Mapping Novel Frataxin Mitochondrial Networks Through Protein- Protein Interactions.

Friedreich's Ataxia (FRDA) is a neuromuscular degenerative disorder caused by trinucleotide expansions in the first intron of the frataxin (FXN) gene, resulting in insufficient levels of functional FNX protein. Deficits in FXN involve mitochondrial disruptions including iron-sulfur cluster synthesis and impaired energetics. These studies were to identify unique protein-protein interactions with FXN to better understand its function and design therapeutics. Two complementary approaches were employed, BioID and Co-IP, to identify protein interactions with FXN at the direct binding, indirect binding, and non-proximal levels. Forty-one novel protein interactions were identified by BioID and IP techniques. The FXN protein landscape was further analyzed incorporating both interaction type and functional pathways using a maximum path of 6 proteins with a potential direct interaction between FXN and NFS1. Probing the intersection between FXN-protein landscape and biological pathways associated with FRDA, we identified 41 proteins of interest. Peroxiredoxin 3 (Prdx3) was chosen for further analysis because of its role in mitochondrial oxidative injury. Our data has demonstrated the strengths of employing complementary methods to identify a unique interactome for FXN. Our data provides new insights into FXN function and regulation, a potential direct interaction between FXN and NFS1, and pathway interactions between FXN and Prdx3.

Selenium Deficiency Exacerbates Hyperoxia-Induced Lung Injury in Newborn C3H/HeN Mice.

Extremely preterm infants are often treated with supraphysiological oxygen, which contributes to the development of bronchopulmonary dysplasia (BPD). These same infants exhibit compromised antioxidant capacities due in part to selenium (Se) deficiency. Se is essential for basal and inducible antioxidant responses. The present study utilized a perinatal Se deficiency (SeD) mouse model to identify the combined effects of newborn hyperoxia exposure and SeD on alveolarization and antioxidant responses, including the identification of affected developmental pathways. Se-sufficient (SeS) and SeD C3H/HeN breeding pairs were generated, and pups were exposed to room air or 85% O2 from birth to 14 d. Survival, antioxidant protein expression, and RNA seq analyses were performed. Greater than 40% mortality was observed in hyperoxia-exposed SeD pups. Surviving SeD pups had greater lung growth deficits than hyperoxia-exposed SeS pups. Gpx2 and 4 protein and Gpx activity were significantly decreased in SeD pups. Nrf2-regulated proteins, Nqo1 and Gclc were increased in SeD pups exposed to hyperoxia. RNA seq revealed significant decreases in the Wnt/ß-catenin and Notch pathways. Se is a biologically relevant modulator of perinatal lung development and antioxidant responses, especially in the context of hyperoxia exposure. The RNA seq analyses suggest pathways essential for normal lung development are dysregulated by Se deficiency.

Effects of DNA methylase inhibitors in a murine model of severe BPD.

DNA methylation is necessary for developmental gene regulation, but adverse environments result in aberrant methylation and gene silencing. The current pilot study tested the hypothesis that treatment with DNA methylation inhibitors (decitabine; RG108) would improve alveolarization in a newborn murine model of severe bronchopulmonary dysplasia. Newborn mice exposed to maternal inflammation (LPS) and neonatal hyperoxia (85% O2) were treated with decitabine (p3, 0.1 mg/kg; p2, 4, 6, 0.1 mg/kg; or p2, 4, 6, 0.15 mg/kg) or RG108 (p3, 0.0013 mg/kg) delivered intranasally. Modest improvements in alveolarization were observed with decitabine, but no differences were observed with RG108. Attenuated phospho-SMAD2/3 levels and greater surfactant protein C protein levels compared to vehicle were observed with some tested doses. No detrimental side effects were observed with the doses used in this study. In summary, our pilot investigations identified a safe dose for intranasal administration of both methylation inhibitors and provides a foundation for further studies into methylation inhibitors in the context of neonatal lung injury.

Transcriptomic-Metabolomic Profiling in Mouse Lung Tissues Reveals Sex- and Strain-Based Differences.

Omics analyses are commonly used for identifying pathways and genes responsible for physiologic and pathologic processes. Though sex is considered a biological variable in rigorous assessments of pulmonary responses to oxidant exposures, the contribution of the murine strain is largely ignored. This study utilized an unbiased integrated assessment of high-resolution metabolomic profiling and RNA-sequencing to explore sex- and strain-dependent pathways in adult mouse lungs. The results indicated that strain exhibited a greater influence than sex on pathways associated with inflammatory and oxidant/antioxidant responses and that interaction metabolites more closely resembled those identified as differentially expressed by strain. Metabolite analyses revealed that the components of the glutathione antioxidant pathway were different between strains, specifically in the formation of mixed disulfides. Additionally, selenium metabolites such as selenohomocystiene and selenocystathionine were similarly differentially expressed. Transcriptomic analysis revealed similar findings, as evidenced by differences in glutathione peroxidase, peroxiredoxin, and the inflammatory transcription factors RelA and Jun. In summary, an multi-omics integrated approach identified that murine strain disproportionately impacts baseline expression of antioxidant systems in lung tissues. We speculate that strain-dependent differences contribute to discrepant pulmonary responses in preclincal mouse models, with deleterious effects on clinical translation.

Effects of Omega-3-6-9 fatty acid supplementation on behavior and sleep in preterm toddlers with autism symptomatology: Secondary analysis of a randomized clinical trial.

BACKGROUND: Children born extremely preterm disproportionately experience sequelae of preterm birth compared to those born at later gestational ages, including higher prevalence of autism spectrum disorder (ASD) and associated behaviors. AIM: Explore effects of combined dietary docosahexaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, and oleic acid (omega 3-6-9) on caregiver-reported behavior and sleep in toddlers born at ≤29 weeks' gestation who were exhibiting symptoms commonly seen with ASD. STUDY DESIGN: 90-day randomized (1:1), double blinded, placebo-controlled trial. SUBJECTS: Thirty-one children aged 18-38 months received omega 3-6-9 (n = 15) or canola oil placebo (n = 16). OUTCOME MEASURES: Mixed effects regression analyses followed intent to treat and explored treatment effects on measures of caregiver-reported behavior (Child Behavior Checklist 1.5-5, Toddler Behavior Assessment Questionnaire - Short Form, Vineland Adaptive Behavior Scales, 2nd Edition) and sleep (Children's Sleep Habits Questionnaire, Brief Infant Sleep Questionnaire). RESULTS: Twenty-nine of 31 (94%; ntx = 13, nplacebo = 16) children randomized had data available for at least one outcome measure, 27 (87%; ntx = 12, nplacebo = 15) had complete outcome data. Children randomized to omega 3-6-9 experienced a medium magnitude benefit of supplementation on anxious and depressed behaviors (ΔDifference = -1.27, d = -0.58, p = 0.049) and internalizing behaviors (ΔDifference = -3.41, d = -0.68, p = 0.05); and a large magnitude benefit on interpersonal relationship adaptive behaviors (ΔDifference = 7.50, d = 0.83, p = 0.01), compared to placebo. No effects were observed on other aspects of behavior or sleep. CONCLUSIONS: Findings provide preliminary support for further exploration of omega 3-6-9 during toddlerhood to improve socioemotional outcomes among children born preterm, especially for those showing early symptoms commonly seen with ASD. Results need to be replicated in a larger sample. TRIAL REGISTRATION: Registered with ClinicalTrials.gov: NCT01683565.

DHA Supplementation Attenuates Inflammation-Associated Gene Expression in the Mammary Gland of Lactating Mothers Who Deliver Preterm.

BACKGROUND: In a randomized trial of DHA supplementation to lactating mothers who delivered preterm, there were significant increases in DHA status in the mother and her infant. OBJECTIVES: Our objective here was to characterize the mammary gland transcriptomes from the above study. We hypothesized that proinflammatory gene expression would be attenuated in the increased DHA group compared with the standard DHA group. METHODS: In the original trial, mothers delivering at <29 wk gestation at the University of Cincinnati Medical Center and intending to express their milk were randomly assigned to supplementation with 200 mg/d DHA (standard group: STD) or 1000 mg/d DHA (experimental group: EXP) within 7 d of delivery. Here, we conducted RNA-seq transcriptome analysis of n = 5 EXP and n = 4 STD extracellular mammary mRNA samples extracted from the fat layer of milk samples obtained 4 wk postenrollment. Transcripts were assessed for differential expression (false discovery rate adjusted P value <0.05) and clustering between EXP compared with STD groups. Ontological analysis of all differentially expressed genes (DEGs) was performed with Toppcluster. RESULTS: There were 409 DEGs. We observed 5 main groups of biological processes that were upregulated, including those associated with improved immune regulation and management of oxidative stress; and 3 main groups of biological processes that were downregulated, including 1 associated with immune dysregulation. For example, we observed upregulation of inflammation-inhibiting genes including NFKB inhibitor alpha (NFKBIA; fold-change (FC), adjusted P value: FC = 1.70, P = 0.007) and interleukin-18 binding protein (IL18BP: FC = 2.2, adjusted P = 0.02); and downregulation of proinflammatory genes including interleukin 7 receptor (IL7R: FC = -1.9, adjusted P = 0.02) and interleukin 1 receptor like 1 (IL1RL1: FC = -13.0, adjusted P = 0.02). CONCLUSIONS: Increased DHA supplementation during lactation can modulate the expression of inflammation-related genes within the mammary gland. This might translate to milk composition with a more optimal inflammasome profile. Future research with a larger clinical trial and greater interrogation of clinical outcomes is warranted.

Randomized Controlled Trial of Omega-3 and -6 Fatty Acid Supplementation to Reduce Inflammatory Markers in Children with Autism Spectrum Disorder.

This double-blind, randomized controlled trial, tested fatty acid (FA) supplementation in children (ages 2- < 6 years) recently diagnosed with Autism Spectrum Disorder (ASD). Participants received daily oral FA supplement containing omega-3 and omega-6 FA, or a placebo for 90 days based on participant weight. Erythrocyte FAs and the cytokines, IL-1ß, IL-2, IFNγ, were measured in plasma obtained from serial blood collections. Treatment increased omega-3 and omega-6 FA levels (1.40 mol% for EPA and 1.62 mol% for DHA) and reduced IL-2 levels compared to placebo (- 0.17 pg/mL, 95% CI - 0.31, - 0.02, d = - 0.62). Omega 3-6 treatment was tolerable and adherence was greater than 70%. Future research will assess the effects of Omega 3-6 treatment on ASD symptoms. Registered on 06/08/2018 with ClinicalTrials.gov: NCT03550209.

Cyclooxygenase-2 deficiency attenuates lipopolysaccharide-induced inflammation, apoptosis, and acute lung injury in adult mice.

Many lung diseases are caused by an excessive inflammatory response, and inflammatory lung diseases are often modeled using lipopolysaccharide (LPS) in mice. Cyclooxygenase-2 (COX-2) encoded by the Ptgs2 gene is induced in response to inflammatory stimuli including LPS. The objective of this study was to test the hypothesis that mice deficient in COX-2 (Ptgs2-/-) will be protected from LPS-induced lung injury. Wild-type (WT; CD1 mice) and Ptgs2-/- mice (on a CD1 background) were treated with LPS or vehicle for 24 h. LPS treatment resulted in histological evidence of lung injury, which was attenuated in the Ptgs2-/- mice. LPS treatment increased the mRNA levels for tumor necrosis factor-α, interleukin-10, and monocyte chemoattractant protein-1 in the lungs of WT mice, and the LPS-induced increases in these levels were attenuated in the Ptgs2-/- mice. The protein levels of active caspase-3 and caspase-9 were lower in the LPS-treated lungs of Ptgs2-/- mice than in LPS-treated WT mice, as were the number of terminal deoxynucleotide transferase dUTP nick end labeling-positive cells in lung sections. LPS exposure resulted in a greater lung wet-to-dry weight ratio (W/D) in WT mice, suggestive of pulmonary edema, while in LPS-treated Ptgs2-/- mice, the W/D was not different from controls and less than in LPS-treated WT mice. These results demonstrate that COX-2 is involved in the inflammatory response to LPS and suggest that COX-2 not only acts as a downstream participant in the inflammatory response, but also acts as a regulator of the inflammatory response likely through a feed-forward mechanism following LPS stimulation.

Higher-Dose DHA Supplementation Modulates Immune Responses in Pregnancy and Is Associated with Decreased Preterm Birth.

Pregnancy and parturition involve extensive changes in the maternal immune system. In our randomized, multi-site, double-blind superiority trial using a Bayesian adaptive design, we demonstrated that 1000 mg/day of docosahexaenoic acid (DHA) was superior to 200 mg/day in preventing both early preterm birth (less than 34 weeks' gestation) and preterm birth (less than 37 weeks' gestation). The goal of this secondary study is to compare the effects of 1000 mg/day versus 200 mg/day on maternal inflammation, a possible mechanism by which DHA may prevent preterm birth. Maternal blood samples were collected at enrollment (12-20 weeks' gestation) and at delivery. Red blood cell DHA levels were measured by gas chromatography, and plasma concentrations of sRAGE, IL-6, IL-1ß, TNFα, and INFγ were measured by ELISA. Data were analyzed for associations with the DHA dose, gestational age at birth, and preterm birth (<37 weeks). Higher baseline and lower delivery levels of maternal sRAGE were associated with a greater probability of longer gestation and delivery at term gestation. Higher-dose DHA supplementation increased the probability of a smaller decrease in delivery sRAGE levels. Higher IL-6 concentrations at delivery were associated with the probability of delivering after 37 weeks, and higher-dose DHA supplementation increased the probability of greater increases in IL-6 concentrations between enrollment and delivery. These data provide a proposed mechanistic explanation of how a higher dose of DHA during pregnancy provides immunomodulatory regulation in the initiation of parturition by influencing sRAGE and IL-6 levels, which may explain its ability to reduce the risk of preterm birth.

Oxidative Stress Promotes Corticosteroid Insensitivity in Asthma and COPD.

Corticosteroid insensitivity is a key characteristic of patients with severe asthma and COPD. These individuals experience greater pulmonary oxidative stress and inflammation, which contribute to diminished lung function and frequent exacerbations despite the often and prolonged use of systemic, high dose corticosteroids. Reactive oxygen and nitrogen species (RONS) promote corticosteroid insensitivity by disrupting glucocorticoid receptor (GR) signaling, leading to the sustained activation of pro-inflammatory pathways in immune and airway structural cells. Studies in asthma and COPD models suggest that corticosteroids need a balanced redox environment to be effective and to reduce airway inflammation. In this review, we discuss how oxidative stress contributes to corticosteroid insensitivity and the importance of optimizing endogenous antioxidant responses to enhance corticosteroid sensitivity. Future studies should aim to identify how antioxidant-based therapies can complement corticosteroids to reduce the need for prolonged high dose regimens in patients with severe asthma and COPD.

Differences in clinical and laboratory biomarkers for short and long-term respiratory outcomes in preterm neonates.

BACKGROUND: Pulmonary outcome of premature neonates has focused more on short-term than long-term respiratory morbidities. OBJECTIVE: Describe risk factors/biomarkers associated with short-term (bronchopulmonary dysplasia [BPD]) (supplemental oxygen use at 36 weeks postmenstrual age [PMA]) and longer-term (chronic respiratory morbidity [CRM]) (respiratory related symptoms, medications, medical/emergency visits, hospitalizations at 6-12 months corrected gestational age [CGA]) respiratory outcomes in a longitudinal cohort. DESIGN/METHODS: Neonates born at 24-29-week gestation were prospectively followed to 6-12-month CGA. Associations between clinical and laboratory risk factors/biomarkers of BPD and CRM were explored. RESULTS: Of 86 subjects, 94% survived. Outcomes were available for 89% at 36-week PMA (BPD present in 42% of infants) and 72% at 6-12-month CGA (CRM present in 47% of infants). For the 54 infants with known outcomes for both BPD and CRM, diagnoses were discordant in 41%. BPD was associated with lower birthweight and birthweight Z-score for GA, lower Apgar scores, more surfactant doses, higher SNAPPE-II scores, highest Day 1 inspired oxygen concentration, Day 7 oxygen use, prolonged ventilatory support, bacteremia, necrotizing enterocolitis, and treated patent ductus arteriosus. CRM was associated with lower Apgar scores, Day 7 oxygen use and higher urine vascular endothelial growth factor. Patterns of plasma and urine lipid oxidation products differed in the two outcomes. CONCLUSION: In this hypothesis generating and exploratory study, BPD and CRM were associated with different risk factors/biomarker patterns. Concordance between these two outcomes was weak. Strategies for reducing CRM should be studied in cohorts identified by appropriate early risk factors/biomarkers.

Higher dose docosahexaenoic acid supplementation during pregnancy and early preterm birth: A randomised, double-blind, adaptive-design superiority trial.

BACKGROUND: Several meta analyses have concluded n-3 fatty acids, including docosahexaenoic acid (DHA), reduce early preterm birth (EPB, < 34 weeks), however, the amount of DHA required is unclear. We hypothesized that 1000 mg DHA per day would be superior to 200 mg, the amount in most prenatal supplements. METHODS: This randomised, multicentre, double-blind, adaptive-design, superiority trial was conducted in three USA medical centres. Women with singleton pregnancies and 12 to 20 weeks gestation were eligible. randomization was generated in SAS® by site in blocks of 4. The planned adaptive design periodically generated allocation ratios favoring the better performing dose. Managing study personnel were blind to treatment until 30 days after the last birth. The primary outcome was EPB by dose and by enrolment DHA status (low/high). Bayesian posterior probabilities (pp) were determined for planned efficacy and safety outcomes using intention-to-treat. The study is registered with ClinicalTrials.gov (NCT02626299) and closed to enrolment. FINDINGS: Eleven hundred participants (1000 mg, n = 576; 200 mg, n = 524) were enrolled between June 8, 2016 and March 13, 2020 with the last birth September 5, 2020. 1032 (n = 540 and n = 492) were included in the primary analyses. The higher dose had a lower EPB rate [1.7% (9/540) vs 2.4% (12/492), pp=0.81] especially if participants had low DHA status at enrolment [2.0% (5/249) vs 4.1%, (9/219), pp=0.93]. Participants with high enrolment DHA status did not realize a dose effect [1000 mg: 1.4% (4/289); 200 mg: 1.1% (3/271), pp = 0.57]. The higher dose was associated with fewer serious adverse events (maternal: chorioamnionitis, premature rupture of membranes and pyelonephritis; neonatal: feeding, genitourinary and neurologic problems, all pp>0.90). INTERPRETATION: Clinicians could consider prescribing 1000 mg DHA daily during pregnancy to reduce EPB in women with low DHA status if they are able to screen for DHA. FUNDING: The National Institutes of Health Child Health and Human Development (NICHD) funded the study. Life's DHA™-S oil, DSM Nutritional Products LLC, Switzerland provided all capsules.

Auranofin-Mediated NRF2 Induction Attenuates Interleukin 1 Beta Expression in Alveolar Macrophages.

BACKGROUND: Alveolar macrophages (AMs) are resident inflammatory cells in the lung that serve as early sentinels of infection or injury. We have identified thioredoxin reductase 1 inhibition by gold compounds increases activation of nuclear factor erythroid 2-related factor 2 (NRF2)-dependent pathways to attenuate inflammatory responses. The present studies utilized murine alveolar macrophages (MH-S) to test the hypothesis that the gold compound, auranofin (AFN), decreases interleukin (IL)-1ß expression through NRF2-mediated interactions with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway genes and/or increases in glutathione synthesis. METHODS: MH-S cells were treated with AFN and lipopolysaccharide (LPS) and analyzed at 6 and 24 h. The Il1b promoter was analyzed by chromatin immunoprecipitation for direct interaction with NRF2. RESULTS: Expression of IL-1ß, p-IκBα, p-p65 NF-kB, and NOD-, LRR-, and pyrin domain-containing protein 3 were elevated by LPS exposure, but only IL-1ß expression was suppressed by AFN treatment. Both AFN and LPS treatments increased cellular glutathione levels, but attenuation of glutathione synthesis by buthionine sulfoximine (BSO) did not alter expression of Il-1ß. Analysis revealed direct NRF2 binding to the Il1b promoter which was enhanced by AFN and inhibited the transcriptional activity of DNA polymerase II. CONCLUSIONS: Our data demonstrate that AFN-induced NRF2 activation directly suppresses IL-1ß synthesis independent of NFκB and glutathione-mediated antioxidant mechanisms. NRF2 binding to the promoter region of IL1ß directly inhibits transcription of the IL1ß gene. Collectively, our research suggests that gold compounds elicit NRF2-dependent pulmonary protection by suppressing macrophage-mediated inflammation.

MiR-29b is associated with perinatal inflammation in extremely preterm infants.

BACKGROUND: Inflammation is strongly associated with premature birth and neonatal morbidities. Increases in infant haptoglobin, haptoglobin-related protein (Hp&HpRP), and interleukin-6 (IL-6) levels are indicators of intra-amniotic inflammation (IAI) and have been linked to poor neonatal outcomes. Inflammation causes epigenetic changes, specifically suppression of miR-29 expression. The current study sought to determine whether miR-29b levels in cord blood or neonatal venous blood are associated with IAI, identified by elevated IL-6 and Hp, and subsequent clinical morbidities in the infant. METHODS: We tested 92 cord blood samples from premature newborns and 18 venous blood samples at 36 weeks corrected gestational age. MiR-29b, Hp&HpRP, and IL-6 were measured by polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: Decreased levels of miR-29b were observed in infants exposed to IAI with elevated Hp&HpRP and IL-6 levels and in infants delivered by spontaneous preterm birth. Lower miR-29 levels were also observed in women diagnosed with histological chorioamnionitis or funisitis and in infants with cerebral palsy. Higher levels of miR-29 were measured in infants small for gestational age and in venous samples from older infants. CONCLUSIONS: MiR-29 may be an additional biomarker of IAI and a potential therapeutic target for treating poor newborn outcomes resulting from antenatal exposure to IAI. IMPACT: Decreases in miR-29b are associated with intrauterine inflammation. Hp&HpRP increases are associated with decreased miR-29b. MiR-29b may be an additional biomarker for neonatal outcomes and a potential therapeutic target for intrauterine inflammation.

Glutathione reductase deficiency alters lung development and hyperoxic responses in neonatal mice.

Cellular antioxidants protect against hyperoxic lung injury. The role of the glutathione (GSH) system in lung development and bronchopulmonary dysplasia (BPD) pathogenesis has not been systematically investigated. The current study utilized GSH reductase-deficient (Gsr-KO) neonatal mice to test the hypothesis that early disruption of the GSH system negatively impacts lung development and hyperoxic responses. Lungs from wild-type (Gsr-WT) and Gsr-KO mice were analyzed for histopathology, developmental markers, redox indices, and transcriptome profiling at different developmental stages following exposure to room air or hyperoxia (85% O2) for up to 14 d. Lungs from Gsr-KO mice exhibited alveolar epithelial dysplasia in the embryonic and neonatal periods with relatively normal lung architecture in adulthood. GSH and its oxidized form (GSSG) were 50-70% lower at E19-PND14 in Gsr-KO lungs than in age-matched Gsr-WT. Differential gene expression between Gsr-WT and Gsr-KO lungs was analyzed at discrete developmental stages. Gsr-KO lungs exhibited downregulated cell cycle and DNA damage checkpoint genes at E19, as well as lung lipid metabolism and surfactant genes at PND5. In addition to abnormal baseline lung morphometry, Gsr-KO mice displayed a blunted response to hyperoxia. Hyperoxia caused a more robust upregulation of the lung thioredoxin system in Gsr-KO compared to Gsr-WT. Gsr-dependent, hyperoxia-responsive genes were highly associated with abnormal cytoskeleton, skeletal-muscular function, and tissue morphology at PND5. Overall, our data in Gsr-KO mice implicate the GSH system as a key regulator of lung development, cellular differentiation, and hyperoxic responses in neonatal mice.

Perinatal inflammation alters histone 3 and histone 4 methylation patterns: Effects of MiR-29b supplementation.

Preterm birth is still a major health problem and maternal inflammation has been shown to play a role. The combination of maternal inflammation and neonatal hyperoxia contributes to epigenetic changes that influence gene expression and the development of bronchopulmonary dysplasia (BPD). We have previously demonstrated suppression of miR-29b and increases in DNA methylation in infants with severe BPD and in our mouse model of maternal inflammation and neonatal hyperoxia exposure. The present studies further explored epigenetic changes in the murine model to include histone methylation. We identified a global suppression of histone methylation in exposed mice and validated decreases in expression in well-defined histone modifications, specifically H3K4me3, H3K27me3, H3K36me2, H3K79me2, and H4K20me3. We further tested the hypothesis that restoration of miR-29b expression would restore the histone methylation marks. Using lipid nanoparticle delivery of miR-29b, partial to full methylation was reestablished for H3K4me3, H3K27me3, and H4K20me3; all tri-methylation marks. To identify the causes of decreased methylation in exposed mice, we measured commonly identified methylases and demethylases. We found a decreased expression of SUV40H2, a methylase primarily associated with H4K20me3. Further studies are needed to identify the causes for the decreased global histone methylation and potential therapeutic opportunities.

Optimizing miR-29 measurements in biobanked, heparinized samples.

AIMS: MicroRNAs (miRs) and their importance in development, normal physiology, and disease have become increasingly recognized. Our laboratory is interested in miR-29 and its effects on lung development. These studies set out to identify optimal conditions for the measurement of miR-29 in heparinized, biobanked samples and to compare isoform expression patterns. MATERIALS AND METHODS: The efficiency of three distinct heparinases were tested using reverse transcriptase polymerase chain reaction (RT-PCR): recombinant F. Heparinum heparinase I; recombinant P. heparinus heparinase II; recombinant P. heparinus heparinase III; and heparinase I (B. efferthii-derived). The effects of freeze/thaws, and the relative expression of different miR-29 isoforms were also assessed using RT-PCR. KEY FINDINGS: Our investigations determined that heparinase 1 (recombinant F. Heparinum) and 2 (recombinant P. heparinus) at 1 or 2 h incubation efficiently neutralized heparin activity and prevented interference with the PCR. Also, a single freeze/thaw did not affect the measurement of miR-29-3p but multiple freeze/thaw cycles decreased the measureable miR levels. Finally, the -3p strand was most abundantly expressed in all three isoforms in both human and mouse plasma. SIGNIFICANCE: Our findings illustrate that specific conditions need to be optimized for the particular miR and the type of sample being tested.

Glutathione Reductase Promotes Fungal Clearance and Suppresses Inflammation during Systemic Candida albicans Infection in Mice.

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in redox regulation. We have previously shown that Gsr facilitates neutrophil bactericidal activities and is pivotal for host defense against bacterial pathogens. However, it is unclear whether Gsr is required for immune defense against fungal pathogens. It is also unclear whether Gsr plays a role in immunological functions outside of neutrophils during immune defense. In this study, we report that Gsr-/- mice exhibited markedly increased susceptibility to Candida albicans challenge. Upon C. albicans infection, Gsr-/- mice exhibited dramatically increased fungal burden in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration, histological abnormalities in both the kidneys and heart, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of the glomeruli in the kidneys of Gsr -/- mice but were not found in wild-type mice. Examination of the neutrophils and macrophages of Gsr-/- mice revealed several defects. Gsr -/- neutrophils exhibited compromised phagocytosis, attenuated respiratory burst, and impaired fungicidal activity in vitro. Moreover, upon C. albicans stimulation, Gsr -/- macrophages produced increased levels of inflammatory cytokines and exhibited elevated p38 and JNK activities, at least in part, because of lower MAPK phosphatase (Mkp)-1 activity and greater Syk activity. Thus, Gsr-mediated redox regulation is crucial for fungal clearance by neutrophils and the proper control of the inflammatory response by macrophages during host defense against fungal challenge.

Cervical and systemic concentrations of long acting hormonal contraceptive (LARC) progestins depend on delivery method: Implications for the study of HIV transmission.

Progestin-only long-acting reversible contraceptives (LARCs) are increasingly popular among women seeking contraception; however, recent epidemiological studies suggest that systemically administered medroxyprogesterone acetate (MPA) may increase HIV acquisition. In order to determine the exact mechanisms underlying increases in transmission specific to MPA use and to test safer, alternative contraceptive progestin types and delivery methods, in vitro modeling studies must be performed. To achieve this, it is imperative that accurate hormone concentrations be utilized when modeling progestin-mediated outcomes, as the down-stream effects are dose-dependent. The local concentrations of progestins to which the lower female genital tract tissues are exposed after initiation of LARCs are unknown, but they likely differ from peripheral concentrations, dependent upon the progestin type and delivery method. Here, we measured in vivo endocervical and plasma concentrations of (1) systemically-delivered depo MPA (DMPA), (2) levonorgestrel (LNG) delivered via intrauterine system (IUS) and (3) etonogestrel (ETG) delivered via vaginal ring in women who recently initiated contraception treatment. Levels of ETG and LNG in cervical secretions were 100-200 fold higher than plasma levels. In contrast, measurable MPA levels were approximately 10-fold higher in plasma compared to cervical secretions. These results will inform the design of accurate in vitro studies on the influence of progestins on epithelial cells, tissue explants, and peripheral blood cells, to be able to better predict in vivo outcomes. Subsequent observations will aid in determining how MPA might influence HIV acquisition and may facilitate identification of optimal progestin-containing LARC alternatives for women at high risk for HIV infection.