Sequelae of prematurity triggered by oxidative stress and free radical-mediated tissue damage have coined the term “oxygen radical disease of prematurity”. Caffeine, a potent free radical scavenger and adenosine receptor antagonist, reduces rates of brain damage in preterm infants. In the present study, we investigated the effects of caffeine on oxidative stress markers, anti-oxidative response, inflammation, redox-sensitive transcription factors, apoptosis, and extracellular matrix following the induction of hyperoxia in neonatal rats. The brain of a rat pups at postnatal Day 6 (P6) corresponds to that of a human fetal brain at 28–32 weeks gestation and the neonatal rat is an ideal model in which to investigate effects of oxidative stress and neuroprotection of caffeine on the developing brain. Six-day-old Wistar rats were pre-treated with caffeine and exposed to 80% oxygen for 24 and 48 h. Caffeine reduced oxidative stress marker ), promoted anti-oxidative response , down-regulated pro-inflammatory cytokines, modulated redox-sensitive transcription factor expression , reduced pro-apoptotic effectors (poly (ADP-ribose) polymerase-1 (PARP-1), apoptosis inducing factor (AIF), and caspase-3), and diminished extracellular matrix degeneration  2, and inhibitor of metalloproteinase (TIMP) 1/2). Our study affirms that caffeine is a pleiotropic neuroprotective drug in the developing brain due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Advances in neonatal intensive care have led to a significant increase in the survival rate of premature infants, but extremely premature infants have a higher risk of dying or suffering permanent and serious damage ,2. Up toThere are several reasons for the high susceptibility of preterm infants to oxidative damage: (i) birth is associated with a dramatic change of intrauterine hypoxic milieu to a relatively hyperoxic extrauterine environment, and this relative hyperoxia can be enhanced by supplemental oxygen ,8; (ii) Oxidative stress can be defined as an imbalance between the amount of reactive oxygen species (ROS) and the intracellular and extracellular antioxidant protection systems. The antioxidative defense system undergoes developmental changes during the neonatal period, resulting in a relevantly lower intracellular defense in preterm infants compared to term infants .In addition to the understanding of the pathology of oxidative stress and the associated effects on the development of premature infants, additional strategies must be developed. Recent studies have proposed that caffeine presents antioxidant activity and therefore, protects human against disorders associated with oxidative stress ,12. The Up to date, it is not yet clarified whether caffeine can also act as a free radical scavenger. Shi et al. reported that caffeine may act as an antioxidant scavenger, thus explaining the observed anticarcinogenic properties of caffeine and related methylxanthine compounds . Furtherp < 0.01) compared to litter control mates kept in atmospheric air (p < 0.001). In control animals, caffeine did not affect TBARS levels. Changes in TBARS after 48 h hyperoxia were not significant in comparison to matched normoxic litters. Interestingly, there was also a significant decrease in TBARS in normoxic newborn rats 48 h after the single administration of caffeine .Thiobarbituric acid reactive substances (TBARS) were increased in brain tissue of newborn rats exposed to 24 h of hyperoxia to 180% ± 27.4% (eric air A. This wp < 0.001) which was blocked by caffeine . Treatment with caffeine in animals kept under normoxic conditions had no effect on hydrogen peroxide concentrations.To confirm the induction of oxidative stress by exposure to hyperoxia, we analyzed hydrogen peroxide concentrations through enzyme linked immunosorbent assay (ELISA) measurement in neonatal rodent brains after hyperoxia and normoxia with and without caffeine treatment B. Hyperop < 0.001) compared with brain tissue from rats under normoxia (p < 0.001). HO-1 is not increased after 24 h hyperoxia. However, caffeine treatment reduced HO-1 levels after 48 h in control litters kept in room air .Heme oxigenase-1 (HO-1) protein levels were markedly increased in brain tissue after 48 h of hyperoxic stimulation  (p < 0.01) and after 48 h  of hyperoxia, which was blocked by caffeine . Contrary to Nrf2, gene expression of Kelch-like ECH-associated protein 1 . Caffeine did not influence the expression of Keap1 at atmospheric air.NFE2-related factor 2 (Nrf2) D was sig1 Keap1; E was notGCLC (p < 0.001) and after 48 h  of hyperoxia, which were significantly reduced by caffeine . Single treatment with caffeine in animals kept under normoxic conditions had no effect on GCLC expression.The glutamate-cysteine ligase (GCL) consists of two separate coded subunits, a catalytic (GCLC) and a modifier (GCLM), which catalyzes the rate-limiting phase of cellular antioxidant glutathione (GSH). GCLC F mRNA exSOD3). SOD1 (SOD2 (SOD3 (p < 0.05) at 24 h. In comparison to hyperoxia without caffeine, we found significant increases for SOD1 to 111% ± 14.4% (p < 0.05) and 129% ± 18.5% (p < 0.01), for SOD3 to 159% ± 13.1% (p < 0.001) and 171% ± 19.3% (p < 0.001) at 24 h and 48 h, respectively, and for SOD2 to 217% ± 25.5% (p < 0.001) at 48 h. Under 48 h normoxic conditions, a single administration of caffeine leads to an increase of SOD2  and SOD3 . Correspondingly, increasing peroxiredoxin (Prx) 1 and sulfiredoxin (Srx) 1 expression were observed at both time points. Prx1 protein expression (p < 0.001) at 24 h and to 145% ± 6.2% (p < 0.001) at 48 h, and Srx1 protein expression (p < 0.001) and 150% ± 9.3% (p < 0.001), respectively. The administration of caffeine showed a significant decrease to 109% ± 6.2% (p < 0.01) and 75% ± 5.9% (p < 0.001) for Prx1, and to 99% ± 8.7% (p < 0.001) and 104% ± 9.3% (p < 0.001) for Srx1 to both times examined. Interestingly, there was a reduction in Prx1 at 48 h under normoxia to 56% ± 2.6% (p < 0.001) and an increase of protein expression after 24 h under normoxia to 126% ± 3.6% (p < 0.01).Acute exposure to high oxygen leads to exhaustion of superoxide dismutases 3 (3). SOD1 A does reD1  in the brain. Using qPCR for gene expression and ELISA for protein expression analysis, we determined levels of tumor necrosis factor α (TNFα), interleukin (IL)-1β, interferon γ (IFNγ), and IL-18 as pro-inflammatory cytokines, IL-12 as early pro- and late anti-inflammatory cytokine, and iNOS. Overall, significant differences between normoxia control group and the hyperoxia group were detected for all cytokines at 24 and/or 48 h .iNOS (p < 0.01), and significantly decreased at 48 h , which was blocked by caffeine at 24 h .Oxidative stress is usually thought to be responsible for tissue injury associated with a range of brain injury, inflammation, and degenerative processes. Moreover, inflammatory target proteins, including iNOS, are associated with oxidative stress induced by pro-inflammatory factors such as cytokines. Expression of iNOS A was sigTNFα mRNA  was found after 24 h of hyperoxia (p < 0.001). The administration of caffeine drastically reduced TNFα mRNA expression under hyperoxic conditions at 24 h  and at 48 h , which was also observed under normoxic conditions . On translational protein levels, caffeine caused a marked reduction in the expression of TNFα protein at 48 h . However, TNFα protein was not affected by caffeine in newborn rats kept at atmospheric air  and 48 h  of hyperoxia exposure. A single dose of caffeine diminished this expression at both time points. At 24 and 48 h, there is a significant reduction in protein expression to 363% ± 24.1% (p < 0.001) and to 150% ± 18.5% (p < 0.05), respectively. Similarly, caffeine affects under hyperoxia IFNγ on the mRNA level with an attenuation to 83% ± 8.3% (p < 0.001) after 24 h and 96% ± 6.8% (p < 0.001) after 48 h. Remarkably, administration of caffeine in control animals showed a high increase of the IFNγ protein expression at 24 h  compared to untreated control litters, whereas no changes were observed at the mRNA level.In relation to the cytokines measured in these experiments, IFNγ showed the largest changes in mRNA and protein levels in the hyperoxic brain D,E. A sip < 0.001) as compared to controls (p < 0.001). Caffeine administration alone under normoxia also showed a dramatic increase in the protein level after 24 h . The mRNA expression significantly decreased during normoxia and caffeine administration after 24 h . Hyperoxia exposure resulted in a non-significant increase (122% ± 7.4%) at 24 h, and a significant increase after 48 h hyperoxia exposure . Here also, mRNA expression under hyperoxia is significantly reduced below the normoxic level by caffeine administration at 24 h  and 48 h .As a further indication of pro-inflammatory responses, 24 h hyperoxia caused a large increase of IL-1β protein expression  and 48 h to 150% ± 8.6% (p < 0.01) compared with rat pups exposed to normoxia (p < 0.001) and to 55% ± 12.6% (p < 0.001), respectively. Caffeine under non-hyperoxic conditions resulted in a significant reduction in IL-18 expression at 48 h .Exposure to hyperoxia increased the concentration of normoxia H. The apIL-12 mRNA expression  is detected after 24 and 48 h of hyperoxia, respectively. Under hyperoxia exposure with prior application of caffeine, the expression increased significantly at 24 h at 66% ± 3.6% (p < 0.05) and at 48 h at 244% ± 21.5% (p < 0.001). In contrast, the expression of IL-12 under normoxia with caffeine was reduced after 24 h  and increased after 48 h .Interestingly, a drastic reduction in p < 0.001, and 204% ± 36.7%; p < 0.001, respectively) and at 48 h  in the cytosol compared to the control group. There were no changes under hyperoxic exposure alone, while the alteration in protein level in the nucleus indicated an up-regulation of NFκB under hyperoxic condition at both time points . In the nuclear protein fraction, a single dose of caffeine significantly reduced NFκB expression at 24 h to 114% ± 20.8% (p < 0.001), and at 48 h to 74% ± 4.1% (p < 0.001).As shown in NFκB1 and NFκB2 corresponded with the cytosolic protein data (NFκB forms (NFκB1 and 2) at both time points. Caffeine with hyperoxia exposure always led to an increase in mRNA expression (NFκB1 at 24 h from 75% ± 6.5% to 154% ± 11.3% (p < 0.001), and at 48 h from 68% ± 6.1% to 115% ± 4.7% (p < 0.01); NFκB2 at 24 h from 72% ± 4.0% to 114% ± 12.1% (p < 0.01), and at 48 h from 38% ± 3.5% to 79% ± 7.7% (p < 0.01)). NFκB1 expression was bolstered by caffeine under normoxia after 24 h .The mRNA expression ratios of ein data B,C. Herep < 0.001; 48 h with 188% ± 14.5%; p < 0.001) and no changes observed in cytosolic fraction. Caffeine reduced this increase to the normoxic level (p < 0.001). High oxygen also induced cytosolic apoptosis inducing factor (AIF) protein at 24 h (p < 0.001), increased caspase-3 cleavage (p < 0.001) and at 48 h , and enhanced caspase-3 mRNA expression at 24 h (p < 0.001). For the targets investigated, the application of caffeine resulted in a significant reduction of protein and/or mRNA expression (AIF at 24 h from 145% ± 4.9% to 103% ± 4.6% (p < 0.001); cleaved caspase-3 at 24 h from 143% ± 12.3% to 91% ± 5.6% (p < 0.001), and at 48 h from 133% ± 6.0% to 109% ± 2.0% (p < 0.05); caspase-3 mRNA expression at 24 h from 159% ± 9.0% to 108% ± 3.7% (p < 0.001), and at 48 h from to 74% ± 2.6% (p < 0.05)).We measured the cytosolic and nuclear expression of cleaved poly (ADP-ribose) polymerase-1 (PARP-1) A. Hyperocleavage C at 24 hp < 0.001). After 48 h hyperoxia pro-MMP2 was increased  and significantly decreased with caffeine application . Caffeine alone under normoxic exposure showed a higher level on pro-MMP2 after 24 h  and a lower level after 48 h . At both time points acute hyperoxia exposure led to a drastic increase of active MMP2 , which was again mitigated by caffeine . Caffeine application at normoxia reduced the processing of active MMP2 . Contrary to MMP2, no active MMP9 band was detected in any samples.Gel gelatin zymography was used to investigate the effect of oxidative stress and caffeine on the gelatinases expressions A. In theTIMP1: 54% ± 6.5%; p < 0.001; TIMP2: 68% ± 7.9%; p < 0.01) and 48 h . Caffeine increased the mRNA expression of both inhibitors only at 48 h . Caffeine alone had no significant influence. The tissue plasminogen activator (tPa) generates the active protease, plasmin, which is capable of degrading numerous substrates. Here  and a single dose of caffeine reduced tPa to normoxia level .The inhibitors of these metalloproteinases, especially tissue inhibitor of metalloproteinase (TIMP) 1/2 B,C, are es. Here D, hyperotransforming growth factor-(TGF)-β (p < 0.001) of hyperoxia. Caffeine strongly reduced TGF-β mRNA under normoxia  as compared to control, as well as hyperoxic conditions compared to the hyperoxic group without caffeine  at both time points.The gene expression of -(TGF)-β E is highNeuronal injury and neurological maldevelopment in preterm infants is commonly ascribed to perinatal infection/inflammation and to oxidative stress hitting the immature brain at a vulnerable phase of development. In our neonatal rat model, oxidative stress is induced by early exposure to high oxygen levels for 24 and 48 h, in which neural cell injury has been shown to occur in a way that is comparable to the human neonatal situation .In preterm infants, caffeine is used to stimulate breathing activity and prevent the onset of apnoea . Based oOxidative stress is caused by excess of free radical formation and/or an overproduction of ROS, and is a major factor of injury in the developing brain leading to neurological sequelae of preterm birth ,34. In oThe increased HO-1 expression suggests an activation of protective mechanisms in response to unwanted cellular oxidative conditions . Anti-oxNrf2 mRNA increase confirms the induction of antioxidant genes under hyperoxic conditions while the expression of Keap1 is unchanged.Nrf2 is a redox-sensitive transcription factor that mediates protection against oxidative stress via the transcriptional activation of several antioxidant enzymes through the antioxidant response element (ARE). Nrf2 is negatively regulated by Keap1 thereby providing inducible antioxidant defense. Under basal conditions Nrf2 is regulated by Keap1, but under oxidative stress, the Nrf2 pathways can also be regulated independently of Keap1 . The expGlutathione (GSH) plays an important role for antioxidant defense and for the regulation of intracellular redox homeostasis. The rate-limiting step of cellular antioxidant GSH is catalyzed by glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit . GCLC isPeroxiredoxin (Prx) and sulfiredoxin (Srx) are important proteins of the thioredoxin family and a key regulator of genes involved in oxidant defense, such as SOD3, Prx1, Srx1, and HO-1, and redox signaling. It is known that non-physiological oxygen concentrations change the balance of the Srx/Prx system in the developing brain . Prx and2O2), and in the relevant context the expression of HO-1 and Nrf2. In the second line, caffeine improved SOD RNA expression and suppressed Prx1 and Srx1 protein expressions. These results point to a broad interaction of caffeine with the antioxidant defense network. The reduced SOD expression under hyperoxic conditions could be explained by an exhaustion of the anti-oxidant system which was successfully counteracted by caffeine. Ahotupa et al. showed an initial decrease of SOD in hyperoxic rat brains, followed by a slight induction of its activity at a later time point [An antioxidant activity of caffeine has been assumed in recent studies in which neuroprotection was demonstrated in patients suffering from neurodegenerative disorders ,12. In ome point . AltogetiNOS, IL-1β, and IL-18 were induced. An interaction of oxidative stress and inflammatory responses has previously been described [Dependent on the level of generated ROS, different redox-sensitive transcription factors are activated. Moderate oxidative stress is related to Nrf2 activation and intermediate amount of ROS initiates an inflammatory response through activation of NFκB pathway . Our resescribed . The proescribed  and is aescribed . Excessiescribed . Oxidatiescribed . Activatescribed  demonstrescribed .IL-12 mRNA expression was dramatically reduced under hyperoxia, which was blocked by caffeine. IL-12 is produced by microglia [SOD2/3 and protein expression of Srx1 and pro-inflammatory cytokines (IL-1β and IFNγ) increased after caffeine administration at normoxia. Leon-Carmona and Galano disclosed in their functional studies that caffeine may act as radical scavenger [In our study, caffeine significantly reduced inflammatory responses. Surprisingly, icroglia , and proicroglia ,59. It hicroglia . Caffeincavenger . Caffeincavenger . In micecavenger . Cytokincavenger ,65. Chavcavenger .High levels of ROS have been shown to induce apoptosis in the immature brain after hyperoxia . In our tPa transcript expression as found in our study, thus leading to less processing of active MMP. TGF-β is a multifunctional cytokine that regulates a broad diversity of physiological and pathological processes. Plasmin releases latent forms of growth factors such as TGF-β [TIMP mRNA expression, and active MMP2 and MMP9 were reduced by protective treatment with erythropoietin [Free radicals and ROS seem to activate matrix metalloproteinases (MMPs), with the biological function to degrade extracellular matrix of cerebral blood vessels and neurons , and actas TGF-β . TGF-β eas TGF-β , so caffopoietin .For the clinical situation, it is important to precisely define the effects of caffeine on the otherwise healthy brain in order to avoid harm in preterm infants. The increase of apoptotic factors in normoxic newborn pups coincides with higher activity of MMP2 and also with increased NFκB protein expression in these animals. Since the injection of caffeine was performed 15 min before the beginning of hyperoxia exposure, it is possible that a certain degree of cellular stimulation is caused by caffeine that functions as a protective preconditioning in those animals challenged by hyperoxia later on. Preconditioning has been characterized as a protective procedure in brain injury models involving inflammatory stimulation , ischemiWistar rats from time-pregnant dams were obtained from Charité-Universitätsmedizin Berlin (Germany) and randomly assigned to cages and treatment. Animals were housed under controlled temperature and light conditions with food and water ad libitum. Experimental procedure and caffeine administration were carried out as described previously [All procedures were approved by the state animal welfare authorities (LAGeSo G-0307/09) and followed institutional guidelines. Six-day-old eviously . The ratAfter 24 h (P7) or 48 h (P8) of exposure, rats were transcardially perfused with normal saline (pH 7.4) under anesthesia (i.p.) of ketamine (50 mg/kg), xylazine (10 mg/kg), and acepromazine (2 mg/kg), then decapitated, the olfactory bulb and cerebellum were removed, and brain hemispheres were snap-frozen in liquid nitrogen and stored at −80 °C.g (4 °C) for 10 min, the microsomal fraction was subsequently centrifuged at 17,000× g (4 °C) for 20 min, and stored at −80 °C until further analysis. After collecting the supernatant, protein concentrations were determined using the Pierce BCA kit  with 30 min incubation at 37 °C prior to spectrophotometry at 562 nm.Protein was extracted as described in ,44. Briev/v) horse serum in Tris-buffered saline /0.1% (v/v) Tween 20 (TBST); AIF, cleaved Casp3, NFκB, and PARP-1 with 5% (w/v) bovine serum albumin  in TBST). Equal loading and transfer of proteins was confirmed by staining the membranes with Ponceau S solution . The membranes were incubated overnight at 4 °C with the following antibodies: rabbit polyclonal anti-AIF , rabbit monoclonal anti-cleaved caspase-3 , rabbit polyclonal anti-NFκB p65 , rabbit monoclonal anti-PARP-1 , goat polyclonal anti-peroxiredoxin 1 , polyclonal goat anti-sulfiredoxin 1  diluted in 0.5% (v/v) horse serum or 1% (w/v) bovine serum albumin in TBST, corresponding to the blocking solution. As reference controls, were used monoclonal mouse anti-β-actin  and rabbit polyclonal anti-α-actinin  diluted in 1% (w/v) bovine serum albumin in TBST. Secondary incubations were performed with horseradish peroxidase-linked polyclonal donkey anti-goat , polyclonal rabbit anti-mouse , or polyclonal donkey anti-rabbit  antibodies, diluted in 1% (w/v) bovine serum albumin in TBST, respectively. Positive signals were visualized using enhanced chemiluminescence  and quantified using a ChemiDoc™ XRS+ system and the software Image Lab™ (Bio-Rad). Each experiment was repeated three times.Western blotting was performed as previously described ,66 for f2, 0.05% (v/v) Brij 35, 1% (v/v) Triton X-100, 0.02% (w/v) NaN3, 50 mM Tris-HCl pH 7.4) on ice, followed by centrifugation for 5 min at 12,000× g. The protein concentration of the supernatant was measured with the Pierce BCA kit (Pierce/Thermo Scientific). One milligram of protein was used for enzyme enrichment and purification with Gelatin Sepharose™ 4B  and eluted in 150 µL working buffer containing 10% (v/v) dimethylsulfoxide. Protein samples mixed with an equal volume of Novex® SDS-sample buffer , and incubated for 5 min at room temperature prior to loading to 10% Novex® gelatin-ready zymogram gel (Life Technologies GmbH). Gels were run at 125 V for 2 h. The gels were incubated in renaturation buffer  under gentle agitation for 30 min. They were then equilibrated for 30 min in developing buffer  and incubated in fresh developing buffer at 37 °C overnight, but at least for 18 h. Gels were washed three times with deionized water under gentle agitation for 5 min and stained with Coomassie Blue G 250  for 30 min, and washed with deionized water for 30 min. Areas of protease activity appear as clear bands against a dark blue background where the protease has digested the gelatin substrate. Quantification of inverted matrix metalloproteinases (MMP) 2 band density was carried out using a ChemiDoc™ XRS+ system and the software Quantity One® (Bio-Rad).Snap-frozen tissue was homogenized in working buffer  for 10 min. The supernatant was transferred on a 96 well plate and absorbance was read at 530 nm in a microplate reader. TBARS concentration as a measure for lipid peroxidation was calculated from a malondialdehyde (MDA) standard curve and normalized to the amount of total protein.Concentrations of markers of lipid peroxidation thiobarbituric acid reactive substances (TBARS) was determined using the TBARS assay kit  according to manufacturer’s instructions. Briefly, first a mixture of sample or standard and SDS solution was prepared. A color reagent was added to the mixture and boiled for one hour. The reaction was stopped on ice for 10 min and centrifuged at 1600× v/v) Tween 20. Afterwards, 100 µL of antibody-POD conjugate solution was added and incubated at room temperature for one hour. The sample solution was removed and ensuing washed four times with 400 µL of PBS, aspirating thoroughly between washes. Substrate solution (100 µL) was added and incubated for 15 min at room temperature. The reaction was stopped by addition of 100 µL stop solution. The absorbance was read at 450 nm in a microplate reader. HO-1 concentration was determined by comparing sample to the standard curve and values were expressed as nanograms per milligram of protein.HO-1 concentration was analyzed in samples of brain homogenate using Rat Heme Oxygenase-1 EIA Kit  according to manufacturer’s instructions. Briefly, 100 µL of sample or standard was first loaded and incubated for one hour at room temperature. Then the sample solution was removed and the wells washed three times with 400 µL of PBS containing 0.1% (2O2) was measured in brain homogenates using OxiSelect Hydrogen peroxide assay kit  according to manufacturer’s instructions. Briefly, 25 µL of sample or standard was first added to the microtiter plate wells and then mixed thoroughly with 250 µL aqueous working reagent. Afterwards samples were incubated on a shaker for 30 min at room temperature and the absorbance was read at 540 nm. The peroxide content in unknown samples was determined by comparison with a predetermined H2O2 standard curve.Hydrogen peroxide (H2O2 and tetramethylbenzidine (R&D Systems GmbH), was added. Samples were incubated in the dark for 20 min and the reaction was stopped using 50 µL 2 N H2SO4. Plates were read at 450 nm and TNFα, IL-1β, and IFN-γ concentrations were estimated from the standard curve and expressed as picograms per milligram protein.Tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interferon-γ (IFN-γ) concentrations were analyzed in samples of brain homogenate using rat TNFα/TNFSF1A, rat IL-1β/IL-1F2, and rat IFN-γ DuoSet ELISA  according to manufacturer’s instructions. Briefly, 96-well plates were coated with 100 µL of capture antibody, incubated overnight, washed with 400 µL wash buffer and incubated for 1 h for blocking with 300 µL of reagent diluent. After washing in wash buffer, 100 µL of samples or standards, diluted in reagent diluent, were added and incubation preceded for 2 h. Samples were then washed and incubated for 2 h in the presence of 100 µL of biotinylated detection antibody at room temperature. The supernatants were aspirated, the wells washed and 100 µL of horseradish peroxidase-conjugated streptavidin was added and incubation continued for 20 min. Samples were washed and 100 µL of substrate solution, a 1:1 mixture of HCasp3), glutamate-cysteine ligase catalytic subunit (GCLC), interferon-γ (IFN-γ), interleukin-1β (IL-1β), interleukin-12b (IL-12b), interleukin-18 (IL-18), inducible nitric oxide synthase (iNOS), Kelch-like ECH-associated protein 1 (Keap1), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 and 2 (NFκB1 and NFκB2), NFE2-related factor 2 (Nrf2), superoxide dismutase 1, 2 and 3 , tissue inhibitor of metalloproteinase 1 and 2 (TIMP1 and TIMP2), tumor necrosis factor α (TNFα), tissue plasminogen activator (tPa), and hypoxanthine-guanine phosphoribosyltransferase (HPRT) were quantified in real time, using dye-labeled fluorogenic reporter oligonucleotide probes and primers  according to the 2−ΔΔCt method [The FAM spectral data were collected from reactions carried out in separate tubes using the same stock of cDNA to avoid spectral overlap between FAM/TAMRA and limitations of reagents. PCR and detection were performed in triplicate and repeated two times for each sample in 11 µL reaction mix, which contained 5 µL of 2× KAPA PROBE FAST qPCR Master mix (PEQLAB Biotechnologie), 2.5 µL of 1.25 µM oligonucleotide mix, 0.5 µL (0.5 µM) of probe , and 3 to 17 ng of cDNA template with t method .p-value of <0.05 was considered significant. Results of the analyses were represented normalized as 100% value of control . For the real-time PCR data, the 100% value of control represented the CT values, while CT was defined as the threshold cycle number of PCRs at which amplified product was first detected. For protein data , the 100% value of control represented the protein concentration or molarity (ELISA), intensity ratio of pixel size (immunoblotting), or inverted intensity of pixel size (gelatin zymography). All graphics and statistical analyses were performed using the GraphPad Prism 6.0 software for Windows .All data are expressed as the mean ± standard error of the mean (SEM). Differences between the control group and experimental groups (oxygen exposure and/or caffeine application) were analyzed using a two-way analysis of variance (ANOVA) with the factors hyperoxia exposure, caffeine, and their interaction. To determine differences between individual groups, each two-way ANOVA was followed by a Bonferroni post-hoc test. A two-sided In summary, our study reveals that single administration of caffeine in oxygen-induced brain injury leads to improved neuroprotective response. Exposure to high oxygen in the developmental brain of six-day-old rat pups resulted in increased levels of lipid peroxidation, enhanced generation of hydrogen peroxidase and heme oxygenase-1, decreased anti-oxidative response, up-regulated pro-inflammatory cytokines expression, changed balanced redox-sensitive reply, and promoted extracellular matrix degeneration and apoptotic cascade. All these consequences of hyperoxia can be reversed by caffeine treatment.Our study suggests that caffeine is a drug that exerts neuroprotective effects on the developing brain due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Future research should focus on the investigation of caffeine for preconditioning and further enhance the knowledge about safety of caffeine use in the immature brain.


It also induced mitochondrial apoptosis by increasing the expression of Bax, caspase-9, and poly(ADP-ribose) polymerase and mitochondrial membrane potential loss and leaks of cytochrome c (Cyt C) from mitochondria in MCF-7 cells and decreasing the expression of mitochondrial Bcl-2. We further demonstrated whether chloroquine (CQ), which inhibits the degradation of autophagosome induced by NBT, affects the proliferation of MCF-7 cells compared with NBT. The experiments inferred that the combination of NBT and CQ significantly promoted MCF-7 cell mitochondria to divide and Cyt C to be released from mitochondria to the cytoplasm, resulting in an increased apoptosis rate. The in vivo experiments showed that NBT inhibited the growth of MCF-7 tumor via the apoptosis pathway, and its effect was similar to 5-fluorouracil.Betulin (BT), a pentacyclic lupine-type triterpenoid natural product, possesses antitumor activity in various types of cancers. However, its clinical development was discouraged due to its low biological activities and poor solubility. We prepared lup-20(29)-en-3β,28-di-yl-nitrooxy acetate (NBT), a derivative of BT, that was chemically modified at position 3 of ring A and C-28 by introducing a NO-releasing moiety. This study mainly explored the mechanism of NBT in treating breast cancer through the crosstalk between apoptosis and autophagy in mitochondria. NBT possessed a potent antiproliferative activity in MCF-7 cells both in vitro and in vivo. Mechanically, NBT affected cell death through the mitochondrial apoptosis pathway and autophagy. NBT induced cell cycle arrest in the G In addition, the antitumor activity of BT has been observed in a broad range of cancer cell lines, and it has demonstrated potent inhibition of proliferation in solid tumors by activating the mitochondrial apoptosis pathway characterized by the cleavage of caspases and poly(ADP-ribose) polymerase (PARP), attenuation of Bcl-2, mitochondrial depolarization, and chromatin condensation8. Despite reports of good efficacy and safety of BT in tumor therapy, its clinical application is discouraged because of its low bioavailability and poor solubility. We focused on the modification of BT at the C-3 and/or C-28 positions as modifications at these positions have been reported to improve its antitumor and antimicrobial activities and hydrosolubility9. Nitric oxide (NO), an important endogenously produced cell signaling and target molecule involved in many physiological and pathological reactions, plays a significant anticancer role via the toxicity of macrophage to tumor cells, inhibition of angiogenesis and metastasis, proliferation inhibition, and apoptosis of tumor cells in various types of cancer cells12. We introduced a NO-releasing moiety into BT by targeting position 3 of ring A and C-28 to synthesize a library of different NO-releasing derivatives of BT by considering the evidence that NO at high concentrations exhibits tumoricidal activity, whereas at low concentrations it stimulates tumor proliferation13 and mediates apoptosis via intrinsic apoptotic signaling by down-regulating Bcl-2 expression14. Among the various derivatives, lup-20(29)-en-3β,28-di-yl-nitrooxy acetate (NBT)  Fig.  is a natBT) Fig.  was the 18. Apoptosis, a major way of killing cancer cells by anticancer agents, includes two kinds of pathways: caspase-dependent and caspase-independent. The caspase-dependent pathway mostly occurs through extrinsic or intrinsic pathways19. Mitochondria are of great significance in intrinsic apoptosis. Autophagy is a conserved process that is involved in turning over organelles, protein degradation, and differentiation20. It begins with the trimer formation of beclin 1, PI3KC3 (Vps34), and Atg 14, with beclin 1 constantly increasing autophagy-related proteins. Light chain 3-II (LC3-II) plays an important role in the elongation of the double membrane until formation of the autolysosome, through the fusion of mature autophagosome and lysosome21. Atg5 is required for LC3 lipidation in autophagy and switches autophagy to apoptosis22. p62, a multifunctional protein, combines with ubiquitinated protein and binds to LC3 II proteins to form a complex that is eventually degraded by enzymes in the lysosome when autophagy occurs24. Hence, it is constantly consumed with increasing levels of autophagy. Therefore, Atg-5, beclin-1, LC 3-II, and p62 are major indicators in the development of autophagy 26.Apoptosis and autophagy participate in cellular degradation pathways for maintaining cellular homeostasis and are involved in the protection of organisms from cancer28. In this study, we sought to evaluate the effect of NBT on inhibiting the proliferation of MCF-7 cells in vitro and in vivo and attempted to elucidate its anticancer mechanisms in terms of apoptosis, autophagy, and the relationship between apoptosis and autophagy.Autophagy can evidently reduce the potency of therapeutic agents for cancers via increasing cellular survival in stress conditions13C NMR : δ 38.5 (C-1), 23.6 (C-2), 83.6 (C-3), 40.8 (C-4), 55.5 (C-5), 18.1 (C-6), 34.6 (C-7), 43.3 (C-8), 51.0 (C-9), 37.1 (C-10), 21.5 (C-11), 22.6 (C-12), 37.6 (C-13), 51.0 (C-14), 28.2 (C-15), 31.9 (C-16), 37.9 (C-17), 52.1 (C-18), 48.8 (C-19), 144.1 (C-20), 29.3(C-21), 34.9 (C-22), 27.9 (C-23), 16.7 (C-24), 16.5 (C-25), 15.5 (C-26), 14.1 (C-27), 67.0 (C-28), 109.6 (C-29), 21.0 (C-30), 165.6 , 67.4 ; 1H NMR : δ 0.83, 0.85, 0.86, 0.87, 0.89, 1.04 , 2.25 , 4.61 , 4.64 , 3.83 , 4.89, 4.88 ; IR (KBr) cm−1: 2918, 2850, 1742, 1655, 1467, 1384, 1292.NMR Fig.  and IR  were use50 values of NBT for HeLa, A549, and MCF-7 were 24.50 ± 2.50, 49.30 ± 7.90, and 10.83 ± 0.54 μM, respectively, for 48 h. The IC50 value of BT for HeLa was 288.21 ± 12.30 μM, and the A549 and MCF-7 treated with BT at 500 μM exhibited ≤40% inhibition . Bax was up-regulated and Bcl-2 was down-regulated in MCF-7 cells treated with NBT  released from mitochondriaNBT Fig. . In addiNBT Fig. .31. Western blot results showed that NBT increased DRP1 and Fis1 but decreased Mfn2 in a dose-dependent manner  images indicated that NBT induced autophagy and apoptotic changes in MCF-7 cells Fig. . Three kTo assess the autophagic flux, MCF-7 cells were transiently transfected with the mRFP-GFP-LC3 vector. mRFP is stable under acidic conditions, whereas GFP is not present in the acid environment of autolysosome. Rapamycin (Rapa), which induces autophagy by inhibiting the mTOR pathway, was chosen as the positive agent. Chloroquine (CQ), which inhibits the fusion of autophagosome and lysosome, was used for autophagic flux detection. Rapa treatment induced yellow and red puncta, which respectively denote autophagosomes and autolysosomes Fig. . MCF-7 cThe cotreatment of NBT and CQ for 24 h in MCF-7 cells resulted in a marked increase in apoptosis according to the annexin V/propidium iodide (PI) staining test Fig. . A 10% iFurthermore, Rapa increased Atg5 and LC3B-II levels, which were further increased by CQ Fig. . Rapa deThe antitumor activities of NBT and BT in vivo were evaluated. After administering NBT at 100 mg/kg i.p. and BT at 100 mg/kg i.p. for 11 days, NBT or 5-fluorouracil (5-FU) led to a significant reduction in tumor growth compared with the control, whereas BT treatment caused minimal reduction in tumor growth Fig. . No signHE staining results showed that fibrosis, necrosis, and inflammatory cell infiltration were observed in tumors treated with NBT or BT  of NBT for a long time (48 h), BEAS-2B cells were found to be sensitive to NBT, exhibiting 50% inhibition. Therefore, 24 h was selected as the optimal time point to perform further experiments. The results meant a possible side effect of NBT on lung function.Breast cancer causes the most cancer-related deaths among females36. In this study, we observed that NBT induced cell cycle arrest in the G0/G1 phase by down-regulating the gene and protein expression levels of cyclin D1 in MCF-7 cells. In addition, NBT augmented a dose-dependent apoptosis induction. Mitochondria play a crucial role in the caspase-dependent apoptosis, which has been validated by evaluating the expression of Bcl-2 family proteins37. The expression of Bcl-2 is up-regulated in breast cancer38, and the high expression of Bax initiates apoptosis39. In the present study, NBT enhanced the Bax/Bcl-2 ratio, increased the loss of ΔΨm, and increased Cyt C release from mitochondria to cytosol. These results confirmed the involvement of mitochondria in NBT-induced apoptosis in MCF-7 cells. Moreover, NBT increased the activity of caspase-9 and cleaved-PARP, suggesting that the NBT-induced cell cycle arrest in the G0/G1 phase preceded apoptosis. The loss of ΔΨm via the modulation of Bcl-2 family protein expression and Cyt C resulted in the disruption of mitochondrial function. Furthermore, the activation of caspase-9 suggested that a mitochondrial-dependent factor was responsible for its apoptotic potential. Subsequently, the activation of the caspase cascade led to apoptosis after formatting the cleaved products of PARP in MCF-7 cells. These results were consistent with the anticancer mechanism of BT. DRP1 catalyzes mediated mitochondrial fission and is recruited to mitochondria40. Mfn1 and Mfn2, which are tethered to the outer mitochondrial membrane to function, drive mitochondrial fusion42. Our results suggested that mitochondrial dynamic proteins participated in NBT-induced apoptosis in MCF-7 cells. The NBT-induced mitochondrial fission led to the loss of ΔΨm, increased Cyt C in the cytoplasm, activation of caspase-9, and degradation of PARP, which preceded apoptosis.Different molecular mechanisms have been implicated in the anticancer activities of chemotherapeutics. Novel molecules selectively inhibit the proliferation of cancer cells via the PI3K/AKT signaling pathway, which induces cell cycle progression and regulates cell survival and apoptosis43. Hypoxia and limited nutrients have been reported to activate autophagy, which may help solid tumor cell survival44. In addition, autophagy promotes the survival of parts of tumor cells to defend themselves against cancer treatment, and eventually, they become blocks for cancer treatment46. However, autophagy also has been shown to induce cell death because of excessive stress. NBT treatment resulted in the accumulation of Atg5 and LC3-II levels and decreased the expression of p62 in MCF-7 cells, indicating that it induced autophagy in MCF-7 cells. In addition, our findings clearly indicated that NBT treatment induced the autophagic flux in MCF-7 cells, where the numbers of autophagosome and LC3 puncta accumulated. Previous studies have shown that autophagy induced by inhibiting p53 led to cell cycle arrest in the G1 phase48. We inferred that NBT might induce cell cycle arrest in the G1 phase by inhibiting the expression level of p53 in the MCF-7 cells.Autophagy, an adaptation response to stress, regulates the turnover and recycling of organelles in the cytoplasm50. Moreover, a study reported that blocking the autophagic flux in breast cancer MCF-7 cells can induce cell death51. In the present study, NBT not only induced apoptosis but also elicited the complete autophagic process in MCF-7 cells. The results indicated that disturbing the autophagic flux enhanced the apoptosis induced by NBT in MCF-7 cells. 3-MA affects autophagy at an early stage via inhibiting its formation, whereas CQ inhibits the fusion of autophagy with lysosome at a later stage52. Currently, the clinical drugs for inhibiting autophagy only include CQ and hydroxychloroquine53. CQ and 3-MA were chosen as autophagic inhibitors to test the effect of inhibiting autophagy on NBT-mediated apoptosis in MCF-7 cells. In our experiments, p62 level was reduced by 60% with Rapa, an initiator of autophagy, treatment alone at 0.25 μM and was increased by 75% with CQ treatment at 40 μM  three times within 24 h. Then, after 11 days of NBT and BT administration, the results showed that NBT or BT was safe. The overall toxicological effects of NBT were not examined in this study but will be examined in our future study. The pharmacodynamical results indicated that NBT demonstrated higher antitumor potential than BT in vivo. In addition, hematoxylin and eosin (H&E) staining and TUNEL assay showed that NBT induced more necrotic and apoptotic cells than BT. Therefore, NBT was useful and safe in treating breast cancer, specifically by targeting the mitochondrial apoptosis pathway.In summary Fig. , cotreatCell Counting Kit-8 (CCK-8) was from Dojindo . The Annexin V-FITC Apoptosis Detection Kit, Cell Cycle Detection Kit (R.T.U), and Caspase-9 Colorimetric Assay Kit were purchased from KeyGen Biotech . Dimethyl sulfoxide (DMSO), Hoechst 33258, 3-MA, and CQ diphosphate salt were from Sigma-Aldrich . The cell lysis buffer for Western blot and IP, Prestained Dual Color Protein Molecular Weight Marker, and BeyoECL Plus were from Beyotime . The anti-cyclin D1 , anti-Bcl-2 , anti-Bax , anti-cytochrome , anti-LC3B , anti-DRP1 , anti-mitofusin 2 , anti-TTC11 , and COX4I1  were from Abcam Technology . Anti-Atg5 , anti-p62 , anti-caspase-9 , anti-PARP , and anti-rabbit IgG, HRP-linked antibody  were from Cell Signaling Technology .2 humidified incubator  at 37 °C with 5% CO2. BT  and NBT were dissolved in DMSO stored at 4 °C until use .MCF-7, HeLa, A549, BEAS-2B, MCF10A, and L-02 cell lines were purchased from the American Type Culture Collection (ATCC) . Cells were maintained in RPMI Medium 1640 basic/DMEM/high-glucose DMEM containing 10% fetal bovine serum, penicillin, and streptomycin  in a CO3 cells/100 μL for 24 h, and then various concentrations of BT or NBT were added for 24 or 48 h. The growth inhibition of BT and NBT was tested with 10 μL CCK-8 per well. Optical density was measured at 450 nm by using a multimode plate reader .Cells were grown in 96-well plates at a density of 5 × 105 cells/2 mL for 24 h and exposed to 6, 12, and 24 μM concentrations of NBT. After 24 h, the cells were washed once with PBS , collected using 0.25% trypsin/0.02% EDTA , and centrifuged at 1500 r.p.m. for 5 min. Approximately 106 cells were fixed in 70% cold ethanol at 4 °C overnight. Ethanol-fixed cells were again washed with PBS and resuspended in 100 μL RNAse A in a 37 °C water bath for 30 min and then were incubated with 400 μL PI at 4 °C for 30 min blocking the light ray and analyzed by using FACS .MCF-7 cells were seeded in six-well plates at a density of 2 × 10MCF-7 cells were harvested after treatment with 6, 12, and 24 μM concentration of NBT for 24 h. Total RNAs were extracted by using TriZol . Next, 1 μg total RNA samples was reverse transcribed into cDNA by using PrimeScript™ RT Master Mix  and amplified by using SYBR Premix Ex Taq™ II  of cyclin D1 and GAPDH.Cyclin D1, forward: 5′-CGTGTAGCTATGGAAGTTGCA-3′,reverse: 5′-CCCGAATGAGAGTCCTACAG-3′;GAPDH, forward: 5′-CGG AGTCAACGGATTTGGTCGTAT-3′,reverse: 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ . RT-PCR was conducted under the following conditions: 95 °C for 5 s, annealing for 30 s at 60 °C, and cycling for 40 times. The RT-PCR results were normalized with GAPDH using ΔΔCT analysis.MCF-7 cells were seeded in a coverslip, treated, fixed in 4% paraformaldehyde at 4 °C for 20 min, washed twice with PBS, and then incubated with Hoechst 33258 fluorescent dye (10 μg/mL) at room temperature for 10 min. Images were taken with a fluorescence microscope .5 cells were resuspended in 500 μL binding buffer and then incubated with 5 μL annexin V-FITC with 5 μL PI at room temperature for 5–15 min. The samples were analyzed by flow cytometry using the FL1 (FITC) and FL3 (PI) laser lines.MCF-7 cells treated with NBT for 24 h were collected by using 0.25% trypsin and washed twice with cold PBS . Approximately 1–5 × 10MCF-7 cells treated with NBT and/or CQ for 24 h were harvested, washed twice with cold PBS, and then fractionated into cytosolic and mitochondrial fractions by using a Qproteome Mitochondria Isolation Kit  following the manufacturer’s protocol.MCF-7 cells treated with NBT and/or CQ for 24 h were lysed in cell lysis buffer containing 1% PMFS for 30 min on ice and centrifuged at 12,000 r.p.m. for 5 min at 4 °C. Then, the protein was quantified by using a BCA Protein Assay Kit .6 MCF-7 cells treated with NBT for 24 h were harvested, washed twice with PBS at 2000 r.p.m. for 5 min, lysed in cold lysis buffer on ice for 20–60 min, and centrifuged at 10,000 r.p.m. for 1 min at 4 °C. A total of 100 μg of protein (BCA method) were mixed with 50 μL 2× reaction buffer containing 0.5 μL DTT and 5 μL caspase-9 substrate. The mixture was incubated at 37 °C in the dark and then measured with a multimode plate reader at 405 nm.Caspase-9 activity assay was conducted by using a caspase-9 colorimetric assay kit. Approximately 3–5 × 10Total cell extracts (50 μg) were separated by using SDS-PAGE at 120 V for 1.5 h at room temperature and then eletrotransferred onto PVDF membranes at 300 mA for 1 h at 4 °C. The membranes were washed three times with TBST each for 10 min, blocked with 5% nonfat dry milk in TBST for 2 h at room temperature, rinsed with TBST, incubated with primary antibodies overnight at 4 °C, washed three times with TBST, incubated with HRP-linked anti-IgG for 1 h at room temperature, and then washed three times as before. At the same time, β-actin, GAPDH, and tubulin acted as the control. Afterward, the chemiluminescent bands were analyzed using Image Lab™ Software on ChemiDoc XRS + .6 MCF-7 cells treated with NBT for 24 h were collected, washed twice with PBS at 2000 r.p.m. for 5 min, and incubated with JC-1 for 20 min in a CO2 incubator at 5% CO2/37 °C. Then, the cells were washed twice in 1× incubation buffer at room temperature  and resuspended in 500 μL 1× incubation buffer. The changes in mitochondrial membrane potential (Δψm) were measured with a flow cytometer in FITC and PI channels .5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine odide (JC-1) retention in mitochondria is driven by ΔΨm, which determines cell population with integrated mitochondrial functions. However, JC-1 is released from mitochondria when its membrane potential depolarizes. Less than 1 × 10Cells were collected with 1 mL serum-free medium and fixed in 2.5% glutaraldehyde at room temperature for 1 h and then 4 °C for 3 h, where glutaraldehyde was replaced with PBS. The samples were examined by using a TEM.4 of MCF-7 cells cultured on coverslips were transduced with 0.8 μg of mRFP-GFP-LC3 plasmid for 48 h and then treated with NBT or NBT + CQ or Rapa or Rapa + CQ for another 24 h. Then, the cells were stained with Hoechst 33258 fluorescent dye. Images were taken by using a fluorescence microscope .Transfection was achieved using Lipofectamine 2000 Transfection Reagent  according to the manufacturer’s protocol. A total of 1 × 106) were injected subcutaneously into the nude mice under the left axillae. The mice were randomly divided into four different groups (n = 8), namely, normal saline (NS) group (0.2 mL i.p.), 5-FU-positive control group (22 mg/kg i.p.), BT group (100 mg/kg i.p.), and NBT group (100 mg/kg i.p.), when the tumor size reached 80 mm3 after 5 days of inoculation of MCF-7 cells in nude mice. The mice were treated with NS, 5-Fu, BT, and NBT every day for 11 days. Tumors were measured every other day with the help of a vernier caliper  = Length (mm) × Width (mm)2/2). After the last treatment, the tumor of each mouse was isolated and fixed in 10% formalin.β-Estradiol (0.03 mg/0.2 mL PBS solution) was intraperitoneally injected to female BALB/c nude mice. After 5 days, MCF-7 cells (10 × 10Tumor samples were fixed in 10% formalin, paraffinized, and sectioned into 4-μm sections, which were stained with H&E according to standard methods.Tumor samples were fixed in 10% formalin, paraffinized, and sectioned into 4-μm sections, which were stained with FITC-POD according to standard methods.


Desulfovibrio vulgaris Hildenborough to salt stress was observed during experimental evolution. In order to identify key metabolites important for salt tolerance, a clone, ES10-5, which was isolated from population ES10 and allowed to experimentally evolve under salt stress for 5,000 generations, was analyzed and compared to clone ES9-11, which was isolated from population ES9 and had evolved under the same conditions for 1,200 generations. These two clones were chosen because they represented the best-adapted clones among six independently evolved populations. ES10-5 acquired new mutations in genes potentially involved in salt tolerance, in addition to the preexisting mutations and different mutations in the same genes as in ES9-11. Most basal abundance changes of metabolites and phospholipid fatty acids (PLFAs) were lower in ES10-5 than ES9-11, but an increase of glutamate and branched PLFA i17:1ω9c under high-salinity conditions was persistent. ES9-11 had decreased cell motility compared to the ancestor; in contrast, ES10-5 showed higher cell motility under both nonstress and high-salinity conditions. Both genotypes displayed better growth energy efficiencies than the ancestor under nonstress or high-salinity conditions. Consistently, ES10-5 did not display most of the basal transcriptional changes observed in ES9-11, but it showed increased expression of genes involved in glutamate biosynthesis, cation efflux, and energy metabolism under high salinity. These results demonstrated the role of glutamate as a key osmolyte and i17:1ω9c as the major PLFA for salt tolerance in D. vulgaris. The mechanistic changes in evolved genotypes suggested that growth energy efficiency might be a key factor for selection.Rapid genetic and phenotypic adaptation of the sulfate-reducing bacterium  Desulfovibrio vulgaris Hildenborough is a model sulfate-reducing bacterium (SRB) that is important in biogeochemical cycling of sulfur, carbon, and nitrogen, potentially for bio-corrosion, and for bioremediation of toxic heavy metals and radionuclides. The coexistence of SRB and high salinity in natural habitats and heavy metal-contaminated field sites laid the foundation for the study of salt adaptation of D. vulgaris Hildenborough with experimental evolution. Here, we analyzed a clone that evolved under salt stress for 5,000 generations and compared it to a clone evolved under the same condition for 1,200 generations. The results indicated the key roles of glutamate for osmoprotection and of i17:1ω9c for increasing membrane fluidity during salt adaptation. The findings provide valuable insights about the salt adaptation mechanism changes during long-term experimental evolution.High salinity  is a stressor that affects many organisms. Salt tolerance, a complex trait involving multiple cellular pathways, is attractive for experimental evolutionary studies.  Microbial experimental evolution coupled with whole-genome sequencing has been widely used to identify key functional genes involved in desired traits, to improve the industrial efficiencies of microbes, and to address fundamental evolutionary questions –9. Howev–Desulfovibrio vulgaris Hildenborough has been used as a model sulfate-reducing bacterium (SRB) for studying the complex physiology and stress responses  have been propagated under a mild salt stress condition. Analysis of the genotype of ES9-11, which was isolated from a 1,200-generation (gen) population of ES9, demonstrated distinct basal and salt stress response changes and genomic changes compared to the ancestor and strains allowed to evolve under nonstress conditions as controls  is an environmental factor that affects many organisms. The well-known strategies of microorganisms for coping with salt stress include intracellular accumulation of organic solutes and changes of membrane PLFA composition to compensate for the membrane fluidity decrease –13. Desu–esponses  due to ihia coli , D. vulgan acute  or chronan acute , dependicontrols , 19. As E. coli under conditions with new carbon sources  that had been allowed to evolve for 5,000 gen under mild salt stress conditions. The growth phenotype was tested in a defined lactate-sulfate (LS4D) medium with 250 mM NaCl, a condition that led to an approximate 50% final biomass reduction in ancestral D. vulgaris Hildenborough (600] ranged from 1.0 to 1.2) and average growth rates (~0.12 h−1) were observed in the 5,000-gen and 1,200-gen ES populations, the 5,000-gen ES populations had shorter lag phases and less variance among populations. The results suggested that salt adaptation involved a rapidly improved growth rate and growth efficiency and a gradual decrease in the lag phase time.We first evaluated the salt tolerance of six nborough , 17 and nborough , 19. Thenborough . Althoug10.1128/mBio.01780-17.1FIG S1 FIG S1, TIF file, 0.8 MB.(A) Growth curves of 5,000-generation ES populations in LS4D (○) or LS4D plus 250 mM NaCl (●). (B) Growth curves of clones isolated from 5,000-generation ES10 population in LS4D , LS4D plus 300 mM NaCl , or LS4D plus 400 mM NaCl . Download Copyright © 2017 Zhou et al.2017Zhou et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the + in total. No growth was observed in LS4D plus 500 mM NaCl. Due to its best growth performance with the shortest lag phase and the highest growth rate in LS4D plus 400 mM NaCl, ES10-5 was chosen as a representative 5,000-gen genotype representing the best adaptation to salt stress, and this strain was compared to the representative genotype ES9-11 strain, which was isolated from a 1,200-gen ES9 strain by similar means . Download Copyright © 2017 Zhou et al.2017Zhou et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01780-17.6TABLE S2 TABLE S2, DOCX file, 0.02 MB.Details of the genes deleted from the plasmid due to the deletion from bp 51979 to 157737. Download Copyright © 2017 Zhou et al.2017Zhou et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01780-17.7TABLE S3 TABLE S3, DOCX file, 0.02 MB.PCR primers used to confirm mutations with Sanger sequencing. Download Copyright © 2017 Zhou et al.2017Zhou et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Intracellular accumulation of organic solutes is one of the major strategies to counteract external osmotic pressure. About 80 metabolites were detected by quadrupole time-of-flight (Q-TOF) analysis, and 13 were identified . Among t10.1128/mBio.01780-17.8TABLE S4 TABLE S4, DOCX file, 0.04 MB.Metabolites detected by Q-TOF. Download Copyright © 2017 Zhou et al.2017Zhou et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.05).Membrane PLFA composition changes, such as increased percentages of unsaturated PLFAs or branched PLFAs, compensate for decreases in membrane fluidity under high salinity. Analysis of the relatively abundant (>5%) PLFAs demonstrated that different sets of PLFAs displayed abundance changes under high-salinity conditions in the ancestor or the evolved strains . An incrP < 0.05) (A higher unsaturation index (UI) and increased percentages of branched PLFAs indicate a more fluid membrane. Under nonstress conditions, ES10-5 had the highest UI and ES9-11 had the highest percentage of branched PLFAs. Under high salinity, all strains had similar UIs, but ES10-5 had the highest percentage of branched PLFAs  . These r10.1128/mBio.01780-17.2FIG S2 P < 0.05). Download FIG S2, TIF file, 0.7 MB.Unsaturation index (A) and percent branched PLFAs (B) in strains ES10-5 and ES9-11 and the ancestral strain (An) under nonstress (LS4D) or high-salinity (LS4D plus 300 mM NaCl) conditions. *, significant change induced by high salinity . An additional 300 mM NaCl in the medium almost completely arrested cell motility. However, ES10-5 showed the best growth, as indicated by the dark color of the colonies generally seen with D. vulgaris. The observed higher cell motility of ES10-5 might be an indicator of better energy management, as cell motility is a process with a high energy cost.Decreased cell motility was observed for ES9-11 . To dete10.1128/mBio.01780-17.3FIG S3 FIG S3, TIF file, 1 MB.Cell motilities of the ancestral strain and the evolved strains ES10-5 and ES9-11 in LS4D (A), LS4D plus 250 mM NaCl (B), and LS4D plus 300 mM NaCl (C). Download Copyright © 2017 Zhou et al.2017Zhou et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To assess overall growth energy efficiency, the total energy usage in each strain was measured by tracking dissipated heat via microcalorimetry, which employs the concept of describing the energy expense of microbial growth in terms of increased enthalpy . In LS4Dr = 0.86, P < 0.001) , the ac< 0.001) . Large-s10.1128/mBio.01780-17.4FIG S4 FIG S4, TIF file, 0.6 MB.(A) Correlation between microarray data and qRT-PCR data for selected genes in strains ES10-5 and ES9-11 compared to those for the ancestor (An) strain in LS4D. (B) Gene expression changes with growth in LS4D plus 300 mM NaCl compared to LS4D medium in all strains. Operons are separated by dot lines. Download Copyright © 2017 Zhou et al.2017Zhou et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01780-17.9TABLE S5 TABLE S5, DOCX file, 0.02 MB.Selected genes and gene-specific primers for real-time RT-PCR. Download Copyright © 2017 Zhou et al.2017Zhou et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01780-17.10TABLE S6 TABLE S6, DOCX file, 0.03 MB.Expression of genes affected by large deletions in chromosome or plasmid DNA in LS4D. Download Copyright © 2017 Zhou et al.2017Zhou et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ech hydrogenase genes. For cell motility-related genes, ES9-11 had decreased expression of flagellar biosynthesis genes and increased expression of four methyl-accepting chemotaxis protein (MCP) genes. ES10-5 had increased expression of one MCP gene, DVU0094. Increased expression of genes involved in ion transport , energy metabolism , and transcriptional regulator genes were only observed in ES9-11. These transcriptional changes might be important for early-stage adaptations.Under nonstress conditions , both ESGene expression changes in the following gene categories were examined . The results suggested that both MCP and flagellum biosynthesis genes might contribute to the cell motility decrease under salt stress but that decreased flagellum biosynthesis is important for early-stage adaptation.Both ES10-5 and ES9-11 had increased expression of MCP gene DVU0094 and decreased expression of three MCP genes. ES9-11 had decreased expression of three additional MCP genes and a flagellum biosynthesis gene, 2+ transport domain and drug efflux pump genes DVU0525 to -0526 and shows decreased expression of DVU0079, which is potentially involved in zinc transport, and DVU0567, which is potentially involved in efflux of tellurium ions. In addition, ES10-5 had increased expression of a TonB-dependent receptor gene, DVU0100, and cation ABC transporter genes DVU0102 to -0104; higher basal expression of these genes was also found in ES9-11, suggesting the importance of these genes at both early and later stages during adaptation. Decreased expression of genes involved in transport of phosphate, phosphonate, or potassium was observed only in ES9-11.Both ES10-5 and ES9-11 had increased expression of a TonB domain-containing protein gene, DVU0099, that is potentially involved in uptake of cobalamin and iron, as DVU0949 contains an ABC-type Coc genes and periplasmic [Ni-Fe] hydrogenase genes. ES10-5 had increased expression of hmc genes, while ES9-11 had decreased expression of ech genes. As ES10-5 had higher basal expression of ech genes, the results suggested that ES10-5 had an overall higher expression of energy metabolism genes. Expression of [Ni-Fe] hydrogenase genes DVU2525 and -2526 increased under nonstress conditions but decreased under high-salinity conditions in ES9-11, suggesting that expression of these genes might be unfavorable for overall energy efficiency.Both ES10-5 and ES9-11 had decreased expression of cytochrome Both ES10-5 and ES9-11 had increased expression of a GntR family transcriptional regulator gene, DVU2086, and decreased expression of the two-component system genes DVU0330 and -0331, DVU0721 and -0722, and DVU0743 and -0744. In addition, ES10-5 had increased expression of the sigma-54-dependent transcriptional regulator gene DVU3142 and an AraC family transcriptional regulator gene, DVU2378. AraC and GntR family transcriptional regulators have been shown to be involved in stress responses in other microorganisms , 29. ES9D. vulgaris Hildenborough   . Diminisevisiae) , 20–23.D. vulgaris to salt shock . The two genotypes shared five preexisting polymorphism-derived mutations and different alleles in three genes. Surprisingly, their growth performances were similar, except that ES10-5 entered the stationary growth phase about 12 h earlier when grown in LS4D plus 250 mM NaCl. The superior salt tolerance of ES10-5 became obvious when tested in LS4D plus 300 mM NaCl . The resD. vulgaris strains allowed to evolve for a different number of generations revealed glutamate as the key osmolyte and i17:1ω9c as a key PLFA for salt tolerance and the mechanistic changes associated with improved salt tolerance. Future studies will focus on analysis of gene function and gene regulatory networks in multiple populations to dissect the molecular mechanisms of salt adaptation.In conclusion, comparison of Desulfovibrio vulgaris Hildenborough populations  were allowed to experimentally evolve in defined LS4D medium (60 mM lactate as the electron donor and 50 mM sulfate as the electron acceptor) supplemented with 100 mM NaCl  were revived in LS4D (10 ml). After 48 h of growth, a 1% inoculum was transferred into production vessels or Hungate tubes for biomass production or growth phenotype tests.D. vulgaris Hildenborough reference sequences (NC_002937.3 [chromosome] and NC_005863.1 [plasmid]) and mutation calls  were performed with Geneious 9.1.5 (Biomatters Ltd.). Final calls of mutations were manually checked and confirmed by PCR and Sanger sequencing  with a cold methanol method  and analLate-exponential-phase cultures (four replicates) were harvested for PLFA assays  . Briefly600 of ~0.4) were applied to the surfaces of 0.4% (wt/vol) soft agar LS4D plates supplemented with 0, 250 mM, or 300 mM NaCl. After 4 days at 37°C, the colony diameters were measured.To test cell motility, mid-exponential-phase cell cultures , which measures the heat flow between a reaction cell and reference cell –44. Prio–600, ~0.4) or LS4D plus 300 mM NaCl  were harvested by spinning 50-ml cell cultures at 6,000 × g for 10 min at 4°C. The pellets were frozen in liquid nitrogen and stored at −80°C.Triplicate cultures grown in LS4D . qRT-PCR of 36 genes . Labeling of total RNA (2 µg per sample) with Cy3 and of genomic DNA  with Cy5 was conducted as described previously . Cy3-cDN36 genes  was cond36 genes  to validGSE103797.The microarray data were deposited in the NCBI GEO database under accession number


Adolescents with anxiety disorders attain poorer outcomes following extinction-based treatment compared to adults. Extinction deficit during adolescence has been identified to involve immaturity in the medial prefrontal cortex (mPFC). Findings from adult rodents suggest extinction involves dopamine signaling in the mPFC. This system changes dramatically during adolescence, but its role in adolescent extinction is unknown. Therefore, we investigated the role of prefrontal dopamine in extinction using Pavlovian fear conditioning in adolescent and adult rats. Using quantitative PCR (qPCR) analyses, we measured changes in dopamine receptor gene expression in the mPFC before and after extinction. We then enhanced dopamine 1 receptor (D1R) or dopamine 2 receptor (D2R) signaling in the infralimbic cortex (IL) of the mPFC using agonists at the time of extinction. Adolescent rats displayed a deficit in extinction retention compared to adults. Extinction induced a reduction in D1R compared to D2R gene expression in adolescent rats, whereas an increase of D1R compared to D2R gene expression was observed in adult rats. Acutely enhancing IL D1R signaling using SKF-81297 had no effect on extinction at either age. In contrast, acutely enhancing IL D2R signaling with quinpirole significantly enhanced long-term extinction in adolescents, and impaired within-session extinction in adults. Our results suggest a dissociated role for prefrontal dopamine in fear extinction during adolescence compared to adulthood. Findings highlight the dopamine system as a potential pharmacological target to improve extinction-based treatments for adolescents. Anxiety disorders are the most frequent mental illness experienced by adolescents worldwide  is a critical neural region underlying extinction deficits in adolescent rodents  40, then decline into adulthood across P60, P80, P100 and P120, with D1R declining more dramatically compared to D2R  analyses, we then measured extinction-related changes in mPFC D1R and D2R gene expression in adolescent and adult rats. Building on these results, we then acutely enhanced D1R or D2R signaling in IL during extinction using SKF-81297 or quinpirole, respectively. Dopamine receptor agonists were chosen so that parallels can be made to existing FDA-approved dopamine receptor agonists, which are more readily administered compared to dopamine receptor antagonists in adolescent humans Kirino, . Thus, rN = 142) were bred in-house at the Florey Institute of Neuroscience and Mental Health. Rats were housed 2–4 per cage in individually ventilated cages, maintained on a 12-h light/dark cycle (lights on at 7 a.m.) with food and water available ad libitum. Rats were handled daily for 3 days prior to the commencement of behavioral experiments. Rats were P35 (±1) or P88 (±1) on extinction day  bilaterally targeting the IL  was implanted stereotaxically . These coordinates were identified by pilot surgeries involving ink microinfusions using the rat brain atlas  consisted of either vehicle , SKF-81297 , or quinpirole  into the IL over 2 min. These doses were chosen based on previous studies that showed consistent effects specifically with these doses , using previously published protocol  before each session. The two contexts served as conditioning or extinction/test contexts in a counterbalanced manner.On day 1 of behavioral experimentation, rats were placed in the chambers and their baseline level of freezing was recorded for 2 min. The CS tone (80 dB) was then presented for 10 s and co-terminated with a 1 s footshock (0.6 mA). There were three CS-US pairings and the inter-trial interval (ITI) between each pairing was between 85–135 s. Following the last CS-US pairing, rats remained in the chambers for 2 min before returning to their home cages.On day 2, rats received extinction in the context different to that in which conditioning took place. Baseline freezing was measured for the first 2 min, followed by 30 CS alone trials with a 10 s ITI.On day 3, rats were tested in the same context as extinction. Baseline freezing was measured for the first minute, followed by a 2 min presentation of the CS alone. Rats remained in the chambers for 1 min before returning to their home cages.World Precision Instruments, Sarasota, FL, USA) under RNase-free conditions. The mPFC was micro-dissected  2 h following handling (Pre-extinction) or extinction (Post-extinction). Previously published protocol was used to measure mRNA levels . Three housekeeping genes  were assessed for stability in adult and adolescent mPFC. Hprt1 was the least variable between groups and was used for all subsequent analyses. Primers were designed using Primer3 , then reverse transcribed into cDNA using TaqMan Reverse Transcription reagents as per the manufacturer’s protocol (Applied Biosystems). Gene expression was analyzed by qPCR using SYBR Green Mastermix (Applied Biosystems) on a ViiAHprt1   forward   5′-CTGGTGAAAAGGACCTCTCG-3′; Hprt1   reverse   5′-TCCACTTTCGCTGATGACAC-3′, Drd1   forward   5′-CCTTCGATGTGTTTGTGTGG-3′, Drd1   reverse   5′-GGGCAGAGTCTGTAGCATCC-3′; Drd2   forward   5′-TCCTGTCCTTCACCATCTCC-3′, Drd2   reverse   5′-GACCAGCAGAGTGACGATGA-3′.−ΔCT and 2−ΔΔCT methods  and 2−ΔΔCT (ΔΔCTD1R/D2R = ΔCTD1R/D2R−(CTD1R average Pre-extinction − CTD2R average Pre-extinction)).Data were interpreted using 2p ≤ 0.05. Data were analyzed using one-way or repeated-measures analysis of variance  appropriate to each experimental design. For analyses of within-session extinction, data were collapsed into six blocks of five CS presentations per block (Extinction blocks). Significant interactions were followed by post hoc per factor ANOVA (Tukey HSD multiple comparisons), or t-tests (when only two groups were in a factor) as described previously , with acceptance for significance at ps > 0.05).Analyses of baseline freezing for the first experiment showed no effect of age at conditioning, extinction or test. For the second experiment, there was no age effect on baseline at conditioning. There was an effect of age at extinction, however RM ANCOVA revealed that when baseline was controlled for, there was no effect of age on freezing during extinction. There was no effect of age on baseline at test. There was no effect in baseline freezing at any phase of the final experiment (n = 11) and adult (n = 13) rats were conditioned with three trials of tone (CS) paired with an electric footshock (US). CS-elicited freezing during conditioning was similar across age groups  = 23.9, p < 0.05), with no effect of age  = 2.1, p = 0.2) and no interaction  = 1.9, p = 0.2). The next day, rats received extinction training consisting of 30 CS-alone trials. Both age groups showed initial high levels of freezing to the CS that decreased over the 30 CS-alone trials  = 30.1, p < 0.05), with no effect of age (F < 1) and no interaction  = 1.6, p = 0.2). Adolescents reinstated CS-elicited freezing when tested the next day, whereas adults did not  = 6.1, p < 0.05), and a significant interaction between day and age  = 9.9, p < 0.05), with no effect of age (F < 1). Post hoc tests found a significant difference in freezing levels at extinction vs. test for adolescents (t(10) = 3.0, p < 0.05), but not for adults (t < 1). Together, these findings indicate that long-term extinction is age-related, with adolescent rats displaying impaired extinction compared to adults.The first experiment aimed to elucidate behavioral differences in fear extinction across adolescence Figure . Adolescs Figure . RM ANOVs Figure . RM ANOVt Figure . RM ANOVn = 8) and adult (n = 12) rats received conditioning as described in the first experiment. All rats showed a significant increase in CS-elicited freezing during conditioning, however, the overall freezing levels were different between age groups  = 34.4, p < 0.05) and an effect of age  = 6.4, p = 0.02), but no interaction  = 2.1, p = 0.1). Twenty-four hours after fear conditioning, half the rats were handled for 2 min without exposure to behavioral chambers (Pre-extinction). Remaining animals underwent extinction as per the first experiment (Post-extinction). Tissue was collected 2 h later. Adolescent and adult rats that received extinction showed comparable within-session extinction  = 3.4, p < 0.05), with no effect of age  = 1.2, p = 0.3), and no effect of baseline  = 2.7, p = 0.1). These results were the same with RM ANOVA.We then measured changes in prefrontal D1R vs. D2R gene expression pre- or post-extinction  = 3.1, p < 0.05; Figure t < 1; Figure t(8) = 5.7, p < 0.05; Figure Adolescents showed higher prefrontal D1R mRNA expression than adults pre-extinction (F < 1), no effect of age  = 2.4, p = 0.1) and no interaction  = 1.1, p = 0.3; Figure Fs < 1; Figure F = 9.4, p < 0.05) and a significant interaction between treatment and age  = 8.5, p < 0.05), and no effect of treatment  = 0.3, p = 0.6). Post hoc tests found that adolescent D1R/D2R mRNA ratio was significantly decreased following extinction (t(6) = 2.6, p < 0.05), while adult D1R/D2R mRNA ratio was significantly increased (t(10) = 2.3, p < 0.05; Figure There was no change in D1R mRNA expression at post-extinction relative to pre-extinction for either age, with ANOVA showing no effect of treatment  = 13.5, p < 0.05). The next day, all adolescents showed comparable extinction with no differences between drug groups  = 16.9, p < 0.05), with no effect of drug and no interaction (Fs < 1). Interestingly, quinpirole prevented the return of extinguished fear at test  = 6.3, p < 0.05) and an interaction between day and drug  = 3.5, p < 0.05), and no effect of drug (F < 1). Post hoc tests found a significant difference in freezing levels at extinction vs. test for vehicle (t(18) = 3.6, p < 0.05) and SKF-81297 (t(7) = 2.6, p < 0.05), but not for quinpirole (t < 1). Thus, acutely enhancing IL D2R signaling at the time of extinction improved long-term extinction in adolescents.Adolescents displayed a significant increase in CS-elicited freezing across conditioning  = 75.1, p < 0.05). The next day, acutely manipulating IL D1R or D2R signaling in adults had a transient effect on within-session extinction, however all adults inhibited freezing to a comparable level by the end of extinction training  = 24.9, p < 0.05) and a block × drug interaction  = 2.0, p < 0.05), but no overall effect of drug  = 1.6, p = 0.2). When the interaction was examined with one-way ANOVA of individual extinction blocks (with Tukey HSD multiple comparisons), an effect of drug at extinction block 5 only was revealed  = 3.3, p < 0.05), with quinpirole group freezing higher than the other two groups (ps < 0.05). This result indicates that quinpirole delayed within-session extinction for adults. RM ANOVA of extinction vs. test showed an effect of day  = 13.0, p < 0.05), with no effect of drug  = 1.0, p = 0.4), and no interaction  = 1.4, p = 0.2). Thus increasing IL D1R or D2R signaling at the time of extinction had no effect on long-term extinction in adults  database reveals that D1R and D2R share 77% of their amino acid sequence  from the National Health and Medical Research Council (NHMRC) of Australia awarded to AJL, NHMRC Career Development Fellowship (grant number APP1083309) awarded to JHK and NHMRC Project grant (APP1063140) awarded to JHK and AJL. Australian Research Council Discovery Grant (DP150102496) awarded to JHK.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Space travel is associated with an exposure to low-dose rate ionizing radiation and the microgravity environment, both of which may lead to impairments in cardiac function. We used a mouse model to determine short- and long-term cardiac effects to simulated microgravity , continuous low-dose rate γ-irradiation, or a combination of HU and low-dose rate γ-irradiation.Cardiac tissue was obtained from female, C57BL/6J mice 7 days, 1 month, 4 months, and 9 months following the completion of a 21 day exposure to HU or a 21 day exposure to low-dose rate γ-irradiation , or a 21 day simultaneous exposure to HU and low-dose rate γ-irradiation. Immunoblot analysis, rt-PCR, high-performance liquid chromatography, and histology were used to assess inflammatory cell infiltration, cardiac remodeling, oxidative stress, and the methylation potential of cardiac tissue in 3 to 6 animals per group.The combination of HU and γ-irradiation demonstrated the strongest increase in reduced to oxidized glutathione ratios 7 days and 1 month after treatment, but a difference was no longer apparent after 9 months. On the other hand, no significant changes in 4-hydroxynonenal adducts was seen in any of the groups, at the measured endpoints. While manganese superoxide dismutase protein levels decreased 9 months after low-dose γ-radiation, no changes were observed in expression of catalase or Nrf2, a transcription factor that determines the expression of several antioxidant enzymes, at the measured endpoints. Inflammatory marker, CD-2 protein content was significantly decreased in all groups 4 months after treatment. No significant differences were observed in α-smooth muscle cell actin protein content, collagen type III protein content or % total collagen.This study has provided the first and relatively broad analysis of small molecule and protein markers of oxidative stress, T-lymphocyte infiltration, and cardiac remodeling in response to HU with simultaneous exposure to low-dose rate γ-radiation. Results from the late observation time points suggest that the hearts had mostly recovered from these two experimental conditions. However, further research is needed with larger numbers of animals for a more robust statistical power to fully characterize the early and late effects of simulated microgravity combined with exposure to low-dose rate ionizing radiation on the heart. Exposure to high doses of ionizing radiation may cause cardiac toxicity that involves pericarditis, fibrosis, conduction abnormalities, coronary heart disease and atherosclerosis ,2. WhileOne mechanism by which ionizing radiation may lead to cardiac toxicity is through increased oxidative stress. One characteristic of ionizing radiation is the formation of free radicals, e.g. reactive oxygen species (ROS) ,11. An iOxidation of glutathione (GSH) to form glutathione disulfide (GSSG) is an important buffering mechanism to reduce ROS levels. Hence, the ratio of tissue levels of GSH to GSSG is used as one indicator of oxidative stress.L-methionine (SAM), a major methyl donor [Another mechanism by which radiation may affect the heart is through DNA methylation, an important mechanism of maintenance of cellular homeostasis ,19. Cardyl donor . Impairmyl donor . O’learyyl donor . While myl donor  and 2) syl donor . ImportaIn addition to space radiation as an occupational hazard, microgravity also presents a unique challenge to an astronaut. Microgravity has been shown to play a role in epigenetics, or DNA methylation, in breast cancer cell lines, human lymphocytes, and lymphoblastoid cells. –29. FurtSince, the combined effects of low-dose rate radiation exposure and microgravity on the heart are largely unknown, the purpose of the current study was to evaluate indicators of oxidative stress and tissue remodeling in cardiac tissue obtained from the mice in the study of Mao et al. .All mice were handled and sacrificed by our co-author, XW Mao, at Loma Linda University under AUP 8130028 in a previous study . All datad libitum and were housed on a 12:12 hour light:dark cycle. Mice were allowed normal ambulation after completion of the 21 day treatment period and were sacrificed 7 days, 1 month, 4 months, or 9 months later. Hearts were collected, either fixed in 4% paraformaldehyde or flash frozen in liquid nitrogen, and shipped to the University of Arkansas for Medical Sciences for analysis. Mice were monitored daily during HU, γ-irradiation, or the combined HU+ γ-irradiation treatment and every other day following completion of their 21 day treatment. While the study began with a total 6 animals per group, HU is not always tolerated well by animals, and a total of 4 mice were removed from the study due to significant decreases in body weight (weight loss greater than 15% within 2 weeks). Additional samples were depleted through biochemical analyses, which limited the n size for some of the analyses. Sample size for each experimental group is indicated in Figure legends.All mice were provided standard mice chow and water 18 Shisheido column (Phenomenex® Inc.) via ESA Autosampler (ESA Inc.). HPLC was performed to assess levels of reduced glutathione (GSH), oxidized glutathione (GSSG), methionine, S-adenosyl-L-methionine (SAM), and S-adenosyl-L-homocysteine (SAH) and expressed in nmol/mg total protein. Glutathione oxidation was expressed as a ratio of GSH:GSSG and methylation potential was expressed as a ratio of SAM:SAH.Markers of oxidative stress and methylation state were measured by high-performance liquid chromatography with electrochemical detection (HPLC-ECD), as previously described ,39. BrieImmunoblot analysis was performed as previously described ,41. In b-ΔΔCT, where ΔΔCT = ΔCTsample− ΔCTcontrol and ΔCT represented the difference in cycle thresholds of a gene of interest and the housekeeping gene (18s rRNA).RNA isolation and rt-PCR was performed as previously detailed ,42. In sMouse hearts were excised and placed in 4% paraformaldehyde (7.4 pH) for 24 hours, rinsed with PBS and dehydrated with ethanol. After dehydration, mouse hearts where embedded in paraffin wax and sectioned (5 μm) for histology, conducted as previously described ,43.To assess inflammatory cell infiltration, longitudinal heart sections were rehydrated and incubated with 0.5% Toluidine Blue in 0.5 N HCL for 3 days at room temperature, followed with a 10 minute wash in 0.7 N HCL, and were counterstained with Eosin (1:4). Mast cells were counted using an Axioskop transmitted light microscope (Carl Zeiss).Cardiac remodeling was evaluated by collagen deposition and was determined using Sirius Red/Fast Green staining. Two series of sections were obtained from each heart, with the second series of histology samples sectioned approximately 50 μm deeper into the embedded mouse hearts, to provide a measure of collagen deposition throughout the heart. Heart sections were rehydrated and incubated with Picro Sirius Red supplemented with Fast Green. Sections were scanned using a ScanScope CS2 slide scanner, analyzed with ImageScope 12 (Leica Biosystems), and collagen deposition was determined as a percentage of the total tissue area, i.e. the relative area of collagen (Pico Sirius Red staining) to the total area of the tissue.t-test conducted within an ANOVA framework. With 3 mice per group, we had 0.80 power to detect a difference of 3.3 SDs. Here, SD is the root mean square error from the ANOVA.All values are means ± standard error of the mean (SEM). NCSS 8  was used to perform repeated measures analysis of variance (ANOVA) on collagen deposition in histology or one-way ANOVA for all other parameters, followed by Tukey-Kramer post-hoc tests of differences among individual experimental groups. Statistical significance was defined as p < 0.05. With 6 mice per group, we had 0.80 power to detect a difference of 1.7 SDs between an irradiated and sham group within a time point, based on a .05 significance level Cardiac tissue GSH  and GSSGA small reduction in cardiac MnSOD expression was observed 9 months after low-dose rate irradiation, but no alterations were seen in MnSOD at 4 months, or GPX 1/2 at 4 or 9 months . FurtherA small reduction in SAM  and incrSince cardiac adverse remodeling typically occurs late after exposure to ionizing radiation, indicators of remodeling and inflammatory infiltration were examined at the observation time points of 4 and 9 months. Histological staining did not reveal significant group differences in total % collagen, in the first series of sections obtained from each heart, the second series, or in a repeated measures analysis of all sections . AdditioMast cells were examined by histological staining with Toluidine Blue and by measuring mast cell tryptase content through immunoblot analysis. Histology did not reveal any significant differences in mast cell counts ; howeverCardiac CD-2 content was used as an indicator of T-lymphocyte infiltration. A significant decrease in CD-2 protein content was detected in all treatment groups compared to CTL in cardiac tissue collected 4 months after completion of HU, low-dose rate γ-irradiation or a combination of the two, but not in cardiac tissue collected at 9 months .The purpose of this study was to evaluate the impact of simulated microgravity and low-dose/dose-rate γ-irradiation on cardiac tissue. HU was used in this study as it is a widely accepted, terrestrial model of the mechanical unloading and fluid shifts that occur during exposure to microgravity ,37. HU wGlutathione, through its oxidation to glutathione disulfide, plays an important role in alleviating oxidative stress and has been used as an index of total tissue oxidative stress . In the -/- mice [Although large doses of γ-radiation have been shown to decrease SOD activity in rat hearts , low dos-/- mice . The stu-/- mice . Further-/- mice . In contSince the anti-oxidant glutathione may be modulated by the methionine cycle ,25,49, wHigh doses of radiation exposure are associated with structural remodeling of the heart, in part through increases in collagen content ,50,51. PWe have previously shown that cardiac remodeling due to high-dose x-ray exposure is associated with increased cardiac mast cell numbers . While tT-lymphocytes are an important modulator of the immune response, are highly sensitive to ionizing radiation , and amoIn summary, exposure to 21 days of HU, low-dose γ-irradiation, or a combination HU + low-dose γ-irradiation decreased GSH:GSSG ratios, SAM:SAH ratios, and the inflammatory marker CD-2 protein content. These data suggest that exposure to both low-dose rate radiation and microgravity decreases methylation potential of the heart. The anti-oxidant protein MnSOD showed a small decrease 9 months after exposure to low-dose γ-radiation. However, this difference was not reflected in the other oxidative stress proteins we evaluated. Our analyses of collagen expression and α-SMC actin revealed no significant cardiac remodeling. It is possible that our study was limited by sample size and that we did not assess the appropriate parameters that indicate cardiac remodeling after HU or low-dose/dose-rate γ-irradiation. Further research is required to fully characterize the early and late effects of simulated microgravity combined with low-dose rate radiation on cardiac tissue.S1 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or a combined HU + γ irradiation. Sample sizes: CTL for all time points, n = 6; HU for 7 day time-point, n = 4; HU for 1 month time-point, n = 5; HU for 9 month time-point, n = 6; 57Co for 7 day and 9 month time-points, n = 5; 57Co for 1 month time-point, n = 6; HU+57Co for 7 day time-point, n = 6; HU+57Co for 1 and 9 month time-points, n = 5. Values are means ± SEM. * Significantly different than CTL, p < 0.05.HPLC was utilized to determine methionine concentration in mouse heart a.) 7 days, b.) 1 month, or c.) 9 month after a 21 day exposure to HU, γ irradiation ((TIF)Click here for additional data file.S2 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or a combined HU + γ irradiation. Sample sizes: CTL for all time points, n = 6; HU for 7 day time-point, n = 4; HU for 1 month time-point, n = 5; HU for 9 month time-point, n = 6; 57Co for 7 day and 9 month time-points, n = 5; 57Co for 1 month time-point, n = 6; HU+57Co for 7 day time-point, n = 6; HU+57Co for 1 and 9 month time-points, n = 5. Values are means ± SEM. * Significantly different than CTL, p < 0.05; t Significantly different than HU, p < 0.05; # Significantly different than 57Co, p < 0.05.HPLC was utilized to determine methionine concentration in mouse heart a.) 7 days, b.) 1 month, or c.) 9 month after a 21 day exposure to HU, γ irradiation ((TIF)Click here for additional data file.S3 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or a combined HU + γ irradiation. Sample sizes: CTL for 4 month time-point n = 3; CTL for 9 month time-point n = 5; HU for 4 month time-point n = 5, HU for 9 month time-point n = 6; 57Co for 4 month time-point n = 6; 57Co for 9 month time-point n = 5; HU+57Co for 4 month time-point n = 4, HU+57Co for 9 month time-point n = 3. Values are means ± SEM. Statistical analysis did not reveal any significant differences.Immunoblot analysis was used to determine 4-HNE protein content a.) 4 months and b.) 9 months after a 21 day exposure to HU, γ irradiation ((TIF)Click here for additional data file.S4 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or combined HU + γ irradiation. All proteins normalized to GAPDH. Immunoblot sample sizes: CTL for 4 month time-point n = 4; CTL for 9 month time-point n = 5; HU for 4 month time-point n = 5, HU for 9 month time-point n = 6; 57Co for 4 month time-point n = 6; 57Co for 9 month time-point n = 5; HU+57Co for 4 month time-point n = 4, HU+57Co for 9 month time-point n = 3. Values are means ± SEM. * Significantly different than CTL, p < 0.05.Immunoblot analysis was used to determine protein content of a.) MnSOD 4 months, b.) MnSOD 9 months c.) GPX1/2 4 months, and d.) GPX1/2 9 months following HU, γ irradiation ((TIF)Click here for additional data file.S5 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or combined HU + γ irradiation. All proteins normalized to GAPDH. Immunoblot sample sizes: CTL for 4 month time-point n = 4; CTL for 9 month time-point n = 5; HU for 4 month time-point n = 5, HU for 9 month time-point n = 6; 57Co for 4 month time-point n = 6; 57Co for 9 month time-point n = 5; HU+57Co for 4 month time-point n = 4, HU+57Co for 9 month time-point n = 3. Values are means ± SEM. * Significantly different than CTL, p < 0.05.Immunoblot analysis was used to determine protein content of a.) Nrf2 4 months, b.) Nrf2 9 months c.) Catalase 4 months, and d.) Catalase 9 months following HU, γ irradiation ((TIF)Click here for additional data file.S6 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or a combined HU + γ irradiation. Sample sizes: CTL for all time points, n = 6; HU for 7 day time-point, n = 4; HU for 1 month time-point, n = 5; HU for 9 month time-point, n = 6; 57Co for 7 day and 9 month time-points, n = 5; 57Co for 1 month time-point, n = 6; HU+57Co for 7 day time-point, n = 6; HU+57Co for 1 and 9 month time-points, n = 5. Values are means ± SEM. * Significantly different than CTL, p < 0.05.HPLC was utilized to determine methionine concentration in mouse heart a.) 7 days, b.) 1 month, or c.) 9 month after a 21 day exposure to HU, γ irradiation ((TIF)Click here for additional data file.S7 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or a combined HU + γ irradiation. Sample sizes: CTL for all time points, n = 6; HU for 7 day time-point, n = 4; HU for 1 month time-point, n = 5; HU for 9 month time-point, n = 6; 57Co for 7 day and 9 month time-points, n = 5; 57Co for 1 month time-point, n = 6; HU+57Co for 7 day time-point, n = 6; HU+57Co for 1 and 9 month time-points, n = 5. Values are means ± SEM. * Significantly different than CTL, p < 0.05; t Significantly different than HU, p < 0.05; # Significantly different than 57Co, p < 0.05.HPLC was utilized to determine methionine concentration in mouse heart a.) 7 days, b.) 1 month, or c.) 9 month after a 21 day exposure to HU, γ irradiation ((TIF)Click here for additional data file.S8 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or a combined HU + γ irradiation. Sample sizes: CTL for all time points, n = 6; HU for 7 day time-point, n = 4; HU for 1 month time-point, n = 5; HU for 9 month time-point, n = 6; 57Co for 7 day and 9 month time-points, n = 5; 57Co for 1 month time-point, n = 6; HU+57Co for 7 day time-point, n = 6; HU+57Co for 1 and 9 month time-points, n = 5. Values are means ± SEM. Statistical analysis did not reveal any significant differences.HPLC was utilized to determine methionine concentration in mouse heart a.) 7 days, b.) 1 month, or c.) 9 month after a 21 day exposure to HU, γ irradiation ((TIF)Click here for additional data file.S9 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or a combined HU + γ irradiation. Sample sizes: all groups n = 4. Values are means ± SEM. Statistical analysis did not reveal any significant differences.rt-PCR was used to determine pro-collagen III fold change 9 months after a 21 day exposure to HU, γ irradiation ((TIF)Click here for additional data file.S10 Fig57Co: 0.01 cGy/h; 0.04 Gy total), or combined HU + γ irradiation. All proteins normalized to GAPDH. Immunoblot sample sizes: CTL for 4 month time-point n = 4; CTL for 9 month time-point n = 5; HU for 4 month time-point n = 5, HU for 9 month time-point n = 6; 57Co for 4 month time-point n = 6; 57Co for 9 month time-point n = 5; HU+57Co for 4 month time-point n = 4, HU+57Co for 9 month time-point n = 3. Values are means ± SEM. * Significantly different than CTL, p < 0.05.Immunoblot analysis was used to determine protein content of a.) α-SMC actin 4 months and b.) α-SMC actin 9 months following HU, γ irradiation ((TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.