Natural Clearance of Chlamydia trachomatis Infection Is Associated With Distinct Differences in Cervicovaginal Metabolites.

BACKGROUND: Natural clearance of Chlamydia trachomatis in women occurs in the interval between screening and treatment. In vitro, interferon-γ (IFN-γ)-mediated tryptophan depletion results in C. trachomatis clearance, but whether this mechanism occurs in vivo remains unclear. We previously found that women who naturally cleared C. trachomatis had lower cervicovaginal levels of tryptophan and IFN-γ compared to women with persisting infection, suggesting IFN-γ-independent pathways may promote C. trachomatis clearance. METHODS: Cervicovaginal lavages from 34 women who did (n = 17) or did not (n = 17) naturally clear C. trachomatis were subjected to untargeted high-performance liquid chromatography mass-spectrometry to identify metabolites and metabolic pathways associated with natural clearance. RESULTS: In total, 375 positively charged metabolites and 149 negatively charged metabolites were annotated. Compared to women with persisting infection, C. trachomatis natural clearance was associated with increased levels of oligosaccharides trehalose, sucrose, melezitose, and maltotriose, and lower levels of indoline and various amino acids. Metabolites were associated with valine, leucine, and isoleucine biosynthesis pathways. CONCLUSIONS: The cervicovaginal metabolome in women who did or did not naturally clear C. trachomatis is distinct. In women who cleared C. trachomatis, depletion of various amino acids, especially valine, leucine, and isoleucine, suggests that amino acids other than tryptophan impact C. trachomatis survival in vivo.

Evaluation of the effects of latanoprost and benzalkonium chloride on the cell viability and nonpolar lipid profile produced by human meibomian gland epithelial cells in culture.

Purpose: The purpose of this study was to explore the effects of a PGF2α analog, latanoprost, and its preservative, benzalkonium chloride (BAK), on the cell viability and lipidomic expression of immortalized human meibomian gland epithelial cells (HMGECs). Methods: Differentiated HMGECs were exposed to latanoprost (0.05 to 50 µg/ml), BAK (0.2 to 200 µg/ml), or combined latanoprost-BAK (0.05-0.2 to 50-200 µg/ml). EP- and FP-type receptors, the cognate receptors of PGE2 and PGF2α, were inhibited, thereby sparing and isolating the function of each receptor to one condition. Cell viability was assessed by ATP quantitation, and lipid extracts were analyzed by ESI-MSMSALL with a Triple TOF 5600 Mass Spectrometer (SCIEX, Framingham, MA) using SCIEX LipidView 1.3. Results: Latanoprost and BAK were found to be lethal to HMGECs at the highest concentrations (p < 0.001 for both). The cytotoxicity of latanoprost was mediated through FP- and EP-independent mechanisms. Both latanoprost and BAK significantly modulated the lipidomic expression of several cholesteryl esters (8% and 30%, respectively) and triacylglycerols (10% and 12%, respectively). The combined latanoprost-BAK agent appeared to be no more toxic and to only negligibly alter the lipid profile relative to its individual components. Conclusions: The use of latanoprost and BAK in glaucoma may alter the viability of the meibomian glands and their lipid expression in vivo. Sublethal concentrations of BAK appear to modulate meibum lipid expression, particularly in relation to sterol biosynthesis. Non-preserved latanoprost had less cytotoxicity at lower doses and fewer lipidomic effects compared to BAK, further strengthening the argument in favor of BAK-free pharmaceutical preparations.

αA and αB peptides from human cataractous lenses show antichaperone activity and enhance aggregation of lens proteins.

Purpose: To identify and characterize properties of αA- and αB-crystallins' low molecular weight peptides (molecular weight [Mr] < 5 kDa) that were present in a 62-year-old human nuclear cataract, but not in normal 62-year-old human lenses. Methods: Low molecular weight peptides (< 5 kDa) were isolated with a trichloroacetic acid (TCA) solubilization method from water-soluble (WS) and water-insoluble (WI) proteins of nuclear cataractous lenses of a 62-year-old donor and normal human lenses from an age-matched donor. Five commercially synthesized peptides (found only in cataractous lenses and not in normal lenses) were used to determine their chaperone and antichaperone activity and aggregation properties. Results: Mass spectrometric analysis showed 28 peptides of αA-crystallin and 38 peptides of αB-crystallin were present in the cataractous lenses but not in the normal lenses. Two αA peptides (named αAP1 and αAP2; both derived from the αA N-terminal domain (NTD) region) and three αB peptides (named αBP3, αBP4, and αBP5, derived from the αB NTD-, core domain (CD), and C-terminal extension (CTE) regions, respectively) were commercially synthesized. αAP1 inhibited the chaperone activity of αA- and αB-crystallins, but the other four peptides (αAP2, αBP3, αBP4, and αBP5) exhibited mixed effects on chaperone activity. Upon incubation with human WS proteins and peptides in vitro, the αBP4 peptide showed higher aggregation properties relative to the αAP1 peptide. During in vivo experiments, the cell-penetrating polyarginine-labeled αAP1 and αBP4 peptides showed 57% and 85% aggregates, respectively, around the nuclei of cultured human lens epithelial cells compared to only 35% by a scrambled peptide. Conclusions: The antichaperone activity of the αAP1 peptide and the aggregation property of the αBP4 peptide with lens proteins could play a potential role during the development of lens opacity.

Gut Microbiome Composition and Serum Metabolome Profile Among Individuals With Spinal Cord Injury and Normal Glucose Tolerance or Prediabetes/Type 2 Diabetes.

OBJECTIVE: To compare the gut microbiome composition and serum metabolome profile among individuals with spinal cord injury (SCI) and normal glucose tolerance (NGT) or prediabetes/type 2 diabetes (preDM/T2D). DESIGN: Cross-sectional design. SETTING: Research university. PARTICIPANTS: A total of 25 adults (N=25) with SCI were included in the analysis and categorized as NGT (n=16) or preDM/T2D (n=9) based on their glucose concentration at minute 120 during a 75-g oral glucose tolerance test. The American Diabetes Association diagnosis guideline was used for grouping participants. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: A stool sample was collected and used to assess the gut microbiome composition (alpha and beta diversity, microbial abundance) via the 16s ribosomal RNA sequencing technique. A fasting serum sample was used for liquid chromatography-mass spectrometry-based untargeted metabolomics analysis, the results from which reflect the relative quantity of metabolites detected and identified. Gut microbiome and metabolomics data were analyzed by the Quantitative Insights into Microbial Ecology 2 and Metaboanalyst platforms, respectively. RESULTS: Gut microbiome alpha diversity (Pielou's evenness index, Shannon's index) and beta diversity (weighted UniFrac distances) differed between groups. Compared with participants with NGT, participants with preDM/T2D had less evenness in microbial communities. In particular, those with preDM/T2D had a lower abundance of the Clostridiales order and higher abundance of the Akkermansia genus, as well as higher serum levels of gut-derived metabolites, including indoxyl sulfate and phenylacetylglutamine (P < .05 for all). CONCLUSIONS: Our results provide evidence for altered gut microbiome composition and dysregulation of gut-derived metabolites in participants with SCI and preDM/T2D. Both indoxyl sulfate and phenylacetylglutamine have been implicated in the development of cardiovascular diseases in the able-bodied population. These findings may inform future investigations in the field of SCI and cardiometabolic health.

Triacylglycerol lipidome from human meibomian gland epithelial cells: Description, response to culture conditions, and perspective on function.

Preliminary work has shown that select triacylglycerols (TAGs) are upregulated in a preclinical model of MGD, suggesting that TAGs may be an important outcome variable in research involving human meibomian gland epithelial cells (HMGECs). The purpose of this study was to explore the HMGEC TAG lipidome in culture conditions known to influence differentiation. HMGECs were differentiated in DMEM/F12 with 10 ng/ml EGF, FBS (2% or 10%), and rosiglitazone (0, 20, or 50 µM) for two or five days. Following culture, lipids were extracted, processed, and directly infused into a Triple TOF 5600 mass spectrometer (SCIEX, Framingham, MA) with electrospray ionization. MS and MS/MSALL spectra were acquired in the positive ion mode and performed with the SWATH technology. Only the TAGs that were present in all 48 samples were included in the analysis. Multiple regression techniques were utilized to assess the effects of each factor (FBS, rosiglitazone, and culture duration) on each expressed TAG. The HMGEC TAG lipidome consisted of 115 TAGs with 42-62 carbons and zero to 10 double bonds. Fatty acyl chains had 14 to 26 carbons and zero to five double bonds. C18:1 (oleic acid, 25/115, 21.7%) and C16:0 (palmitic acid, 16/115, 13.9%) were the most common fatty acids. FBS, rosiglitazone, and culture duration were significant predictors for 93 TAGs (80.9%) with R2 values ranging from 0.20 to 0.77 (p < 0.05). FBS and rosiglitazone achieved significance (p < 0.05) for 80 (69.6%) and 67 TAGs (58.3%), respectively. Rosiglitazone demonstrated a selective upregulation of TAGs containing 16 or 18 carbons. Culture duration reached significance (p < 0.05) for only 36 TAGs (31.3%). When comparing the 10 most abundant C18:1-containing TAGs in meibum, FBS was a negative predictor for five TAGs (mean standardized coefficient [SC] = -0.58, p < 0.001), rosiglitazone was a positive predictor for six TAGs (mean SC = 0.41, p ≤ 0.03), and culture duration weakly influenced one TAG (SC = 0.27, p = 0.008). FBS and rosiglitazone, unlike culture duration, are powerful modulators of the TAG profile. Rosiglitazone induces changes that could be consistent with fatty acid synthesis, suggesting that quantifying the TAG lipidome could be an indirect measure of lipogenesis. Though both have been described as differentiating agents, FBS and rosiglitazone induce opposing effects on meibum-relevant TAGs. Culturing with rosiglitazone is associated with a TAG profile that is more consistent with the expected outcome of lipogenesis and with the profile observed in normal human meibum.

R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells.

BACKGROUND: Loss of the Ras GTPase-activating protein neurofibromin promotes nervous system tumor pathogenesis in patients with neurofibromatosis type 1 (NF1). Neurofibromin loss potentially hyperactivates classic Ras (H-Ras, N-Ras, K-Ras), M-Ras, and R-Ras (R-Ras, R-Ras2/TC21) subfamily proteins. We have shown that classic Ras proteins promote proliferation and survival, but not migration, in malignant peripheral nerve sheath tumor (MPNST) cells. However, it is unclear whether R-Ras, R-Ras2 and M-Ras are expressed and hyperactivated in MPNSTs and, if so, whether they contribute to MPNST pathogenesis. We assessed the expression and activation of these proteins in MPNST cells and inhibited them to determine the effect this had on proliferation, migration, invasion, survival and the phosphoproteome. METHODS: NF1-associated (ST88-14, 90-8, NMS2, NMS-PC, S462, T265-2c) and sporadic (STS-26T, YST-1) MPNST lines were used. Cells were transfected with doxycycline-inducible vectors expressing either a pan-inhibitor of the R-Ras subfamily [dominant negative (DN) R-Ras] or enhanced green fluorescent protein (eGFP). Methodologies used included immunoblotting, immunocytochemistry, PCR, Transwell migration, 3H-thymidine incorporation, calcein cleavage assays and shRNA knockdowns. Proteins in cells with or without DN R-Ras expression were differentially labeled with SILAC and mass spectrometry was used to identify phosphoproteins and determine their relative quantities in the presence and absence of DN R-Ras. Validation of R-Ras and R-Ras2 action and R-Ras regulated networks was performed using genetic and/or pharmacologic approaches. RESULTS: R-Ras2 was uniformly expressed in MPNST cells, with R-Ras present in a major subset. Both proteins were activated in neurofibromin-null MPNST cells. Consistent with classical Ras inhibition, DN R-Ras and R-Ras2 knockdown inhibited proliferation. However, DN R-Ras inhibition impaired migration and invasion but not survival. Mass spectrometry-based phosphoproteomics identified thirteen protein networks distinctly regulated by DN R-Ras, including multiple networks regulating cellular movement and morphology. ROCK1 was a prominent mediator in these networks. DN R-Ras expression and RRAS and RRAS2 knockdown inhibited migration and ROCK1 phosphorylation; ROCK1 inhibition similarly impaired migration and invasion, altered cellular morphology and triggered the accumulation of large intracellular vesicles. CONCLUSIONS: R-Ras proteins function distinctly from classic Ras proteins by regulating distinct signaling pathways that promote MPNST tumorigenesis by mediating migration and invasion. Mutations of the NF1 gene potentially results in the activation of multiple Ras proteins, which are key regulators of many biologic effects. The protein encoded by the NF1 gene, neurofibromin, acts as an inhibitor of both classic Ras and R-Ras proteins; loss of neurofibromin could cause these Ras proteins to become persistently active, leading to the development of cancer. We have previously shown that three related Ras proteins (the classic Ras proteins) are highly activated in malignant peripheral nerve sheath tumor (MPNST) cells with neurofibromin loss and that they drive cancer cell proliferation and survival by activating multiple cellular signaling pathways. Here, we examined the expression, activation and action of R-Ras proteins in MPNST cells that have lost neurofibromin. Both R-Ras and R-Ras2 are expressed in MPNST cells and activated. Inhibition of R-Ras action inhibited proliferation, migration and invasion but not survival. We examined the activation of cytoplasmic signaling pathways in the presence and absence of R-Ras signaling and found that R-Ras proteins regulated 13 signaling pathways distinct from those regulated by classic Ras proteins. Closer study of an R-Ras regulated pathway containing the signaling protein ROCK1 showed that inhibition of either R-Ras, R-Ras2 or ROCK1 similarly impaired cellular migration and invasion and altered cellular morphology. Inhibition of R-Ras/R-Ras2 and ROCK1 signaling also triggered the accumulation of abnormal intracellular vesicles, indicating that these signaling molecules regulate the movement of proteins and other molecules in the cellular interior. Video Abstract.

Relative stabilities of wild-type and mutant glial fibrillary acidic protein in patients with Alexander disease.

Alexander disease (AxD) is an often fatal astrogliopathy caused by dominant gain-of-function missense mutations in the glial fibrillary acidic protein (GFAP) gene. The mechanism by which the mutations produce the AxD phenotype is not known. However, the observation that features of AxD are displayed by mice that express elevated levels of GFAP from a human WT GFAP transgene has contributed to the notion that the mutations produce AxD by increasing accumulation of total GFAP above some toxic threshold rather than the mutant GFAP being inherently toxic. A possible mechanism for accumulation of GFAP in AxD patients is that the mutated GFAP variants are more stable than the WT, an attribution abetted by observations that GFAP complexes containing GFAP variants are more resistant to solvent extraction. Here we tested this hypothesis by determining the relative levels of WT and mutant GFAP in three individuals with AxD, each of whom carried a common but different GFAP mutation (R79C, R239H, or R416W). Mass spectrometry analysis identified a peptide specific to the mutant or WT GFAP in each patient, and we quantified this peptide by comparing its signal to that of an added [15N]GFAP standard. In all three individuals, the level of mutant GFAP was less than that of the WT. This finding suggests that AxD onset is due to an intrinsic toxicity of the mutant GFAP instead of it acting indirectly by being more stable than WT GFAP and thereby increasing the total GFAP level.

Differences in airway microbiome and metabolome of single lung transplant recipients.

BACKGROUND: Recent studies suggest that alterations in lung microbiome are associated with occurrence of chronic lung diseases and transplant rejection. To investigate the host-microbiome interactions, we characterized the airway microbiome and metabolome of the allograft (transplanted lung) and native lung of single lung transplant recipients. METHODS: BAL was collected from the allograft and native lungs of SLTs and healthy controls. 16S rRNA microbiome analysis was performed on BAL bacterial pellets and supernatant used for metabolome, cytokines and acetylated proline-glycine-proline (Ac-PGP) measurement by liquid chromatography-high-resolution mass spectrometry. RESULTS: In our cohort, the allograft airway microbiome was distinct with a significantly higher bacterial burden and relative abundance of genera Acinetobacter & Pseudomonas. Likewise, the expression of the pro-inflammatory cytokine VEGF and the neutrophil chemoattractant matrikine Ac-PGP in the allograft was significantly higher. Airway metabolome distinguished the native lung from the allografts and an increased concentration of sphingosine-like metabolites that negatively correlated with abundance of bacteria from phyla Proteobacteria. CONCLUSIONS: Allograft lungs have a distinct microbiome signature, a higher bacterial biomass and an increased Ac-PGP compared to the native lungs in SLTs compared to the native lungs in SLTs. Airway metabolome distinguishes the allografts from native lungs and is associated with distinct microbial communities, suggesting a functional relationship between the local microbiome and metabolome.

Increased Association of Deamidated αA-N101D with Lens membrane of transgenic αAN101D vs. wild type αA mice: potential effects on intracellular ionic imbalance and membrane disorganization.

We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. METHODS: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice. RESULTS: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. CONCLUSIONS: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Vaporized E-Cigarette Liquids Induce Ion Transport Dysfunction in Airway Epithelia.

Cigarette smoking is associated with chronic obstructive pulmonary disease and chronic bronchitis. Acquired ion transport abnormalities, including cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, caused by cigarette smoking have been proposed as potential mechanisms for mucus obstruction in chronic bronchitis. Although e-cigarette use is popular and perceived to be safe, whether it harms the airways via mechanisms altering ion transport remains unclear. In the present study, we sought to determine if e-cigarette vapor, like cigarette smoke, has the potential to induce acquired CFTR dysfunction, and to what degree. Electrophysiological methods demonstrated reduced chloride transport caused by vaporized e-cigarette liquid or vegetable glycerin at various exposures (30 min, 57.2% and 14.4% respectively, vs. control; P < 0.0001), but not by unvaporized liquid (60 min, 17.6% vs. untreated), indicating that thermal degradation of these products is required to induce the observed defects. We also observed reduced ATP-dependent responses (-10.8 ± 3.0 vs. -18.8 ± 5.1 µA/cm2 control) and epithelial sodium channel activity (95.8% reduction) in primary human bronchial epithelial cells after 5 minutes, suggesting that exposures dramatically inhibit epithelial ion transport beyond CFTR, even without diminished transepithelial resistance or cytotoxicity. Vaporizing e-cigarette liquid produced reactive aldehydes, including acrolein (shown to induce acquired CFTR dysfunction), as quantified by mass spectrometry, demonstrating that respiratory toxicants in cigarette smoke can also be found in e-cigarette vapor (30 min air, 224.5 ± 15.99; unvaporized liquid, 284.8 ± 35.03; vapor, 54,468 ± 3,908 ng/ml; P < 0.0001). E-cigarettes can induce ion channel dysfunction in airway epithelial cells, partly through acrolein production. These findings indicate a heretofore unknown toxicity of e-cigarette use known to be associated with chronic bronchitis onset and progression, as well as with chronic obstructive pulmonary disease severity.

Truncating PKHD1 and PKD2 mutations alter energy metabolism.

Deficiency in polycystin 1 triggers specific changes in energy metabolism. To determine whether defects in other human cystoproteins have similar effects, we studied extracellular acidification and glucose metabolism in human embryonic kidney (HEK-293) cell lines with polycystic kidney and hepatic disease 1 ( PKHD1) and polycystic kidney disease (PKD) 2 ( PKD2) truncating defects along multiple sites of truncating mutations found in patients with autosomal recessive and dominant PKDs. While neither the PKHD1 or PKD2 gene mutations nor their position enhanced cell proliferation rate in our cell line models, truncating mutations in these genes progressively increased overall extracellular acidification over time ( P < 0.001 for PKHD1 and PKD2 mutations). PKHD1 mutations increased nonglycolytic acidification rate (1.19 vs. 1.03, P = 0.002), consistent with an increase in tricarboxylic acid cycle activity or breakdown of intracellular glycogen. In addition, they increased basal and ATP-linked oxygen consumption rates [7.59 vs. 5.42 ( P = 0.015) and 4.55 vs. 2.98 ( P = 0.004)]. The PKHD1 and PKD2 mutations also altered mitochondrial morphology, resembling the effects of polycystin 1 deficiency. Together, these data suggest that defects in major PKD genes trigger changes in mitochondrial energy metabolism. After validation in in vivo models, these initial observations would indicate potential benefits of targeting energy metabolism in the treatment of PKDs.

Detection of Lipid Mediators of Inflammation in the Human Tear Film.

PURPOSE: Lipid mediators of inflammation are a group of signaling molecules produced by various cells under physiological conditions and modulate the inflammatory process during various pathologic conditions. Although eicosanoids and F2-isoprostanes are recognized lipid mediators of inflammation, there is no consensus yet on the extraction and mass spectrometry (MS) method for their analysis in individual human tear samples. Thus, the aim of this study was to develop an optimal method for extraction of lipid mediators of inflammation in the tear film and evaluate MS techniques for their analysis. METHODS: Basal tears were collected from each eye of 19 subjects using glass microcapillaries. Lipid extraction was performed using either varying concentrations of acidified methanol, a modified Folch method, or solid-phase extraction. Initially, an untargeted analysis of the extracts was performed using SCIEX TripleTOF 5600 mass spectrometer to identify any lipid mediators of inflammation (eicosanoids) and later a targeted analysis was performed using the SCIEX 6500 Qtrap to identify and quantify prostaglandins and isoprostanes. Mass spectra and chromatograms were analyzed using Peakview, XCMS, and Multiquant software. RESULTS: Prostaglandins and isoprostanes were observed and quantified using the Qtrap mass spectrometer under multiple reaction monitoring (MRM) mode after solid-phase extraction. Extraction with acidified methanol along with the Folch method produced cleaner spectra during MS with the Triple time of flight (TOF) mass spectrometer. Lipid mediators of inflammation were not observed in any of the tear samples using the Triple TOF mass spectrometer. CONCLUSIONS: Solid-phase extraction may be the method of choice for extraction of prostaglandins and isoprostanes in low volumes of tears. The SCIEX Qtrap 6500 in MRM mode may be suitable to identify and quantify similar lipid mediators of inflammation.

Early airway microbial metagenomic and metabolomic signatures are associated with development of severe bronchopulmonary dysplasia.

The pathogenesis of bronchopulmonary dysplasia (BPD) is not well understood. We previously identified differences in the airway microbiome at birth between preterm infants who were BPD predisposed versus those who were BPD resistant. In this study, we attempted to identify mechanisms by which the airway microbiome could modify the risk for BPD. We used a software-based method to predict the metagenome of the tracheal aspirate (TA) microbiome from 16S rRNA sequencing data in preterm infants and to identify functional ortholog genes that were differentially abundant in BPD-predisposed and BPD-resistant infants. We also identified metabolites that were differentially enriched in these samples by use of untargeted mass spectrometry and mummichog to identify the metabolic pathways involved. Microbial metagenome analysis identified specific pathways that were less abundant in the functional metagenome of the microbiota of BPD-predisposed infants compared with BPD-resistant infants. The airway metabolome of BPD-predisposed infants was enriched for metabolites involved in fatty acid activation and androgen and estrogen biosynthesis compared with BPD-resistant infants. These findings suggest that in extremely preterm infants the early airway microbiome may alter the metabolome, thereby modifying the risk of BPD. The differential enrichment of sex steroid metabolic pathways supports previous studies suggesting a role for sexual dimorphism in BPD risk. This study also suggests a role for metabolomic and metagenomic profiles to serve as early biomarkers of BPD risk.

Comprehensive profiling of prostaglandins in human ovarian follicular fluid using mass spectrometry.

Prostaglandins are formed by enzymatic and non-enzymatic mechanisms. They have been detected in human ovarian follicular fluid (HFF), a medium rich in growth factors and nutrients important for oocyte growth and fertility. However, the comprehensive identification of HFF prostaglandins has not been addressed. Here we use hybrid triple quadrupole time-of-flight and triple quadrupole mass spectrometers to comprehensively analyze prostaglandins in HFF. We identified PGE1, PGE2, PGF2α, and other prostaglandins synthesized via prostaglandin-endoperoxide synthase (i.e. Cox) cascades. We also identified specific PGF2α isomers (F2-isoprostanes) and PGF3α analogs whose structures are inconsistent with Cox-dependent formation. A prospective cohort pilot study of infertility patient subtypes revealed two potential associations. F2-isoprostanes are decreased in the diminished ovarian reserve subtype and elevated PGF2α may be associated with decreased live birth. Other than PGF2α, only body mass index >25kg/m2 correlated with poor in vitro fertilization outcome. Our studies suggest that HFF contains prostaglandins formed from at least two mechanisms, which may correlate with distinct clinical parameters.

Lower Levels of Cervicovaginal Tryptophan Are Associated With Natural Clearance of Chlamydia in Women.

Chlamydiatrachomatis (Ct) infection causes significant morbidity. In vitro studies demonstrate that Ct growth inhibition occurs by interferon-gamma (IFN-γ)-mediated depletion of intracellular tryptophan, and some Ct strains utilize extracellular indole to restore tryptophan levels. Whether tryptophan levels are associated with Ct infection clearance in humans remains unknown. We evaluated tryptophan, indole, and IFN-γ levels in cervicovaginal lavages from women with either naturally cleared or persisting Ct infection. Women who cleared infection had significantly lower tryptophan levels and trended toward lower IFN-γ levels compared to women with persisting infection. Due to its volatility, indole was not measurable in either group.

The Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor Augments Mucociliary Clearance Abrogating Cystic Fibrosis Transmembrane Conductance Regulator Inhibition by Cigarette Smoke.

Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to chronic obstructive pulmonary disease pathogenesis and is a potential therapeutic target. We sought to determine the acute effects of cigarette smoke on ion transport and the mucociliary transport apparatus, their mechanistic basis, and whether deleterious effects could be reversed with the CFTR potentiator ivacaftor (VX-770). Primary human bronchial epithelial (HBE) cells and human bronchi were exposed to cigarette smoke extract (CSE) and/or ivacaftor. CFTR function and expression were measured in Ussing chambers and by surface biotinylation. CSE-derived acrolein modifications on CFTR were determined by mass spectroscopic analysis of purified protein, and the functional microanatomy of the airway epithelia was measured by 1-µm resolution optical coherence tomography. CSE reduced CFTR-dependent current in HBE cells (P < 0.05) and human bronchi (P < 0.05) within minutes of exposure. The mechanism involved CSE-induced reduction of CFTR gating, decreasing CFTR open-channel probability by approximately 75% immediately after exposure (P < 0.05), whereas surface CFTR expression was partially reduced with chronic exposure, but was stable acutely. CSE treatment of purified CFTR resulted in acrolein modifications on lysine and cysteine residues that likely disrupt CFTR gating. In primary HBE cells, CSE reduced airway surface liquid depth (P < 0.05) and ciliary beat frequency (P < 0.05) within 60 minutes that was restored by coadministration with ivacaftor (P < 0.005). Cigarette smoking transmits acute reductions in CFTR activity, adversely affecting the airway surface. These effects are reversible by a CFTR potentiator in vitro, representing a potential therapeutic strategy in patients with chronic obstructive pulmonary disease with chronic bronchitis.

Composition of Rosenthal Fibers, the Protein Aggregate Hallmark of Alexander Disease.

Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.

Early lipid changes in acute kidney injury using SWATH lipidomics coupled with MALDI tissue imaging.

Acute kidney injury (AKI) is one of the leading causes of in-hospital morbidity and mortality, particularly in critically ill patients. Although our understanding of AKI at the molecular level remains limited due to its complex pathophysiology, recent advances in both quantitative and spatial mass spectrometric approaches offer new opportunities to assess the significance of renal metabolomic changes in AKI models. In this study, we evaluated lipid changes in early ischemia-reperfusion (IR)-related AKI in mice by using sequential window acquisition of all theoretical spectra (SWATH)-mass spectrometry (MS) lipidomics. We found a significant increase in two abundant ether-linked phospholipids following IR at 6 h postinjury, a plasmanyl choline, phosphatidylcholine (PC) O-38:1 (O-18:0, 20:1), and a plasmalogen, phosphatidylethanolamine (PE) O-42:3 (O-20:1, 22:2). Both of these lipids correlated with the severity of AKI as measured by plasma creatinine. In addition to many more renal lipid changes associated with more severe AKI, PC O-38:1 elevations were maintained at 24 h post-IR, while renal PE O-42:3 levels decreased, as were all ether PEs detected by SWATH-MS at this later time point. To further assess the significance of this early increase in PC O-38:1, we used matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) to determine that it occurred in proximal tubules, a region of the kidney that is most prone to IR injury and also rich in the rate-limiting enzymes involved in ether-linked phospholipid biosynthesis. Use of SWATH-MS lipidomics in conjunction with MALDI-IMS for lipid localization will help in elucidating the role of lipids in the pathobiology of AKI.