Maternal oxidative stress during pregnancy associated with emotional and behavioural problems in early childhood: implications for foetal programming.

Childhood mental disorders, including emotional and behavioural problems (EBP) are increasingly prevalent. Higher maternal oxidative stress (OS) during pregnancy (matOSpreg) is linked to offspring mental disorders. Environmental factors contribute to matOSpreg. However, the role of matOSpreg in childhood EBP is unclear. We investigated the associations between (i) matOSpreg and offspring EBP; (ii) social and prenatal environmental factors and matOSpreg; and (iii) social and prenatal factors and childhood EBP and evaluated whether matOSpreg mediated these associations. Maternal urinary OS biomarkers, 8-hydroxyguanosine (8-OHGua; an oxidative RNA damage marker) and 8-hydroxy-2'-deoxyguanosine (8-OHdG; an oxidative DNA damage marker), at 36 weeks of pregnancy were quantified by liquid chromatography-mass spectrometry in a population-derived birth cohort, Barwon Infant Study (n = 1074 mother-infant pairs). Social and prenatal environmental factors were collected by mother-reported questionnaires. Offspring total EBP was measured by Child Behavior Checklist Total Problems T-scores at age two (n = 675) and Strengths and Difficulties Questionnaire Total Difficulties score at age four (n = 791). Prospective associations were examined by multivariable regression analyses adjusted for covariates. Mediation effects were evaluated using counterfactual-based mediation analysis. Higher maternal urinary 8-OHGua at 36 weeks (mat8-OHGua36w) was associated with greater offspring total EBP at age four (ß = 0.38, 95% CI (0.07, 0.69), P = 0.02) and age two (ß = 0.62, 95% CI (-0.06, 1.30), P = 0.07). Weaker evidence of association was detected for 8-OHdG. Five early-life factors were associated with both mat8-OHGua36w and childhood EBP (P-range < 0.001-0.05), including lower maternal education, socioeconomic disadvantage and prenatal tobacco smoking. These risk factor-childhood EBP associations were partly mediated by higher mat8-OHGua36w (P-range = 0.01-0.05). Higher matOSpreg, particularly oxidant RNA damage, is associated with later offspring EBP. Effects of some social and prenatal lifestyle factors on childhood EBP were partly mediated by matOSpreg. Future studies are warranted to further elucidate the role of early-life oxidant damage in childhood EBP.

Early life environmental factors associated with autism spectrum disorder symptoms in children at age 2 years: A birth cohort study.

LAY ABSTRACT: Mounting evidence indicates the contribution of early life environmental factors in autism spectrum disorder. We aim to report the prospective associations between early life environmental factors and autism spectrum disorder symptoms in children at the age of 2 years in a population-derived birth cohort, the Barwon Infant Study. Autism spectrum disorder symptoms at the age of 2 years strongly predicted autism spectrum disorder diagnosis by the age of 4 years (area under curve = 0.93; 95% CI (0.82, 1.00)). After adjusting for child's sex and age at the time of behavioural assessment, markers of socioeconomic disadvantage, such as lower household income and lone parental status; maternal health factors, including younger maternal age, maternal pre-pregnancy body mass index, higher gestational weight gain and prenatal maternal stress; maternal lifestyle factors, such as prenatal alcohol and environmental air pollutant exposures, including particulate matter < 2.5 µm at birth, child secondhand tobacco smoke at 12 months, dampness/mould and home heating with oil, kerosene or diesel heaters at 2 years postnatal. Lower socioeconomic indexes for area, later birth order, higher maternal prenatal depression and maternal smoking frequency had a dose-response relationship with autism spectrum disorder symptoms. Future studies on environmental factors and autism spectrum disorder should consider the reasons for the socioeconomic disparity and the combined impact of multiple environmental factors through common mechanistic pathways.

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis.

Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

Daily Oral Administration of the Novel Androgen 11ß-MNTDC Markedly Suppresses Serum Gonadotropins in Healthy Men.

BACKGROUND: 11ß-methyl-19-nortestosterone (11ß-MNT) is a modified testosterone (T) with androgenic and progestational activity. A single oral dose of the prodrug, 11ß-MNT dodecylcarbonate (11ß-MNTDC), was well tolerated in healthy men. METHODS: We conducted a randomized, double-blind study at 2 academic medical centers. 42 healthy men (18-50 years) were randomized to receive oral placebo or 11ß-MNTDC, 200 or 400 mg daily, for 28 consecutive days. Primary outcome (safety and tolerability) measures were assessed twice per week. Subjects underwent serial blood sampling over 24 hours on days 1 and 28 to assess secondary outcomes: pharmacokinetics (serum drug concentrations); pharmacodynamics of 11ß-MNTDC (serum sex steroids and gonadotropins); and mood and sexual function (via validated questionnaires). RESULTS: There were no serious adverse events. No participants discontinued because of an adverse event or laboratory test abnormality. 11ß-MNTDC resulted in a dose-related increase in serum 11ß-MNTDC and 11ß-MNT concentrations sustained over 24 hours. Administration of 11ß-MNTDC resulted in a marked suppression of serum gonadotropins, T, calculated free T, estradiol, and SHBG over the treatment period (P < 0.01). Adverse effects that may be related to 11ß-MNTDC included weight gain, acne, headaches, fatigue, and mild mood changes, with 5 men reporting decreased libido and 3 decreased erectile/ejaculatory function. Serum low-density lipoprotein cholesterol, weight (~2 kg), hematocrit, and hemoglobin increased and serum high-density lipoprotein cholesterol decreased in both 11ß-MNTDC groups. CONCLUSION: Daily oral 11ß-MNTDC for 28 days in healthy men markedly suppressed serum gonadotropin and T concentrations without serious adverse effects. These results warrant further evaluation of 11ß-MNTDC as a potential male oral contraceptive.

Terminal Hydride Species in [FeFe]-Hydrogenases Are Vibrationally Coupled to the Active Site Environment.

A combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calculations was used to observe and characterize Fe-H/D bending modes in CrHydA1 [FeFe]-hydrogenase Cys-to-Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H-cluster from a -SH to -OH, thus altering the proton transfer pathway. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H-cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe-H/D normal modes. We observed a significant shift to higher frequency in an Fe-H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)-substituted CrHydA1. On the basis of DFT calculations, we propose that this shift is caused by the stronger interaction of the -OH group of C169S with the bridgehead -NH- moiety of the active site, as compared to that of the -SH group of C169 in the native enzyme.

Reaction Coordinate Leading to H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory.

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (Hhyd) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, and the Fe-H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that Hhyd is the catalytic state one step prior to H2 formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H2 bond formation by [FeFe]-hydrogenases.

Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy.

[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site ("H-cluster") consists of a [4Fe-4S]H cluster linked through a bridging cysteine to a [2Fe]H subsite coordinated by CN- and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fed). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe-H species fully consistent with widely accepted models of the catalytic cycle.

Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity.

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2',7'-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H-Cluster.

Three maturase enzymes-HydE, HydF, and HydG-synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)](-) species is a key intermediate in H-cluster maturation.

Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster.

Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-L-methionine family of enzymes that transforms Fe and L-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the "dangler" Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that L-cysteine (Cys) binds the auxiliary [4Fe-4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe-4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release.