Gut microbiota Turicibacter strains differentially modify bile acids and host lipids.

Bacteria from the Turicibacter genus are prominent members of the mammalian gut microbiota and correlate with alterations in dietary fat and body weight, but the specific connections between these symbionts and host physiology are poorly understood. To address this knowledge gap, we characterize a diverse set of mouse- and human-derived Turicibacter isolates, and find they group into clades that differ in their transformations of specific bile acids. We identify Turicibacter bile salt hydrolases that confer strain-specific differences in bile deconjugation. Using male and female gnotobiotic mice, we find colonization with individual Turicibacter strains leads to changes in host bile acid profiles, generally aligning with those produced in vitro. Further, colonizing mice with another bacterium exogenously expressing bile-modifying genes from Turicibacter strains decreases serum cholesterol, triglycerides, and adipose tissue mass. This identifies genes that enable Turicibacter strains to modify host bile acids and lipid metabolism, and positions Turicibacter bacteria as modulators of host fat biology.

Improved Chromatography and MS-Based Detection of Glyphosate and Aminomethylphosphonic Acid Using iTrEnDi.

Glyphosate (GLY), a synthetic, nonselective systemic herbicide that is particularly effective against perennial weeds, is the most used weedkiller in the world. There are growing concerns over GLY accumulation in the environment and the attendant human health-associated risks, and despite increased attention in the media, GLY and its breakdown product aminomethylphosphonic acid (AMPA) remain elusive to many analytical strategies. Chemical derivatization coupled with high-performance liquid chromatography-mass spectrometry (HPLC-MS) addresses the challenge of quantifying low levels of GLY and AMPA in complex samples. Here we demonstrate the use of in situ trimethylation enhancement using diazomethane (iTrEnDi) to derivatize GLY and AMPA into permethylated products ([GLYTr]+ and [AMPATr]+, respectively) prior to analysis via HPLC-MS. iTrEnDi produced quantitative yields and resulted in a 12-340-fold increases in HPLC-MS-based sensitivity for [GLYTr]+ and [AMPATr]+, respectively, compared with underivatized counterparts. The limits of detection of derivatized compounds were found to be 0.99 ng/L for [GLYTr]+ and 1.30 ng/L for [AMPATr]+, demonstrating significant sensitivity improvements compared to previously established derivatization techniques. iTrEnDi is compatible with the direct derivatization of Roundup formulations. Finally, as proof of principle, a simple aqueous extraction followed by iTrEnDi enabled the detection of [GLYTr]+ and [AMPATr]+ on the exterior of field-grown soybeans that were sprayed with Roundup. Overall, iTrEnDi ameliorates issues relating to low proton affinity and chromatographic retention, boosting HPLC-MS-based sensitivity and enabling the elucidation of elusive analytes such as GLY and AMPA within agricultural systems.

Tissue-engineered vocal fold replacement in swine: Methods for functional and structural analysis.

We have developed a cell-based outer vocal fold replacement (COVR) as a potential therapy to improve voice quality after vocal fold (VF) injury, radiation, or tumor resection. The COVR consists of multipotent human adipose-derived stem cells (hASC) embedded within a three-dimensional fibrin scaffold that resembles vocal fold epithelium and lamina propria layers. Previous work has shown improved wound healing in rabbit studies. In this pilot study in pigs, we sought to develop methods for large animal implantation and phonatory assessment. Feasibility, safety, and structural and functional outcomes of the COVR implant are described. Of eight pigs studied, six animals underwent COVR implantation with harvest between 2 weeks and 6 months. Recovery of laryngeal tissue structure was assessed by vibratory and histologic analyses. Recovery of voice function was assessed by investigating acoustic parameters that were derived specifically for pigs. Results showed improved lamina propria qualities relative to an injured control animal at 6 months. Acoustic parameters reflected voice worsening immediately after surgery as expected; acoustics displayed clear voice recovery in the animal followed for 6 months after COVR. These methods form the basis for a larger-scale long-term pre-clinical safety and efficacy study.

Small molecules disaggregate alpha-synuclein and prevent seeding from patient brain-derived fibrils.

The amyloid aggregation of alpha-synuclein within the brain is associated with the pathogenesis of Parkinson's disease (PD) and other related synucleinopathies, including multiple system atrophy (MSA). Alpha-synuclein aggregates are a major therapeutic target for treatment of these diseases. We identify two small molecules capable of disassembling preformed alpha-synuclein fibrils. The compounds, termed CNS-11 and CNS-11g, disaggregate recombinant alpha-synuclein fibrils in vitro, prevent the intracellular seeded aggregation of alpha-synuclein fibrils, and mitigate alpha-synuclein fibril cytotoxicity in neuronal cells. Furthermore, we demonstrate that both compounds disassemble fibrils extracted from MSA patient brains and prevent their intracellular seeding. They also reduce in vivo alpha-synuclein aggregates in C. elegans. Both compounds also penetrate brain tissue in mice. A molecular dynamics-based computational model suggests the compounds may exert their disaggregating effects on the N terminus of the fibril core. These compounds appear to be promising therapeutic leads for targeting alpha-synuclein for the treatment of synucleinopathies.

Genistein Inhibits Clostridioides difficile Infection via Estrogen Receptors and Lysine-Deficient Protein Kinase 1.

BACKGROUND: Clostridioides difficile infection (CDI) is a debilitating nosocomial disease. Postmenopausal women may have an increased risk of CDI, suggesting estrogen influence. Soybean products contain a representative estrogenic isoflavone, genistein. METHODS: The anti-inflammatory and antiapoptotic effects of genistein were determined using primary human cells and fresh colonic tissues. The effects of oral genistein therapy among mice and hamsters were evaluated. RESULTS: Within 10 days of CDI, female c57BL/6J mice in a standard environment (regular diet) had a 50% survival rate, while those with estrogen depletion and in an isoflavone-free environment (soy-free diet) had a 25% survival rate. Oral genistein improved their 10-day survival rate to 100% on a regular diet and 75% in an isoflavone-free environment. Genistein reduced macrophage inflammatory protein-1α (MIP-1α) secretion in fresh human colonic tissues exposed to toxins. Genistein inhibited MIP-1α secretion in primary human peripheral blood mononuclear cells, abolished apoptosis and BCL-2-associated X (BAX) expression in human colonic epithelial cells, and activated lysine-deficient protein kinase 1 (WNK1) phosphorylation in both cell types. The anti-inflammatory and antiapoptotic effects of genistein were abolished by inhibiting estrogen receptors and WNK1. CONCLUSIONS: Genistein reduces CDI disease activity by inhibiting proinflammatory cytokine expression and apoptosis via the estrogen receptor/G-protein estrogen receptor/WNK1 pathways.

Low-dose exposure to PBDE disrupts genomic integrity and innate immunity in mammary tissue.

The low-dose mixture hypothesis of carcinogenesis proposes that exposure to an environmental chemical that is not individually oncogenic may nonetheless be capable of enabling carcinogenesis when it acts in concert with other factors. A class of ubiquitous environmental chemicals that are hypothesized to potentially function in this low-dose capacity are synthesized polybrominated diphenyl ethers (PBDEs). PBDEs can affect correlates of carcinogenesis that include genomic instability and inflammation. However, the effect of low-dose PBDE exposure on such correlates in mammary tissue has not been examined. In the present study, low-dose long-term (16 weeks) administration of PBDE to mice modulated transcriptomic indicators of genomic integrity and innate immunity in normal mammary tissue. PBDE increased transcriptome signatures for the Nuclear Factor Erythroid 2 Like 2 (NFE2L2) response to oxidative stress and decreased signatures for non-homologous end joining DNA repair (NHEJ). PBDE also decreased transcriptome signatures for the cyclic GMP-AMP Synthase - Stimulator of Interferon Genes (cGAS-STING) response, decreased indication of Interferon Stimulated Gene Factor 3 (ISGF3) and Nuclear Factor Kappa B (NF-κB) transcription factor activity, and increased digital cytometry estimates of immature dendritic cells (DCs) in mammary tissue. Replication of the PBDE exposure protocol in mice susceptible to mammary carcinogenesis resulted in greater tumor development. The results support the notion that ongoing exposure to low levels of PBDE can disrupt facets of genomic integrity and innate immunity in mammary tissue. Such effects affirm that synthesized PBDEs are a class of environmental chemicals that reasonably fit the low-dose mixture hypothesis.

Arachidonic acid metabolism and inflammatory biomarkers associated with exposure to polycyclic aromatic hydrocarbons.

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with systemic inflammation, yet what mechanisms regulate PAHs' inflammatory effects are less understood. This study evaluated the change of arachidonic acid (ARA) metabolites and inflammatory biomarkers in response to increased exposure to PAHs among 26 non-smoking healthy travelers from Los Angeles to Beijing. Traveling from Los Angeles to Beijing significantly increased urinary metabolites of dibenzofuran (800%), fluorene (568%), phenanthrene (277%), and pyrene (176%), accompanied with increased C-reactive protein, fibrinogen, IL-8, and IL-10, and decreased MCP-1, sCD40L, and sCD62P levels in the blood. Meanwhile, the travel increased the levels of ARA lipoxygenase metabolites that were positively associated with a panel of pro-inflammatory biomarkers. Concentrations of cytochrome P450 metabolite were also increased in Beijing and were negatively associated with sCD62P levels. In contrast, concentrations of ARA cyclooxygenase metabolites were decreased in Beijing and were negatively associated with anti-inflammatory IL-10 levels. Changes in both inflammatory biomarkers and ARA metabolites were reversed 4-7 weeks after participants returned to Los Angeles and were associated with urinary PAH metabolites, but not with other exposures such as secondhand smoke, stress, or diet. These results suggested possible roles of ARA metabolic alteration in PAHs-associated inflammatory effects.

Accumulation of Seminolipid in Sertoli Cells Is Associated with Increased Levels of Reactive Oxygen Species and Male Subfertility: Studies in Aging Arsa Null Male Mice.

Seminolipid (also known as sulfogalactosylglycerolipid-SGG), present selectively in male germ cells, plays important roles in spermatogenesis and sperm-egg interaction. The proper degradation of SGG in apoptotic germ cells is also as important. Sertoli cells first phagocytose apoptotic germ cells, then Sertoli lysosomal arylsulfatase A (ARSA) desulfates SGG, the first step of SGG degradation. We have reported that aging male Arsa-/- mice become subfertile with SGG accumulation in Sertoli cell lysosomes, typical of a lysosomal storage disorder (LSD). Since reactive oxygen species (ROS) levels are increased in other glycolipid-accumulated LSDs, we quantified ROS in Arsa-/- Sertoli cells. Our analyses indicated increases in superoxide and H2O2 in Arsa-/- Sertoli cells with elevated apoptosis rates, relative to WT counterparts. Excess H2O2 from Arsa-/- Sertoli cells could travel into testicular germ cells (TGCs) to induce ROS production. Our results indeed indicated higher superoxide levels in Arsa-/- TGCs, compared with WT TGCs. Increased ROS levels in Arsa-/- Sertoli cells and TGCs likely caused the decrease in spermatogenesis and increased the abnormal sperm population in aging Arsa-/- mice, including the 50% decrease in sperm SGG with egg binding ability. In summary, our study indicated that increased ROS production was the mechanism through which subfertility manifested following SGG accumulation in Sertoli cells.

Carbon Monoxide Inhibits Cytochrome P450 Enzymes CYP3A4/2C8 in Human Breast Cancer Cells, Increasing Sensitivity to Paclitaxel.

Paclitaxel (PTX) is a first-line treatment in breast cancer, though resistance develops quickly and frequently. Cytochrome P450 enzymes CYP3A4 and CYP2C8, which metabolically inactivate PTX in hepatic tissue, are overexpressed in malignant breast tissues. CYP3A4 expression correlates with PTX therapy failure and poor outcomes, though no direct evidence of CYP3A4 contributing to PTX sensitivity exists. Because CYP3A4/2C8 is susceptible to carbon monoxide (CO)-mediated inhibition and CO (a gaseous signaling molecule) has previously exhibited drug-sensitizing effects in cancer cells, we hypothesized that CO-mediated inhibition of CYP3A4/2C8 could lead to enhanced drug sensitivity. Using a photo-activated CO-releasing molecule, we have assessed the ability of CO to alter the pharmacokinetics of PTX in breast cancer cells via inhibition of CYP3A4/2C8 and determined that CO does enhance sensitivity of breast cancer cells to PTX. Inhibition of CYP3A4/2C8 by CO could therefore be a promising therapeutic strategy to enhance PTX response in breast cancer.

Electronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative Biomarkers.

[Figure: see text].

A sensitive LC-MS assay using derivatization with boron trifluoride to quantify curcuminoids in biological samples.

A procedure is described to measure curcumin (C), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), tetrahydrocurcumim (TC) and their glucuronidated metabolites (CG, DMCG, and BDMCG) in plasma, brain, liver and tumor samples. The procedure involves converting the analytes to their boron difluoride derivatives and analyzing them by combined liquid chromatography coupled to an ion trap mass spectrometer operating in the negative ion MSn scan mode. The method has superb limits of detection of 0.01 nM for all curcuminoids and 0.5 nM for TC and the glucuroniated metabolites, and several representative chromatograms of biological samples containing these analytes are provided. In addition, the pharmacokinetic profile of these compounds in one human who daily consumed an over-the-counter curcuminoid product shows the peak and changes in circulating concentrations achieved by this mode of administration.

Primary Sertoli Cell Cultures From Adult Mice Have Different Properties Compared With Those Derived From 20-Day-Old Animals.

Cultures of Sertoli cells isolated from 20-day-old mice are widely used in research as substitutes for adult Sertoli cell cultures. This practice is based on the fact that Sertoli cells cease to proliferate and become mature in vivo by 16 to 20 days after birth. However, it is important to verify whether cultured Sertoli cells derived from 20-day-old mice do not proliferate ex vivo and whether they have the same properties as cultured adult Sertoli cells. Herein we described an isolation/culture method of Sertoli cells from 10-week-old adult mice with > 90% purity. Properties of these cultured adult Sertoli cells were then compared with those of cultured Sertoli cells derived from 20-day-old mice (also > 90% purity). By cell counting, bromo-2-deoxyuridine incorporation, and metaphase plate detection, we demonstrated that only adult Sertoli cells did not proliferate throughout 12 culture days. In contrast, Sertoli cells derived from 20-day-old mice still proliferated until Day 10 in culture. The morphology and profiles of intracellular lipidomics and spent medium proteomics of the 2 cultures were also different. Cultured adult Sertoli cells were larger in size and contained higher levels of triacylglycerols, cholesteryl esters, and seminolipid, and the proteins in their spent medium were mainly engaged in cellular metabolism. In contrast, proteins involved in cell division, including anti-Mullerian hormone, cell division cycle protein 42 (CDC42), and collagen isoforms, were at higher levels in Sertoli cell cultures derived from 20-day-old mice. Therefore, cultured Sertoli cells derived from 10-week-old mice, rather than those from 20-day-old animals, should be used for studies on properties of adult Sertoli cells.

The mechanism of ß-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation.

ß-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

Extracellular Vesicles From Auditory Cells as Nanocarriers for Anti-inflammatory Drugs and Pro-resolving Mediators.

Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. We propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to the target cells. The present study provides evidence that extracellular vesicles (EVs) from auditory HEI-OC1 cells may incorporate significant quantities of anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. EVs generated by HEI-OC1 cells were divided by size into two fractions, small (≤150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin D1, and the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with PUFA and the large ones with aspirin, LXA4 and resolvin D1. Importantly, our data indicate that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large EVs from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators.

Pro-Oxidative and Proinflammatory Effects After Traveling From Los Angeles to Beijing: A Biomarker-Based Natural Experiment.

BACKGROUND: Exposure to air pollution increases cardiovascular morbidity and mortality. Preventing chronic cardiovascular diseases caused by air pollution relies on detecting the early effects of pollutants on the risk of cardiovascular disease development, which is limited by the lack of sensitive biomarkers. We have previously identified promising biomarkers in experimental animals but comparable evidence in humans is lacking. METHODS: Air pollution is substantially worse in Beijing than in Los Angeles. We collected urine and blood samples from 26 nonsmoking, healthy adult residents of Los Angeles (mean age, 23.8 years; 14 women) before, during, and after spending 10 weeks in Beijing during the summers of 2014 and 2015. We assessed a panel of circulating biomarkers indicative of lipid peroxidation and inflammation. Personal exposure to polycyclic aromatic hydrocarbons (PAHs), a group of combustion-originated air pollutants, was assessed by urinary PAH metabolite levels. RESULTS: Urinary concentrations of 4 PAH metabolites were 176% (95% CI, 103% to 276%) to 800% (95% CI, 509% to 1780%) greater in Beijing than in Los Angeles. Concentrations of 6 lipid peroxidation biomarkers were also increased in Beijing, among which 5-, 12-, and 15-hydroxyeicosatetraenoic acid and 9- and 13-hydroxyoctadecadienoic acid levels reached statistical significance (false discovery rate <5%), but not 8-isoprostane (20.8%; 95% CI, -5.0% to 53.6%). The antioxidative activities of paraoxonase (-9.8%; 95% CI, -14.0% to -5.3%) and arylesterase (-14.5%; 95% CI, -22.3% to -5.8%) were lower and proinflammatory C-reactive protein (101%; 95% CI, 3.3% to 291%) and fibrinogen (48.3%; 95% CI, 4.9% to 110%) concentrations were higher in Beijing. Changes in all these biomarkers were reversed, at least partially, after study participants returned to Los Angeles. Changes in most outcomes were associated with urinary PAH metabolites (P<0.05). CONCLUSIONS: Traveling from a less-polluted to a more-polluted city induces systemic pro-oxidative and proinflammatory effects. Changes in the levels of 5-, 12-, and 15-hydroxyeicosatetraenoic acid and 9- and 13-hydroxyoctadecadienoic acid as well as paraoxonase and arylesterase activities in the blood, in association with exposures to PAH metabolites, might have important implications in preventive medicine as indicators of increased cardiovascular risk caused by air pollution exposure.

Diesel Exhaust Induces Mitochondrial Dysfunction, Hyperlipidemia, and Liver Steatosis.

OBJECTIVE: Air pollution is associated with increased cardiovascular morbidity and mortality, as well as dyslipidemia and metabolic syndrome. Our goal was to dissect the mechanisms involved. Approach and Results: We assessed the effects of exposure to air pollution on lipid metabolism in mice through assessment of plasma lipids and lipoproteins, oxidized fatty acids 9-HODE (9-hydroxyoctadecadienoic) and 13-HODE (13-hydroxyoctadecadienoic), lipid, and carbohydrate metabolism. Findings were corroborated, and mechanisms were further assessed in HepG2 hepatocytes in culture. ApoE knockout mice exposed to inhaled diesel exhaust (DE, 6 h/d, 5 days/wk for 16 weeks) exhibited elevated plasma cholesterol and triglyceride levels, increased hepatic triglyceride content, and higher hepatic levels of 9-HODE and 13-HODE, as compared to control mice exposed to filtered air. A direct effect of DE exposure on hepatocytes was demonstrated by treatment of HepG2 cells with a methanol extract of DE particles followed by loading with oleic acid. As observed in vivo, this led to increased triglyceride content and significant downregulation of ACAD9 mRNA expression. Treatment of HepG2 cells with DE particles and oleic acid did not alter de novo lipogenesis but inhibited total, mitochondrial, and ATP-linked oxygen consumption rate, indicative of mitochondrial dysfunction. Treatment of isolated mitochondria, prepared from mouse liver, with DE particles and oleic acid also inhibited mitochondrial complex activity and ß-oxidation. CONCLUSIONS: DE exposure leads to dyslipidemia and liver steatosis in ApoE knockout mice, likely due to mitochondrial dysfunction and decreased lipid catabolism.

Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells.

PROBLEM: Sperm are the major cells in semen. Human sperm possess a number of HIV-1 gp120 binding ligands including sulfogalactosylglycerolipid (SGG). However, the mechanisms of how sperm capture HIV-1 onto their surface are unclear. Furthermore, the ability of sperm to deliver HIV-1 to vaginal/cervical epithelial cells lining the lower female reproductive tract, as a first step in HIV-1 transmission, needs to be determined. METHOD OF STUDY: Sperm from healthy donors were incubated with dual-tropic HIV-1CS204 (clinical isolate), and virus capture was determined by p24 antigen ELISA. The involvement of SGG in HIV-1 capture was assessed by determining Kd values of HIV-1 gp120-SGG binding as well as computational docking of SGG to the gp120 V3 loop. The ability of sperm-associated HIV-1 to infect peripheral blood mononuclear cells (PBMCs) and TZM-bl indicator cells was determined. Lastly, infection of vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cells mediated by HIV-1-associated sperm was evaluated. RESULTS: Sperm were able to capture HIV-1 in a dose-dependent manner, and the capture reached a maximum within 5 minutes. Captured HIV-1, however, could be removed from sperm by Percoll-gradient centrifugation. Affinity of gp120 for SGG was substantial, implicating sperm SGG in HIV-1 capture. Sperm-associated HIV-1 could productively infect PBMCs and TZM-bl cells, and was capable of being transmitted into vaginal/cervical epithelial cells. CONCLUSION: Sperm are able to capture HIV-1, which remains infectious and is able to be transmitted into vaginal/cervical epithelial cells, a result indicating the importance of sperm in HIV transmission.

Preliminary Characterization of Extracellular Vesicles From Auditory HEI-OC1 Cells.

OBJECTIVES: Isolate, purify, and characterize extracellular vesicles (EVs) obtained from auditory HEI-OC1 cells, and evaluate their suitability for intracochlear transport and delivery of pharmacological drugs and/or pro-resolution mediators of acute inflammatory processes. METHODS: HEI-OC1 EVs were isolated and purified using the exoEasy Maxi Kit, and their size was evaluated by nanoparticle tracking techniques. Bottom-up proteomics of the EVs, either freshly obtained or stored for up to 4 months at -20°C, was performed by LC-ESI-MS/MS. LC-ESI-MS/MS-MRM was used to measure the loading of dexamethasone inside EVs following co-incubation at room temperature for 1 hour with and without 5 minutes sonication. RESULTS: Routinely, we were able to obtain purified fractions of >2 × 109 EVs/mL, with diameters varying between 50 and 800 nm. Bottom-up proteomics showed that among the most abundant EVs proteins, 19.2% were cytoplasmic, 17.2% were membrane localized, 12.3% were cytosolic, and 14.6% were nucleolar. No significant differences between fresh and stored EVs were detected. Importantly, co-incubation of HEI-OC1 EVs (1 × 108 EVs/mL) with dexamethasone (10 mM) resulted in the incorporation of 10.1 ± 1.9 nM dexamethasone per milliliter of EVs suspension. CONCLUSIONS: Altogether, the results suggest that EVs from HEI-OC1 cells could be advantageously used as biological nanocarriers for the delivery of specific molecules and pharmacological drugs into the inner ear.

Tyrosine aminotransferase is involved in the oxidative stress response by metabolizing meta-tyrosine in Caenorhabditis elegans.

Under oxidative stress conditions, hydroxyl radicals can oxidize the phenyl ring of phenylalanine, producing the abnormal tyrosine isomer meta-tyrosine (m-tyrosine). m-Tyrosine levels are commonly used as a biomarker of oxidative stress, and its accumulation has recently been reported to adversely affect cells, suggesting a direct role for m-tyrosine in oxidative stress effects. We found that the Caenorhabditis elegans ortholog of tyrosine aminotransferase (TATN-1)-the first enzyme involved in the metabolic degradation of tyrosine-is up-regulated in response to oxidative stress and directly activated by the oxidative stress-responsive transcription factor SKN-1. Worms deficient in tyrosine aminotransferase activity displayed increased sensitivity to multiple sources of oxidative stress. Biochemical assays revealed that m-tyrosine is a substrate for TATN-1-mediated deamination, suggesting that TATN-1 also metabolizes m-tyrosine. Consistent with a toxic effect of m-tyrosine and a protective function of TATN-1, tatn-1 mutant worms exhibited delayed development, marked reduction in fertility, and shortened lifespan when exposed to m-tyrosine. A forward genetic screen identified a mutation in the previously uncharacterized gene F01D4.5-homologous with human transcription factor 20 (TCF20) and retinoic acid-induced 1 (RAI1)-that suppresses the adverse phenotypes observed in m-tyrosine-treated tatn-1 mutant worms. RNA-Seq analysis of F01D4.5 mutant worms disclosed a significant reduction in the expression of specific isoforms of genes encoding ribosomal proteins, suggesting that alterations in protein synthesis or ribosome structure could diminish the adverse effects of m-tyrosine. Our findings uncover a critical role for tyrosine aminotransferase in the oxidative stress response via m-tyrosine metabolism.

C/EBPß regulates the M2 transcriptome in ß-adrenergic-stimulated macrophages.

At the M2 terminal of the macrophage activation spectrum, expression of genes is regulated by transcription factors that include STAT6, CREB, and C/EBPß. Signaling through ß-adrenergic receptors drives M2 activation of macrophages, but little is known about the transcription factors involved. In the present study, we found that C/EBPß regulates the signaling pathway between ß-adrenergic stimulation and expression of Arg1 and several other specific genes in the greater M2 transcriptome. ß-adrenergic signaling induced Cebpb gene expression relatively early with a peak at 1 h post-stimulation, followed by peak Arg1 gene expression at 8 h. C/EBPß transcription factor activity was elevated at the enhancer region for Arg 1 at both 4 and 8 h after stimulation but not near the more proximal promoter region. Knockdown of Cebpb suppressed the ß-adrenergic-induced peak in Cebpb gene expression as well as subsequent accumulation of C/EBPß protein in the nucleus, which resulted in suppression of ß-adrenergic-induced Arg1 gene expression. Analysis of genome-wide transcriptional profiles identified 20 additional M2 genes that followed the same pattern of regulation by ß-adrenergic- and C/EBPß-signaling. Promoter-based bioinformatic analysis confirmed enrichment of binding motifs for C/EBPß transcription factor across these M2 genes. These findings pinpoint a mechanism that may be targeted to redirect the deleterious influence of ß-adrenergic signaling on macrophage involvement in M2-related diseases such as cancer.