opening, processes accessible only because of the exocyclic double bond. In addition, oxetane oxocarbenium ions, derived from protonation of the enol ether, can react with nucleophiles to provide 2,2-disubstituted oxetanes. Oxaspirohexanes are readily prepared by Simmons-Smith cyclopropanation of 2-methyleneoxetanes. These unusual systems undergo a variety of substituent dependent rearrangements in the presence of the Lewis acid BF3·Et2O. In addition, upon treatment with Zeise's dimer, oxaspirohexanes are transformed to synthetically useful 3-methylenetetrahydrofurans. Dioxaspirohexanes are easily accessed by dimethyldioxirane oxidation of 2-methyleneoxetanes. Predictably, dioxaspirohexanes react with many nucleophiles to give α-functionalized-ß'-hydroxy ketones. Unexpectedly, 2,2-disubstituted oxetanes can also be selectively produced. This latter pathway has led to further unusual transformations, illuminating computational studies, and novel routes to biologically relevant molecules.

The Nevada peripheral artery disease screening effort in a Medicare Advantage population and subsequent mortality and major adverse cardiovascular event risk.

BACKGROUND: Contemporary estimates of undetected asymptomatic lower extremity peripheral artery disease (PAD) in the community and its association with adverse outcomes in the population are lacking. We investigated the long-term association between previously undetected PAD and subsequent all-cause mortality and major adverse cardiovascular events (MACE) for Medicare Advantage beneficiaries aged ≥65 years in a large metropolitan area characterized by concentrations of atherosclerotic risk factors and a more vulnerable socioeconomic risk profile. METHODS: Data were derived from the patients' electronic medical records and linked with claims outcomes data for 13,971 Medicare Advantage beneficiaries aged ≥65 years who had undergone PAD screening in 2016 as a part of their routine annual health assessment in the greater Las Vegas, Nevada, metropolitan area. PAD screening was performed with their primary care provider using volume plethysmography system methods. The association between PAD screening status and 1- and 3-year all-cause mortality and MACE rates was documented. RESULTS: The cohort had a mean age of 75.3 ± 6.6 years, and 57.7% were women. Of the 13,768 patients, 4351 (31.6%) had had a positive PAD screening result. Almost 60% had had a lower socioeconomic income level, with 15.1% living under the poverty level. The risk estimates associated with a positive vs negative PAD screening result for both all-cause mortality and MACE were as follows: unadjusted hazard ratio (HR) for mortality, 2.17 (95% confidence interval [CI], 1.79-2.63) and unadjusted HR for MACE, 2.00 (95% CI, 1.15-3.49) at 1 year and unadjusted HR for mortality, 2.04 (95% CI, 1.84-2.26) and unadjusted HR for MACE, 1.67 (95% CI, 1.37-2.02) at 3 years. After multivariable adjustment, all associations persisted (P < .001), with HRs ranging from 1.41 to 1.69, except for that for 1-year MACE (similar risk estimate but P = .09). CONCLUSIONS: A positive screening result for previously undetected lower extremity PAD was independently associated with short- and long-term increased risks of mortality and MACE for individuals aged ≥65 years living in a large, metropolitan area.

α-Methylene-ß-Lactone Scaffold for Developing Chemical Probes at the Two Ends of the Selectivity Spectrum.

The utilities of an α-methylene-ß-lactone (MeLac) moiety as a warhead composed of multiple electrophilic sites are reported. We demonstrate that a MeLac-alkyne not only reacts with diverse proteins as a broadly reactive measurement probe, but also recruits reduced endogenous glutathione (GSH) to assemble a selective chemical probe of GSH-ß-lactone (GSH-Lac)-alkyne in live cells. Tandem mass spectrometry reveals that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael or acyl addition. A peptide-centric proteomics platform demonstrates that the proteomic selectivity profiles of orlistat and parthenolide, which have distinct reactivities, are measurable by MeLac-alkyne as a high-coverage probe. The GSH-Lac-alkyne selectively probes the glutathione S-transferase P responsible for multidrug resistance. The assembly of the GSH-Lac probe exemplifies a modular and scalable route to develop selective probes with different recognizing moieties.

Chemical proteomic analysis of palmostatin beta-lactone analogs that affect N-Ras palmitoylation.

S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.

Prognostic value of test(s) for O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation for predicting overall survival in people with glioblastoma treated with temozolomide.

BACKGROUND: Glioblastoma is an aggressive form of brain cancer. Approximately five in 100 people with glioblastoma survive for five years past diagnosis. Glioblastomas that have a particular modification to their DNA (called methylation) in a particular region (the O6-methylguanine-DNA methyltransferase (MGMT) promoter) respond better to treatment with chemotherapy using a drug called temozolomide. OBJECTIVES: To determine which method for assessing MGMT methylation status best predicts overall survival in people diagnosed with glioblastoma who are treated with temozolomide. SEARCH METHODS: We searched MEDLINE, Embase, BIOSIS, Web of Science Conference Proceedings Citation Index to December 2018, and examined reference lists. For economic evaluation studies, we additionally searched NHS Economic Evaluation Database (EED) up to December 2014. SELECTION CRITERIA: Eligible studies were longitudinal (cohort) studies of adults with diagnosed glioblastoma treated with temozolomide with/without radiotherapy/surgery. Studies had to have related MGMT status in tumour tissue (assessed by one or more method) with overall survival and presented results as hazard ratios or with sufficient information (e.g. Kaplan-Meier curves) for us to estimate hazard ratios. We focused mainly on studies comparing two or more methods, and listed brief details of articles that examined a single method of measuring MGMT promoter methylation. We also sought economic evaluations conducted alongside trials, modelling studies and cost analysis. DATA COLLECTION AND ANALYSIS: Two review authors independently undertook all steps of the identification and data extraction process for multiple-method studies. We assessed risk of bias and applicability using our own modified and extended version of the QUality In Prognosis Studies (QUIPS) tool. We compared different techniques, exact promoter regions (5'-cytosine-phosphate-guanine-3' (CpG) sites) and thresholds for interpretation within studies by examining hazard ratios. We performed meta-analyses for comparisons of the three most commonly examined methods (immunohistochemistry (IHC), methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ)), with ratios of hazard ratios (RHR), using an imputed value of the correlation between results based on the same individuals. MAIN RESULTS: We included 32 independent cohorts involving 3474 people that compared two or more methods. We found evidence that MSP (CpG sites 76 to 80 and 84 to 87) is more prognostic than IHC for MGMT protein at varying thresholds (RHR 1.31, 95% confidence interval (CI) 1.01 to 1.71). We also found evidence that PSQ is more prognostic than IHC for MGMT protein at various thresholds (RHR 1.36, 95% CI 1.01 to 1.84). The data suggest that PSQ (mainly at CpG sites 74 to 78, using various thresholds) is slightly more prognostic than MSP at sites 76 to 80 and 84 to 87 (RHR 1.14, 95% CI 0.87 to 1.48). Many variants of PSQ have been compared, although we did not see any strong and consistent messages from the results. Targeting multiple CpG sites is likely to be more prognostic than targeting just one. In addition, we identified and summarised 190 articles describing a single method for measuring MGMT promoter methylation status. AUTHORS' CONCLUSIONS: PSQ and MSP appear more prognostic for overall survival than IHC. Strong evidence is not available to draw conclusions with confidence about the best CpG sites or thresholds for quantitative methods. MSP has been studied mainly for CpG sites 76 to 80 and 84 to 87 and PSQ at CpG sites ranging from 72 to 95. A threshold of 9% for CpG sites 74 to 78 performed better than higher thresholds of 28% or 29% in two of three good-quality studies making such comparisons.

"Running myself ragged": stressors faced by peer workers in overdose response settings.

BACKGROUND: Peer workers or "peers" (workers with past or present drug use experience) are at the forefront of overdose response initiatives, and their role is essential in creating safe spaces for people who use drugs (PWUD). Working in overdose response settings has benefits for peer workers but is also stressful, with lasting emotional and mental health effects. Yet, little is known about the stressors peer workers face and what interventions can be implemented to support them in their roles. METHODS: This project used a community-based sequential mixed-methods research design. Eight peer researcher-led focus groups (n = 31) were conducted between November 2018 and March 2019 to assess needs of peer workers. The transcripts were thematically coded and analysed using interpretative description. These results informed a survey, which was conducted (n = 50) in September 2019 to acquire quantitative data on peer workers' perception of health, quality of life, working conditions and stressors. Frequency distributions were used to describe characteristics of participants. X2 distribution values with Yates correction were conducted to check for association between variables. RESULTS: Five themes emerged from the focus groups that point to stressors felt by peer workers: (1) financial insecurity; (2) lack of respect and recognition at work; (3) housing challenges; (4) inability to access and/or refer individuals to resources; and (5) constant exposure to death and trauma. Consistent with this, the factors that survey participants picked as one of their "top three stressors" included financial situation, work situation, and housing challenges. CONCLUSION: Peer workers are faced with a diversity of stressors in their lives which often reflect societal stigmatization of drug use. Recognition of these systemic stressors is critical in designing interventions to ease the emotional, physical and financial burden faced by peer workers.

A molecular basis for the exquisite CD1d-restricted antigen specificity and functional responses of natural killer T cells.

Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.

Highbush blueberry proanthocyanidins alleviate Porphyromonas gingivalis-induced deleterious effects on oral mucosal cells.

Strong evidence points to Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, as a keystone species in the development of the chronic form of periodontitis. The aim of the present study was to investigate the ability of highbush blueberry proanthocyanidins (PACs) to alleviate the P. gingivalis-induced deleterious effects on oral mucosal cells. We first showed that highbush blueberry PACs protect the integrity of the gingival keratinocyte barrier against P. gingivalis-mediated damage, as determined by measuring the transepithelial electrical resistance and paracellular flux of FITC-conjugated dextran. Moreover, the PACs prevented the translocation of P. gingivalis across the gingival keratinocyte barrier model. The proteinase activity of P. gingivalis was inhibited by the PACs suggesting that they may exert beneficial effects by reducing proteolytic degradation of the epithelial tight junctions. Regulation of gingival fibroblast inflammatory reactions may be one of the ways to prevent and control periodontal disease progression and severity. We showed that PACs significantly reduce IL-6 and IL-8 secretion by P. gingivalis-stimulated gingival fibroblasts. The present study showed the capacity of highbush blueberry PACs to protect the integrity of an in vitro model of gingival keratinocyte barrier against P. gingivalis, and to attenuate the secretion of pro-inflammatory cytokines by gingival fibroblasts infected with P. gingivalis. These results suggest beneficial effects of blueberry PACs thus supporting the need for future clinical trials on the potential of these bioactive molecules for periodontal disease prevention and/or treatment.

Antiviral effects of blueberry proanthocyanidins against Aichi virus.

Blueberry polyphenols are known for their high antioxidant and antimicrobial potential. Aichi virus (AiV) is an emerging human enteric virus that causes gastroenteritis outbreaks worldwide. This study aimed to (1) determine the time- and dose-dependent effects of blueberry proanthocyanidins (B-PAC) against AiV over 24 h at 37 °C; (2) gain insights on their mode of action using pre- and post-treatment of host cells and Transmission Electron Microscopy; and (3) determine their anti-AiV effects in model foods and under simulated gastric conditions. AiV at ∼5 log PFU/ml was incubated with equal volumes of commercial blueberry juice (BJ, pH 2.8), neutralized BJ (pH 7.0), B-PAC (2, 4, and 10 mg/ml) prepared either in 10% ethanol, apple juice (AJ), 2% milk, simulated gastric fluid (SGF, pH 1.5) or simulated intestinal fluid (SIF, pH 7.5), and controls (malic acid (pH 3.0), phosphate buffered saline (pH 7.2), apple juice (pH 3.6) and 2% milk) over 24 h at 37 °C, followed by standard plaque assays. Each experiment was replicated thrice and data were statistically analyzed. Differences in AiV titers with 1 mg/ml B-PAC were 2.13 ±â€¯0.06 log PFU/ml lower after 24 h and ≥3 log PFU/ml (undetectable levels) lower with 2 and 5 mg/ml B-PAC compared to AiV titers in PBS after 24 h and 3 h, respectively. BJ at 37 °C resulted in titer differences (lower titers compared to PBS) of 0.17 ±â€¯0.06, 1.27 ±â€¯0.01, and 1.73 ±â€¯0.23 log PFU/ml after 1, 3, and 6 h and ≥3 log PFU/ml after 24 h. Pre- and post-treatment of host cells with 0.5 mg/ml B-PAC caused titer decreases of 0.62 ±â€¯0.33 and 0.30 ±â€¯0.06 log PFU/ml, respectively suggesting a moderate effect on viral-host cell binding. B-PAC at 2 mg/ml in AJ caused titer differences of ≥3 log PFU/ml after 0.5 h, while differences of 0.84 ±â€¯0.03 log PFU/ml with 5 mg/ml B-PAC in milk, and ≥3 log PFU/ml with B-PAC at 5 mg/ml in SIF after 30 min were obtained. This study shows the ability of BJ and B-PAC to decrease AiV titers to potentially prevent AiV-related illness and outbreaks.

Contact sensitizers trigger human CD1-autoreactive T-cell responses.

Allergic contact dermatitis is a primarily T-cell-mediated inflammatory skin disease induced by exposure to small molecular-weight haptens, which covalently bind to proteins. The abundance of cutaneous T cells that recognize CD1a antigen-presenting molecules raises the possibility that MHC-independent antigen presentation may be relevant in some hapten-driven immune responses. Here we examine the ability of contact sensitizers to influence CD1-restricted immunity. Exposure of human antigen-presenting cells such as monocyte-derived dendritic cells and THP-1 cells to the prototypical contact sensitizer dinitrochlorobenzene potentiated the response of CD1a- and CD1d-autoreactive T cells, which released a vast array of cytokines in a CD1- and TCR-dependent manner. The potentiating effects of dinitrochlorobenzene depended upon newly synthesized CD1 molecules and the presence of endogenous stimulatory lipids. Further examination of a broad panel of contact sensitizers revealed 1,4-benzoquinone, resorcinol, isoeugenol, and cinnamaldehyde to activate the same type of CD1-restricted responses. These findings provide a basis for the antigen-specific activation of skin-associated CD1-restricted T cells by small molecules and may have implications for contact sensitizer-induced inflammatory skin diseases.

T cell receptor CDR2 beta and CDR3 beta loops collaborate functionally to shape the iNKT cell repertoire.

Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.

Kinetics and cellular site of glycolipid loading control the outcome of natural killer T cell activation.

CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

Scaling Proteome-Wide Reactions of Activity-Based Probes.

Unified analysis of complex reactions of an activity-based probe with proteins in a proteome remains an unsolved challenge. We propose a power expression, rate = kobs[Probe]α, for scaling the progress of proteome-wide reactions and use the scaling factor (0 ≤ α ≤ 1) as an apparent, partial order with respect to the probe to measure the "enzyme-likeness" for a protein in reaction acceleration. Thus, α reports the intrinsic reactivity of the protein with the probe. When α = 0, the involved protein expedites the reaction to the maximal degree; when α = 1, the protein reacts with the probe via an unaccelerated, bimolecular reaction. The selectivity (ß) of the probe reacting with two proteins is calculated as a ratio of conversion factors (kobs values) for corresponding power equations. A combination of α and ß provides a tiered system for quantitatively assessing the probe efficacy; an ideal probe exhibits high reactivity with its protein targets (low in α) and is highly selective (high in ß) in forming the probe-protein adducts. The scaling analysis was demonstrated using proteome-wide reactions of HT-29 cell lysates with a model probe of threonine ß-lactone.

Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay.

Lysophosphatidylserines (lyso-PSs) are a class of signaling lipids that regulate immunological and neurological processes. The metabolism of lyso-PSs remains poorly understood in vivo. Recently, we determined that ABHD12 is a major brain lyso-PS lipase, implicating lyso-PSs in the neurological disease polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC), which is caused by null mutations in the ABHD12 gene. Here, we couple activity-based profiling with pharmacological and genetic methods to annotate the poorly characterized enzyme ABHD16A as a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. We describe a small-molecule inhibitor of ABHD16A that depletes lyso-PSs from cells, including lymphoblasts derived from subjects with PHARC. In mouse macrophages, disruption of ABHD12 and ABHD16A respectively increases and decreases both lyso-PSs and lipopolysaccharide-induced cytokine production. Finally, Abhd16a(-/-) mice have decreased brain lyso-PSs, which runs counter to the elevation in lyso-PS in Abhd12(-/-) mice. Our findings illuminate an ABHD16A-ABHD12 axis that dynamically regulates lyso-PS metabolism in vivo, designating these enzymes as potential targets for treating neuroimmunological disorders.

1,4-Dicarbofunctionalization of 4-Fluoroaryl Grignard and Lithium Reagents with Disubstituted Malononitriles.

An efficient one-pot 1,4-dicarbofunctionalization of 4-fluoroaryl Grignard or lithium reagents with 2,2-disubstituted malononitriles is described. The reaction proceeds by sequential transnitrilation and SNAr reactions. Commercial Grignard solutions, Grignard reagents prepared in situ by halogen/magnesium exchange with i-PrMgCl, or aryllithium reagents prepared in situ by bromine/lithium exchange with n-BuLi are compatible with the reaction conditions. Moreover, 2,2-disubstituted malononitriles of diverse structures are accommodated. The reaction provides a unique approach to 1,4-dicarbofunctionalization of activated arenes in a tandem, one-pot transformation.

Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.

Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness.