Differences in P-Type and Type 1 Uropathogenic Escherichia coli Urinary Anti-Adhesion Activity of Cranberry Fruit Juice Dry Extract Product and D-Mannose Dietary Supplement.

BACKGROUND: Urinary tract infection (UTI) prevention benefits of cranberry intake are clinically validated, especially for women and children. To ensure the benefits of cranberry dietary supplement products, the anti-adhesion activity (AAA) against uropathogenic bacteria is routinely used in in vitro bioassays to determine the activity in whole product formulations, isolated compounds, and ex vivo bioassays to assess urinary activity following intake. D-mannose is another dietary supplement taken for UTI prevention, based on the anti-adhesion mechanism. OBJECTIVE: Compare the relative AAA of cranberry and D-mannose dietary supplements against the most important bacterial types contributing to the pathogenesis of UTI, and consider how certain components potentially induce in vivo activity. METHODS: The current study used a crossover design to determine ex vivo AAA against both P- and Type 1-fimbriated uropathogenic Escherichia coli of either D-mannose or a cranberry fruit juice dry extract product containing 36 mg of soluble proanthocyanidins (PACs), using bioassays that measure urinary activity following consumption. AAA of extracted cranberry compound fractions and D-mannose were compared in vitro and potential induction mechanisms of urinary AAA explored. RESULTS: The cranberry dietary supplement exhibited both P-type and Type 1 in vitro and ex vivo AAA, while D-mannose only prevented Type 1 adhesion. Cranberry also demonstrated more robust and consistent ex vivo urinary AAA than D-mannose over each 1-week study period at different urine collection time points. The means by which the compounds with in vitro activity in each supplement product could potentially induce the AAA in urines was discussed relative to the data. CONCLUSIONS: Results of the current study provide consumers and healthcare professionals with additional details on the compounds and mechanisms involved in the positive, broad-spectrum AAA of cranberry against both E. coli bacterial types most important in UTIs and uncovers limitations on AAA and effectiveness of D-mannose compared to cranberry.

Sore throat, fever, and pancytopenia during winter.

Care must be taken to mitigate the effect of cognitive bias in times of frequent common presentations. The etiology of bicytopenias and pancytopenias must always be carefully investigated. Blast cells in low count B ALL may not be seen on a peripheral smear and diagnosis often requires confirmational bone marrow aspirate with flow cytometry and molecular typing.

A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells.

Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure-activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.

Prebiotic proanthocyanidins inhibit bile reflux-induced esophageal adenocarcinoma through reshaping the gut microbiome and esophageal metabolome.

The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammation-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barrett's esophagus, and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague-Dawley rats, with or without reflux induction, received water or C-PAC ad libitum (700 µg/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-κB/TP53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Streptococcus parasanguinis, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory, and immune-implicated proteins and genes, including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1b, Lbp, Lcn2, Myd88, Nfkb1, Tlr2, and Tlr4, aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe, promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation, and cellular damage.

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.

Improved in vitro Hemagglutination Assays Utilizing P-Type and Type 1 Uropathogenic Escherichia coli to Evaluate Bacterial Anti-Adhesion Activity of Cranberry Products.

Cranberries have a long history of use in the prevention of urinary tract infections. Cranberry products vary in proanthocyanidin content, a compound implicated in preventing the adhesion of uropathogenic Escherichia coli (E. coli) to uroepithelial cells. Testing is routinely done by cranberry product formulators to evaluate in vitro bacterial anti-adhesion bioactivity, shelf-life, and potential efficacy of cranberry products for consumer use to maintain urinary tract health. Hemagglutination assays evaluate the anti-adhesion bioactivity of cranberry products by determining how effectively the products prevent agglutination of specific red blood cells with E. coli expressing P-type and Type 1 fimbriae. The current study sought to improve upon an established anti-adhesion assay method by expanding the number of E. coli strains used to broaden potential in vivo efficacy implications and presenting results using photomicrographic data to improve accuracy and build databases on products that are routinely tested. Different lots of cranberry powder ingredient and two formulated products were tested independently for anti-adhesion activity using the established method and the improved method. Positive harmonization of results on the same samples using rigorous controls was achieved and provides the substantiation needed for the cranberry industry to utilize the improved, rapid in vitro testing method to standardize cranberry products for sufficient anti-adhesion bioactivity and maintain consumer confidence.

Cranberry Proanthocyanidins Mitigate Reflux-Induced Transporter Dysregulation in an Esophageal Adenocarcinoma Model.

We recently reported that cranberry proanthocyanidins (C-PACs) inhibit esophageal adenocarcinoma (EAC) by 83% through reversing reflux-induced bacterial, inflammatory and immune-implicated proteins and genes as well as reducing esophageal bile acids, which drive EAC progression. This study investigated whether C-PACs' mitigation of bile reflux-induced transporter dysregulation mechanistically contributes to EAC prevention. RNA was isolated from water-, C-PAC- and reflux-exposed rat esophagi with and without C-PAC treatment. Differential gene expression was determined by means of RNA sequencing and RT-PCR, followed by protein assessments. The literature, coupled with the publicly available Gene Expression Omnibus dataset GSE26886, was used to assess transporter expression levels in normal and EAC patient biopsies for translational relevance. Significant changes in ATP-binding cassette (ABC) transporters implicated in therapeutic resistance in humans (i.e., Abcb1, Abcb4, Abcc1, Abcc3, Abcc4, Abcc6 and Abcc10) and the transport of drugs, xenobiotics, lipids, and bile were altered in the reflux model with C-PACs' mitigating changes. Additionally, C-PACs restored reflux-induced changes in solute carrier (SLC), aquaporin, proton and cation transporters (i.e., Slc2a1, Slc7a11, Slc9a1, Slco2a1 and Atp6v0c). This research supports the suggestion that transporters merit investigation not only for their roles in metabolism and therapeutic resistance, but as targets for cancer prevention and targeting preventive agents in combination with chemotherapeutics.

Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer.

In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding ß-galactosylceramide (ßGalCer) without sulfate. C24:2 induced IFN-γ-dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid's function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.

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.

Prebiotic proanthocyanidins inhibit bile reflux-induced esophageal adenocarcinoma through reshaping the gut microbiome and esophageal metabolome.

The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammatory-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barrett's esophagus and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague Dawley rats, with or without reflux-induction received water or C-PAC ad libitum (700 µg/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis, and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-κB/P53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Clostridium perfringens, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory and immune-implicated proteins and genes including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1ß, Lbp, Lcn2, Myd88, Nfkb1, Tlr2 and Tlr4 aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation and cellular damage.

Teamwork measure relates to provider experience, burnout, and intent to stay.

OBJECTIVES: To develop a brief teamwork measure and determine how teamwork relates to provider experience, burnout, and work intentions. STUDY DESIGN: Survey of clinicians. METHODS: We analyzed data from Optum's 2019 biannual clinician survey, including a validated burnout measure and measures of provider experience and intent to stay. A 6-item measure of team effectiveness (TEAM) focused on efficiency, communication, continuous improvement, and leadership. Construct validity was assessed with content, reliability, and correlation with burnout. Generalized estimating equations with robust SEs determined relationships among TEAM score, provider experience, and intent to stay, controlling for demographics, clustering, and practice factors. RESULTS: Of 1500 physicians and advanced practice clinicians (1387 with complete data; response rate 56%), there were 58% in primary care; 57% were women, and 38% identified as Asian, Black/Hispanic, or another race/ethnicity other than White non-Hispanic. Burnout was present in 30%. The Cronbach α was excellent (0.86), and TEAM correlated with the validated burnout measure (adjusted odds ratio [OR] of lower burnout with high TEAM score, 0.28; 95% CI, 0.19-0.40; P < .0001). Clinicians with TEAM scores of at least 4 were more likely to have positive provider experiences (79% favorable vs 24% with low TEAM score; P < .001), had lower burnout rates (17% vs 44%%; P < .001), and more often intended to stay (93% vs 65%; P < .001). TEAM index score was strongly associated with provider experience (adjusted OR, 11.72; 95% CI, 8.11-16.95; P < .001) and intent to stay (adjusted OR, 7.24; 95% CI, 5.34-9.83; P < .001). CONCLUSIONS: The TEAM index is related to provider experience, burnout, and intent to stay, and it may help organizations optimize clinical work environments.

Role of DNA methylation in the relationship between glioma risk factors and glioma incidence: a two-step Mendelian randomization study.

Genetic evidence suggests glioma risk is altered by leukocyte telomere length, allergic disease (asthma, hay fever or eczema), alcohol consumption, childhood obesity, low-density lipoprotein cholesterol (LDLc) and triglyceride levels. DNA methylation (DNAm) variation influences many of these glioma-related traits and is an established feature of glioma. Yet the causal relationship between DNAm variation with both glioma incidence and glioma risk factors is unknown. We applied a two-step Mendelian randomization (MR) approach and several sensitivity analyses (including colocalization and Steiger filtering) to assess the association of DNAm with glioma risk factors and glioma incidence. We used data from a recently published catalogue of germline genetic variants robustly associated with DNAm variation in blood (32,851 participants) and data from a genome-wide association study of glioma risk (12,488 cases and 18,169 controls, sub-divided into 6191 glioblastoma cases and 6305 non-glioblastoma cases). MR evidence indicated that DNAm at 3 CpG sites (cg01561092, cg05926943, cg01584448) in one genomic region (HEATR3) had a putative association with glioma and glioblastoma risk (False discovery rate [FDR] < 0.05). Steiger filtering provided evidence against reverse causation. Colocalization presented evidence against genetic confounding and suggested that differential DNAm at the 3 CpG sites and glioma were driven by the same genetic variant. MR provided little evidence to suggest that DNAm acts as a mediator on the causal pathway between risk factors previously examined and glioma onset. To our knowledge, this is the first study to use MR to appraise the causal link of DNAm with glioma risk factors and glioma onset. Subsequent analyses are required to improve the robustness of our results and rule out horizontal pleiotropy.

A humanized mouse model for in vivo evaluation of invariant Natural Killer T cell responses.

Invariant natural killer T (iNKT) cells mediate immune responses when stimulated by glycolipid agonists presented by CD1d. In extensive studies of synthetic analogues of α-galactosyl ceramides, we identified numerous examples of significant differences in the recognition of specific glycolipids in wild type mice versus human iNKT cell clones or PBMC samples. To predict human iNKT cell responses more accurately in a mouse model, we derived a mouse line in which compound genetic modifications were used to express a human-like iNKT cell TCR along with human CD1d in place of the endogenous mouse proteins. Detailed transcriptional and phenotypic profiling demonstrated that these partially humanized mice developed an expanded population of T cells recognizing CD1d-presented glycolipid antigens, among which a subset characterized by expression of chemokine receptor CXCR6 had features characteristic of authentic iNKT cells. Responses to iNKT cell activating glycolipids in these mice generated cytokine production in vitro and in vivo that showed a pattern of fine specificity that closely resembled that of cultured human iNKT cell clones. Anti-tumor responses to variants of α-galactosyl ceramide in VαKI mice also correlated with their potency for stimulating human iNKT cells. This genetically modified mouse line provides a practical model for human presentation and recognition of iNKT cell activators in the context of a normally functioning immune system, and may furnish valuable opportunities for preclinical evaluation of iNKT cell-based therapies.

Cranberry Polyphenols in Esophageal Cancer Inhibition: New Insights.

Esophageal adenocarcinoma (EAC) is a cancer characterized by rapidly rising incidence and poor survival, resulting in the need for new prevention and treatment options. We utilized two cranberry polyphenol extracts, one proanthocyanidin enriched (C-PAC) and a combination of anthocyanins, flavonoids, and glycosides (AFG) to assess inhibitory mechanisms utilizing premalignant Barrett's esophagus (BE) and EAC derived cell lines. We employed reverse phase protein arrays (RPPA) and Western blots to examine cancer-associated pathways and specific signaling cascades modulated by C-PAC or AFG. Viability results show that C-PAC is more potent than AFG at inducing cell death in BE and EAC cell lines. Based on the RPPA results, C-PAC significantly modulated 37 and 69 proteins in JH-EsoAd1 (JHAD1) and OE19 EAC cells, respectively. AFG treatment significantly altered 49 proteins in both JHAD1 and OE19 cells. Bioinformatic analysis of RPPA results revealed many previously unidentified pathways as modulated by cranberry polyphenols including NOTCH signaling, immune response, and epithelial to mesenchymal transition. Collectively, these results provide new insight regarding mechanisms by which cranberry polyphenols exert cancer inhibitory effects targeting EAC, with implications for potential use of cranberry constituents as cancer preventive agents.

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.

Trans-ethnic Mendelian-randomization study reveals causal relationships between cardiometabolic factors and chronic kidney disease.

BACKGROUND: This study was to systematically test whether previously reported risk factors for chronic kidney disease (CKD) are causally related to CKD in European and East Asian ancestries using Mendelian randomization. METHODS: A total of 45 risk factors with genetic data in European ancestry and 17 risk factors in East Asian participants were identified as exposures from PubMed. We defined the CKD by clinical diagnosis or by estimated glomerular filtration rate of <60 ml/min/1.73 m2. Ultimately, 51 672 CKD cases and 958 102 controls of European ancestry from CKDGen, UK Biobank and HUNT, and 13 093 CKD cases and 238 118 controls of East Asian ancestry from Biobank Japan, China Kadoorie Biobank and Japan-Kidney-Biobank/ToMMo were included. RESULTS: Eight risk factors showed reliable evidence of causal effects on CKD in Europeans, including genetically predicted body mass index (BMI), hypertension, systolic blood pressure, high-density lipoprotein cholesterol, apolipoprotein A-I, lipoprotein(a), type 2 diabetes (T2D) and nephrolithiasis. In East Asians, BMI, T2D and nephrolithiasis showed evidence of causality on CKD. In two independent replication analyses, we observed that increased hypertension risk showed reliable evidence of a causal effect on increasing CKD risk in Europeans but in contrast showed a null effect in East Asians. Although liability to T2D showed consistent effects on CKD, the effects of glycaemic phenotypes on CKD were weak. Non-linear Mendelian randomization indicated a threshold relationship between genetically predicted BMI and CKD, with increased risk at BMI of >25 kg/m2. CONCLUSIONS: Eight cardiometabolic risk factors showed causal effects on CKD in Europeans and three of them showed causality in East Asians, providing insights into the design of future interventions to reduce the burden of CKD.

Differences in Urinary Bacterial Anti-Adhesion Activity after Intake of Cranberry Dietary Supplements with Soluble versus Insoluble Proanthocyanidins.

A number of clinical trials support the use of standardized cranberry supplement products for prevention of urinary tract infections; however, products that are not well-characterized for sufficient levels of bioactive components may contribute to negative clinical outcomes. Cranberry supplements for consumer use are not regulated and can be formulated different ways using cranberry juice, pomace or various combinations. This can lead to consumer confusion regarding effectiveness of individual products. The current study compared two commercial supplement products, one made from cranberry juice extract and the other from a blend of whole cranberry. The influence of formulation and proanthocyanidin (PAC) solubility on in vitro and ex vivo P-fimbriated Escherichia coli bacterial anti-adhesion activity (AAA) was determined. Both supplement products as well as whole, frozen cranberries were chromatographically separated into crude polyphenolic, sugar and acid fractions. In vitro AAA testing of all fractions confirmed that only those containing soluble PACs elicited activity. The cranberry juice extract product had higher soluble PAC content than the whole cranberry blended product, which contained mainly insoluble PACs. The influence of soluble and insoluble PAC levels in each product on the urinary (ex vivo) AAA was determined following ingestion. The juice extract product was associated with significantly higher urinary AAA than that of the whole berry blended product when consumed once daily over the 1-week intervention period.

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.

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.

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.