Chronic gonadotropin-releasing hormone agonist (GnRHa) treatment is effective for the medical suppression of the hypothalamic-pituitary-gonadal axis in situations like central precocious puberty and gender dysphoria. However, its administration during the peripubertal period could influence normal brain development and function because GnRH receptors are expressed in brain regions that regulate emotions, cognition, motivation and memory. This study used an ovine model to determine whether chronic peripubertal GnRHa-treatment affected the developmental shift from preference of familiarity to novelty. Experimental groups included Controls and GnRHa-treated rams. To differentiate between effects of altered GnRH signaling and those associated with the loss of sex steroids, a group was also included that received testosterone replacement as well as GnRHa (GnRHa + T). Preference for a novel versus familiar object was assessed during 5-min social isolation at 8, 28 and 46 weeks of age. Approach behavior was measured as interactions with and time spent near the objects, whereas avoidance behavior was measured by time spent in the entrance zone and attempts to escape the arena via the entry point. Emotional reactivity was measured by the number of vocalizations, escape attempts and urinations. As Control and GnRHa-treated rams aged, their approach behaviors showed a shift from preference for familiarity (8 weeks) to novelty (46 weeks). In contrast, relative to the Controls the GnRHa + T rams exhibited more approach behaviors towards both objects, at 28 and 46 weeks of age and preferred familiarity at 46 weeks of age. Vocalisation rate was increased in GnRHa treated rams in late puberty (28 weeks) compared to both Control and GnRHa + T rams but this effect was not seen in young adulthood (46 weeks). These results suggest that the specific suppression of testosterone during a developmental window in late puberty may reduce emotional reactivity and hamper learning a flexible adjustment to environmental change. The results also suggest that disruption of either endogenous testosterone signalling or a synergistic action between GnRH and testosterone signalling, may delay maturation of cognitive processes (e.g. information processing) that affects the motivation of rams to approach and avoid objects. As such, its importance in the regulation of pituitary-gonadal function has long been recognized. The presence of GnRH (types I and II) and GnRH receptors in other tissues , includiWithin human and veterinary medicine, GnRH agonists (GnRHa) are used chronically to suppress activity within the hypothalamo-pituitary-gonadal axis (HPG). This is possible, as continuous activation of GnRH receptors leads to G protein receptor uncoupling, followed by internalization and recycling of receptors  and subsUsing an ovine model, we previously reported sex- and age-dependent effects of chronic peripubertal GnRHa-treatment on aspects of cognition, behavior and emotional reactivity . The intNovelty preference behavior can provide valuable insights to cognitive factors that influence motivation, as it reflects the outcome of judging associated risks against possible reward under different test conditions . In mammThe aims of the present study were to 1) investigate the effects of chronic peripubertal GnRHa-treatment on novelty preference behavior as a measure of cognitive processes that influence motivation, and 2) differentiate between the effects of chronically disrupted peripubertal GnRH and gonadal steroid signaling in males before and after puberty.22.1ad libitum access to hay or silage and other nutritional supplements according to standard management practices.The research was conducted at the University of Glasgow Cochno Farm and Research Centre (55° 55′N) in accordance with Home Office Regulations (Project License: 60/4422). Animals were male Scottish Mule Texel crosses born between 23 March and 12 April 2013 . All animals came from single sex litters, to control for any effects of differences in the prenatal steroid environment associated with female siblings. Lambs were maintained with their dams until weaned at approximately 21 weeks of age. All sheep were grazed on pasture, except during behavioral trials, when indoor-housed sheep had n = 60); 2) chronic GnRHa-treated rams ; and 3) chronic GnRHa-treated rams that also received testosterone replacement therapy  to tease apart treatment effects associated with either GnRH or gonadal sex steroids. GnRHa-treatment  commenced at 8 weeks of age  and was administered every fourth week, until 1 year of age. To mimic the endogenous testosterone concentrations expected in the Controls, testosterone was replaced from 16 weeks of age until the end of the experiment via 2-weekly intramuscular injection of testosterone cypionate  dissolved in vegetable oil as previously described  untreated Control rams  and body weight (kg) recorded. Additional daily blood samples were collected from 10 GnRHa + T animals for 14 consecutive days in October (28–30 weeks of age) to characterize circulating testosterone concentrations in response to exogenous testosterone cypionate treatment . Blood samples (3 mL) were collected by jugular venipuncture and placed in heparin-treated tubes. After centrifugation at 5000 escribed , followi2.3Novelty preference behavior was assessed over 2–4 days, at three developmental ages: 8 weeks , 28 weeks  and 46 weeks . At each age, animals from the three groups were assessed in a random order . The indoor test arena  recorded. Emotional reactivity was assessed by counting the number of vocalizations, escape attempts and urinations/defecations. An escape attempt was defined as a proactive effort to move through, over or under the border of the test arena. Revision of video footage was used to determine the time spent in each zone within the test arena. Presence in a zone was defined from when one foreleg was placed within that zone.2.42/kg). Time spent near an object was defined as the time an animal was within the inner and outer zones surrounding each object. Novelty preference was defined as the percentage of time spent near the novel object, relative to the total time spent near both objects, and the number of exploratory interactions with the novel object relative to the total number of object interactions. Statistical analyses were performed with R software  using the RStudio interface . All variables were analyzed with two-factorial ANOVA (Treatment × Age) using linear mixed-effects models with repeated measures (sheep identity specified as random factor) and the data distribution specified as Gaussian for continuous data  or Poisson for count data . To differentiate between the effects of GnRH and testosterone, respectively, two-tailed t-tests (or Wilcoxon signed-rank tests for non-parametric data) were used at each age . All values presented are the mean and standard error of the mean of untransformed data. There were no between group differences, for any variable, prior to the start of the treatment at 8 weeks of age and, therefore, overall population means are presented in tables and figures. For all statistical tests an alpha-level of 0.05 was considered statistically significant and where alpha <0.09, trends are reported.As statistical analysis indicated that body weight influenced testes size, the measure of testes size used in the analyses was calculated as (testes length × circumference)/body weight (cm33.1P < 0.001). After 8 weeks of age, testes size was similar in both GnRHa-treated groups, but significantly (P < 0.001) lower compared to the Controls. While mean circulating testosterone concentrations increased in all groups, this increase was significantly delayed in both GnRHa-treated groups compared to Controls (Treatment × Age P = 0.004). Testosterone concentrations were significantly lower in GnRHa rams (P < 0.05) between 20–32 weeks of age. It should be noted that values in In Control rams, plasma testosterone concentrations and testes size increased from 12 weeks of age  and reac3.23.2.1At all ages, rams spent the most time in the neutral zone . OverallP = 0.335) with age as the novelty preferences across all animals assessed at 8, 28 and 46 weeks of age were 46 ± 2.8, 47 ± 1.8, and 50 ± 1.6%, respectively.The amount of time animals spent near the novel and familiar objects  signific3.2.2P < 0.05; 8 wk: 45 ± 2.7%, 28 wk: 52 ± 2.3%, 46 wk 51 ± 2.0%). There were no statistically significant difference in these parameters between weeks 28 and 46.The number of explorations of the familiar object decreased with age (Tables 1 & 2), whereas it remained unchanged for the novel object , Table 23.2.3P < 0.001) and urinations (P < 0.05) during the testing period with the largest change seen between 8 and 28 weeks of age (P < 0.001) more likely to make escape attempts  and a similar trend (P = 0.076) at 46 weeks of age. At 28 weeks of age the GnRHa+T rams spent more (P < 0.05) time in the outer zone of the novel object than the other groups, which was also reflected by a trend (P = 0.053) for GnRHa+T rams to spend more time near the novel object. However, at 46 weeks of age the GnRHa+T rams spent (P < 0.01) more time near the familiar object  novelty preferences (39±4.2%) than the Control (53±2.4%) and GnRHa (52 ± 2.4%) groups .Comparisons of treatment groups at 28 and 46 weeks of age indicated significant differences with regard to the time spent in different zones and explorations of the objects . The GnRr object  compared3.3.2P = 0.021, Treatment × Age P = 0.333) on object interaction preference, whereby at 46 weeks of age GnRHa+T rams displayed a greater preference . GnRHa rams vocalized more than GnRHa+T rams at 28 weeks of age and tended (P = 0.058) to vocalize more than Control rams  or if the effects in the GnRHa + T rams are the result of maintained high concentrations of exogenous testosterone in this group relative to the Controls.4.3While in the arena, the motivation of animals to approach objects was assessed by the time spent near the objects, which decreased from 8 to 28 weeks of age in Controls. This contrasts with an age dependent increase (between 28 to 46 weeks) in avoidance behavior, which for this study was reflected by the time spent in the entrance zone and the number of escape attempts, where animals expressed typical flight behaviors as they searched for their entry point and/or expressed a strong desire to return to their social peers. It is of note that many animals and humans share elements that regulate the functions of escape behaviors which take precedence over all other activities including social behavior, although some species or prey-predator differences may exist in safety and defense strategies . If not In the present study, the lack of an effect of GnRHa on the post-pubertal increase in avoidance and decrease in approach behaviors was similar to a previous study examining responses of male and female GnRHa treated  sheep to a novel object . However4.4The decrease in the number of vocalizations and urinations with age in Control rams reflects the maturational change in emotional reactivity reported previously  through which GnRHa may affect the neural systems is poorly understood. Results that indicate persisting effects of elevated GnRH on cognitive function when sex steroids are replaced, argue against the hypothesis that these effects are secondary to the loss of sex steroids and this is supported by extensive studies in elderly men and women . Even in5The disruption of both peripubertal GnRH and testosterone signaling interfered with the normal maturational changes expected in certain aspects of cognitive processes that regulate motivation. There appears to be a window during late puberty or early adulthood where suppression of testosterone is associated with lower emotional reactivity, which will be of relevance for patients receiving GnRHa-treatment during this time. Increased motivation to approach objects and preferences for familiarity during young adulthood following peripubertal GnRHa and exogenous testosterone co-treatment may reflect reduced cognitive processes associated with the prefrontal cortex (e.g. reduced information processing or exaggerated approach as seen with adolescent risk-taking). The distinct difference between the GnRHa + T versus the GnRHa and Control groups may have resulted from loss of endogenous patterns of testosterone secretion, or a synergistic action between GnRH and testosterone signaling on selected cognitive processes during the peripubertal period. Imbalance of the fundamental (across species) motivational tendencies for approach and avoidance when encountering novel objects or situations may lead to reduced novelty seeking, and consequently hamper learning and flexible adjustment to environmental change. The findings may be of clinical relevance where chronic peripubertal GnRHa-treatment is administered with or without exogenous sex steroid treatments during this sensitive window as is the case in central precocious puberty and gender dysphoria.Declarations of interest: noneThe authors declare no conflict of interest.


The data presented here corresponds to a comprehensive design of experiments conducted separately for both neat PHBV polymer and wood–PHBV composites, in which the effects of temperature profile, screw speed, feeding rate, feeding method, screw configuration, and wood contents (wood–PHBV composites only) of 10, 20, 30, and 40 wt% wood content were examined. For each processing condition, 5 specimens were tested under uniaxial tensile loading. Here we provide the complete set of extrusion parameters, including the observed screw torque, residence time and material output. Individual stress–strain curves for each specimens are provided, along with their calculated elastic modulus, strength, and strain at maximum load. The data is also provided as support material for the research article: “Extrusion of wood fibre reinforced Poly (PHBV) biocomposites: statistical analysis of the effect of processing conditions on mechanical performance” .This article features a large database on different extrusion processing conditions and the resulting tensile properties of poly(hydroxybutyrate- Specifications tableValue of the data•The database presented here can be used to find optimal processing parameters for neat PHBV polymer and wood–PHBV composites of different wood contents.•The data is useful to understand the effect and contribution of individual extrusion parameters on the mechanical properties of PHBV and wood–PHBV materials.•The calculated tensile properties can serve as a benchmark for the performance of PHBV and wood–PHBV composites, in comparison to other PHA copolymers.•The mean and standard deviation values of mechanical properties can be used to determine design allowables for a component design.1https://doi.org/10.17632/9fzzyp63hp.1.This article features raw stress–strain tensile data with the calculated tensile modulus, strength and strain at maximum load for more than 300 specimens corresponding to PHBV and wood–PHBV materials extruded under different conditions. For each sample the extrusion processing parameters are reported and include: feeding method, feeding rate, fibre ratio, screw configuration, screw speed, temperature profile, observed screw torque, residence time, and material output. Finally, we include a summary table with processed data for neat PHBV and wood–PHBV material separately. The complete data can be found in the file ‘Data in Brief - wood–PHBV Composites.zip’ available in the Mendeley data repository under the following identifier DOI: 22.1Full Design of Experiments’ is the order in which samples were run through the extruder. The labelling nomenclature was 1A to 32A for neat PHBV material, and 1B to 32B for wood–PHBV composites. Within each sample, individual dog bone specimens were labelled 1–5. A color code is used in the file ‘Full Design of Experiments’ to help identify the four different levels of varying extrusion parameters. The recorded output values include screw torque (N m), residence time (s), material output (g/min) during extrusion (shown in Full Design of Experiments’.A design of Experiment (DoE) through a 32-run Taguchi orthogonal array was applied for this investigation. 1 factor at 2 levels and 4 factors at 4 levels (L32 2ss1 4ss4) were used to define experimental runs for wood–PHBV composites. The full test matrix, including manufacturing parameters and the values investigated is shown in shown in , and ten2.2co-3-hydroxyvalerate) (PHBV) with 1 mol% 3- (HV) content. This was purchased from TianAn Biopolymer, China, in powder form under the trade name of ENMAT Y1000 3, and a reported melt flow index (MFI) of 5.2 g/10 min at 180 °C, using a weight of 2.16 kg, according to ISO 1133 The type of polyhydroxyalkanoate (PHA) polymer used was Poly wood fibres from radiata pine wood, were supplied as unbleached by Norske Skog paper mill, Boyer, Tasmania. A contoured weighted average length L(c) of 2.36 mm, fibre width (W) of 33.6 μm, and a fibre wall thickness  of 5.0 μm were obtained through characterisation with a FiberLab2.3A EuroLab 16 XL co-rotating twin screw extruder  with a diameter of 16 mm and a length-to-diameter ratio of 40:1 was used for this work. Residence time was measured for each extrusion run by introducing ~1 g of red tracer and monitoring the time until it was first observed at the die exit. Temperature profiles at 170, 180, 190, and 210 °C were investigated in this study. The individual barrel temperature for these temperature profiles is described in Full Design of Experiments’ and referred to as ‘Standard Mixing zone’ and ‘Aggressive mixing zone’.The screw configurations chosen for this study is shown in 2.4Raw Data for mechanical testing – Samples A’ and ‘Raw Data for mechanical testing – Samples B’ for neat PHBV material and wood–PHBV composites respectively. More specifically the raw data files include values for time (s), extension (mm), load (N), video axial strain (%) and tensile strength (MPa), as well as each specimen dimensions  and the test date. In this work, tensile strength was calculated as the tensile stress at maximum load (in MPa). Tensile modulus (in GPa) was calculated by the linear trend from fitting between 0% and 2.0% strain, while strain  was measured at the point of maximum load. A summary of all the processed data for neat PHBV material and wood–PHBV composites is available in root folder in the files ‘Summary of mechanical testing – Samples A’ and ‘Summary of mechanical testing – Samples B’ for neat PHBV and wood–PHBV composites respectively.Specimens to be tested were conditioned at a controlled temperature of 25 °C and humidity of 40–60%, during two weeks prior to testing. This allows sufficient time for secondary crystallisation of PHBV


Halorubrum sodomense) in pH 8 Tris buffer was studied. The samples were photoexcited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_580 (absorption maximum at λmax ≈ 580 nm), and the retinal Schiff base photoisomerization and protonation state changes were followed by absorption spectra recordings during light exposure and after light exposure. Ret_580 turned out to be composed of two protonated retinal Schiff base isomers, namely Ret_580I and Ret_580II. Photoexcitation of Ret_580I resulted in barrier-involved isomerization to Ret_540 (quantum yield ≈ 0.056) and subsequent retinal proton release leading to Ret_410 deprotonated retinal Schiff base (RSB). In the dark, Ret_410 partially recovered to Ret_580I and partially stabilized to irreversible Ret_400 due to apoprotein restructuring (Ret_410 lifetime ≈ 2 h). Photoexcitation of Ret_580II resulted in barrier-involved isomerization to Ret_640 (quantum yield ≈ 0.00135) and subsequent deprotonation to Ret_370 (RSB). In the dark, Ret_370 partially recovered to Ret_580II and partially stabilized to irreversible Ret_350 due to apoprotein restructuring (Ret_370 lifetime ≈ 10 h). Photocycle schemes and reaction coordinate diagrams for Ret_580I and Ret_580II were developed and photocyle parameters were determined.The retinal photocycle dynamics of the fluorescent voltage sensor QuasAr1 (Archaerhodopsin 3 P60S-T80S-D95H-D106H-F161V mutant from  Tracking membrane potential of cells, especially neurons, using fluorescence methods is of high interest and is an active field of research (change of membrane voltage causes change of fluorescence efficiency) ,7,8,9,10All-optical electrophysiology in neuroscience was achieved by channelrhodopsin based optical perturbation of membrane potentials and the membrane potential readout with fluorescent voltage sensing domains ,27,28.Halorubrum sodomense /l, (l is sample length) were calculated, and absorption coefficients, αa(λ), were determined by subtracting scattering coefficient contributions, αs(λ), according to αa(λ) = α(λ)−αs(λ). The scattering coefficient spectrum was approximated by the empirical relation [0 is selected in the transparency region and γ ≤ 4 is fitted to the experimental attenuation in the transparency region =αsails see ).2, sample thickness along excitation path 1.5 mm, and transmission detection path length 3 mm). The excitation power Pexc was measured with a power meter . In the study of the absorption coefficient spectra development, transmission spectra T(λ) were recorded repeatedly during the period of light exposure and after light switch-off . The temporal development of the absorption behavior of QuasAr1 at selected wavelengths was carried out with a temporal resolution of 12.5 ms.For the absorption spectroscopic photocycle experiments, QuasAr1 samples were excited with light emitting diodes  or with a He-Ne laser emitting at 632.8 nm . The sample cell in the spectrophotometer was irradiated transverse to the transmission detection path (exposed area 3 × 5 mmEF(λ), the fluorescence quantum yields ϕF, and the fluorescence excitation quantum distributions Eex(λ) are given in [Fluorescence spectroscopic measurements immediately after the end of photoexcitation and after excitation recovery were carried out with a spectrophotometer . Details of the determination of the fluorescence quantum distributions given in . The fluHalorubrum sodomense was studied in detail. Its dominant protonated retinal Schiff base Ret_580 absorption band around 580 nm was found to consist of two isomers Ret_580I (likely a cis isomer) and Ret_580II (likely a trans isomer) stabilized by different adjacent apoprotein amino acid arrangements. Their slow barrier-involved photoisomerization dynamics in the tens of picosecond regime and the low quantum efficiency of photoisomerization are thought to be responsible for the high fluorescence efficiency and high membrane voltage sensitivity of QuasAr1.The photocycle dynamics of the Archaerhodopsin 3 based fluorescent voltage sensor QuasAr1 from trans) from the educt Ret_580I, and Ret_640 (likely PRSBcis) from the educt Ret_580II, deprotonate slowly on a time scale of tens of seconds to the neutral retinal Schiff bases Ret_410 and Ret_370, respectively. The long lifetimes of the metastable photoisomers Ret_540 and Ret_640 cause strong excitation intensity dependent back photoisomerization to the primary isomers Ret_580I and Ret_580II.The primary photoisomerization products, Ret_540 (likely PRSBI and Ret_580II occurred on a timescale of several hours. During this long time period, thermal apoprotein restructuring led to a stabilization of the deprotonated retinal Schiff base isomers, Ret_410 to Ret_400 and Ret_370 to Ret_350, leading to an incomplete recovery to the originals Ret_580I and Ret_580II in the photocycle process.The reprotonation and back isomerization of the deprotonated retinal Schiff bases Ret_410 Ret_370 to the original isomers Ret_580The performed photocycle studies on QuasAr1 are hoped to be of value for the application of this fluorescent voltage sensor in cell membrane and neuronal function studies.


It is widely believed that rewarming slowly after therapeutic hypothermia for hypoxic-ischemic (HI) encephalopathy can improve outcomes, but its impact on white matter injury after HI is unclear. Fetal sheep (0.85 gestation) received 30 min ischemia-normothermia (n = 8), or hypothermia from 3–48 h with rapid spontaneous rewarming over 1 h , or 48 h with slow rewarming over 24 h  or 72 h with rapid rewarming . Ischemia was associated with loss of total and mature oligodendrocytes and reduced area fraction of myelin basic protein (MBP) and 2′,3′-cyclic nucleotide 3′-phosphodiesterase  and increased microglia and astrocytes. Total numbers of oligodendrocytes were increased by all hypothermia protocols but only ischemia-72 h hypothermia attenuated loss of mature oligodendrocytes. All hypothermia protocols similarly increased the area fraction of MBP, whereas there was only an intermediate effect on the area fraction of CNPase. Microglia were suppressed by all hypothermia protocols, with the greatest reduction after ischemia-72 h hypothermia, and an intermediate effect after ischemia-slow rewarming. By contrast, induction of astrocytes was significantly reduced only after ischemia-slow rewarming. In conclusion, slow rewarming after hypothermia did not improve oligodendrocyte survival or myelination or suppression of microgliosis compared to fast rewarming, but modestly reduced astrocytosis. Critically, hypothermia was associated with improved long-term neurocognitive and motor impairment in survivors2. Although most of the parameters required for optimal neuroprotection with hypothermia including the window of opportunity, depth and duration are now well understood3, there is no human controlled evidence for the optimal rate of rewarming. There is accumulating evidence from preclinical models that much of the effects of rewarming are mediated by the total duration of hypothermia4. It is important to appreciate that although the focus of most studies of neuroprotection has been on neuronal loss, both clinically and experimentally, HIE at term is associated with significant white matter injury. The effect of the rate of rewarming on white matter injury is unknown.Therapeutic hypothermia initiated in the first 6 hours after birth and continued for 72 h significantly reduces death or moderate to severe disability after hypoxic-ischemic encephalopathy (HIE) in term and near-term infants6. For example, in infants with moderate to severe HIE, abnormal signal intensity in the posterior limb of the internal capsule on magnetic resonance imaging (MRI) is highly associated with impaired independent walking at 2 years of age6. Similarly, in a cohort of 84 term infants with HIE with normal appearance of the basal ganglia and thalami on MRI, the severity of white matter injury was significantly correlated with cognitive, visual, language, behavioral and seizure problems7.The predominant clinical patterns of white matter injury include damage to the posterior limb of the internal capsule and injury of cortical white matter in a watershed distribution9. Therapeutic hypothermia started 90 minutes after global cerebral ischemia significantly improved white matter recovery, but the benefits were markedly attenuated when the initiation of hypothermia was delayed to 3 or 5.5 hours10. We have recently shown that compared with hypothermia starting 3 hours after ischemia and continued until 72 hours, 48 hours of hypothermia was associated with a marked deterioration of electrographic (EEG) power following rewarming and reduced neuronal survival4. For these protocols, fetal sheep rewarmed spontaneously over approximately 1 hour. We then tested the possibility that slower rewarming might alleviate EEG deterioration after the 48 hour protocol11. Intriguingly, after hypothermia for 48 hours, controlled slow rewarming over 24 h at ~0.2 °C/h was associated with improved recovery of EEG power compared to rapid spontaneous rewarming, but inferior grey matter survival compared to rapid spontaneous rewarming at ~5 °C/h. Notably, both fast and slow rewarming after 48 h of hypothermia showed reduced histological neuroprotection compared to 72 hours of hypothermia with rapid rewarming, suggesting that the effect of slow rewarming was simply mediated by the longer total duration of cooling. In the present study, in the same experimental cohort11, we tested the hypothesis that the better electrographic recovery despite no change in neuronal survival with slow rewarming after 48 hours of hypothermia was mediated by improved white matter protection.Consistent with this, 30 minutes of global cerebral ischemia in near-term fetal sheep was associated with profound loss of oligodendrocytes, loss and disruption of myelination and increased numbers of microglia in the intragyral and periventricular white matter11. All procedures and methods were approved by the Animal Ethics Committee of The University of Auckland under the New Zealand Animal Welfare Act, and the Code of Ethical Conduct for animals in research established by the Ministry for Primary Industries, Government of New Zealand. The experiment has been reported in compliance with the ARRIVE guidelines12.We have previously published the pH, blood gas, temperature changes, electrophysiological data and neuronal survival from the same cohort of animals as the present study2) after intubation . Ewes were ventilated and under constant isotonic saline infusion (at approximately 250 mL/h) during surgery. Trained anesthetic staff monitored maternal heart rate, blood pressure and depth of anesthesia. Following sterilization of primary surgical fields, a laparotomy was conducted along the linea alba, followed by a 5–6 centimeter (cm) uterine incision in an area free of cotyledons and in parallel to major uterine blood vessels to expose the fetus.At 125–126 days gestation (term is 145 days), 40 ewes underwent surgery for fetal instrumentation under sterile conditions. Food, but not water, was withdrawn 18 hours before surgery. Holding rooms were maintained at a constant temperature of 16 ± 1 degrees Celsius (°C) and 50 ± 10 percent (%) humidity on a 12 hour (h) light/dark cycle (light hours 06:00–18:00). Ewes were administered long acting oxytetracycline at 1 milliliter (mL)/10 kilogram (kg)  intramuscularly (i.m.) for prophylaxis. Anesthesia was achieved by intravenous (i.v.) propofol  and maintained with 2–3% isoflurane in oxygen  were placed bilaterally around the carotid arteries for induction of cerebral ischemia. Furthermore, the right carotid artery received an ultrasound flow probe  for measuring of carotid blood flow (CaBF).Polyvinyl catheters were placed in both fetal brachial arteries to measure mean arterial blood pressure (MAP). A catheter to the amniotic cavity was secured to the fetal shoulder. Subcutaneous electrocardiogram (ECG) electrodes  were sutured onto the right shoulder and the left fifth intercostal space for monitoring of fetal heart rate (FHR). To prevent vertebral blood supply to the carotid arteries during ischemia, the vertebral-occipital anastomoses were ligatedTwo pairs of electroencephalograph (EEG) electrodes made of 7 stranded stainless steel wire  were placed on either hemisphere over the parasagittal parietal cortex . Electrodes were secured on the dura with cyanoacrylate glue and a reference earth wire sewn over the fetal occiput. Cortical impedance was measured by placement of a third pair of electrodes 5 mm laterally to the EEG electrodes . An additional pair of electrodes (Cooner Wire) was sewn into the nuchal muscle to record fetal electromyogram (EMG) activity. Extradural temperature was measured by inserting a thermistor  over the parasagittal dura (30 mm anterior to the bregma). A second thermistor was placed into the esophagus for measuring of body temperature. A cooling cap made of silicon tubing  was placed onto the fetal head and secured with sutures.The fetus was returned to the uterus and warm, sterile saline (approximately 500 mL at 37 °C) was added to counteract loss of amniotic fluid. All leads were exteriorized through an incision made into the maternal flank. Gentamicin  was administered to the amniotic cavity. Bupivacaine (10 ml of 0.5%) and adrenaline  were administered onto the skin of the maternal laparotomy incision. A catheter was inserted into the maternal long saphenous vein for post-operative care and euthanasia.ad libitum access to food and water. Prophylactic antibiotics were administered i.v. to the ewe daily for 4 days after surgery . Fetal wellbeing was monitored by continuous tracing of MAP, FHR, CaBF, and EEG activity as well as fetal blood sampling. Heparinized saline was continually infused (20 U/mL at 0.2 mL/h) to ensure that fetal catheters were maintained patent, whilst the maternal catheter was flushed daily with heparinized saline.Animals were housed together in individual metabolic cages with Data recording began 24 hours before the start of the experiment and continued for the remainder of the experiment. Data were recorded and saved continuously to disk for off-line analysis using custom data acquisition programs . Arterial blood samples were taken for preductal pH, blood gas, base excess , glucose and lactate measurements . All fetuses had normal baseline biochemical values for their gestational ages.Experiments began at 129 ± 1 day (d) of gestation. Experiments were generally conducted at 9:00 am. Blood samples were taken two, four, and six hours after the end of ischemia, and daily thereafter. Animals were randomized into five groups: sham ischemia followed by normothermia ; ischemia followed by normothermia ; ischemia followed by 72 hours of hypothermia ; ischemia followed by 48 hours of hypothermia ; or ischemia followed by 48 hours of hypothermia and 24 hours of slow rewarming .Cerebral ischemia was achieved by inflation of both balloons of each silicone carotid occluder with varying volumes of sterile saline and double-clamping of balloon inflation lines with clamps. Occlusion was confirmed successful where a rapid fall in EEG activity and in CaBF, as well as a delayed increase in cortical impedance was observed. Occlusion was maintained for 30 minutes and terminated immediately thereafter by release of clamps. The return of at least 80% CaBF was used to confirm successful reversal of occlusion. Successful occlusions also showed continuing suppression of EEG background after occlusion (≤10 dB).Cooling was initiated 3 hours after the end of occlusion by linking the cooling coil over the fetal scalp to a pump  in a cooled water bath and circulating cold water through the cooling coil. The target extradural temperature was 31–33 °C. Cooling was stopped at 72 hours in the ischemia-72 h hypothermia group, and 48 hours in both the ischemia-48 h hypothermia and slow rewarming groups. In the sham-ischemia and ischemia-normothermia groups, the water was not circulated and the cooling coil remained in equilibrium with fetal temperature.In the ischemia-72 h hypothermia and ischemia-48 h hypothermia groups, rapid rewarming was induced by switching off the cooling machine and allowing temperature to spontaneously return to baseline. In the ischemia-slow rewarming group, fetuses were rewarmed over 24 hours with a computer-controlled linear increase in bath temperature up to 39 degrees, at 0.2 °C per hour.Ewes and fetuses were euthanized with an overdose of sodium pentobarbitone  administered into the saphenous vein 7 days after ischemia. Numbers of fetuses per pregnancy, sex, fetal body weight, and individual organ weights  were recorded. The brain was removed and fixed for histological assessment of cerebral injury.In situ fixation of fetal brains was achieved by perfusion of carotid arteries with 500 mL of 0.9% saline solution followed by 1000 mL of 10% phosphate buffered formalin and embedded using a standard paraffin tissue preparation13. Coronal slices of 10 micrometers (μm) in thickness were cut and mounted on chromalum-gelatine coated slides. Slices were obtained from the level where the dorsal hippocampus becomes apparent.20 450 ml, citric acid 8 mL and sodium citrate 42 mL). Sections were washed in PBS (3 × 5 min each). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide in methanol for 30 minutes at room temperature covered by tin foil to prevent exposure to light. Sections were washed in PBS (3 × 5 min each) and blocked with 300 microliters (μL) of 3% normal goat serum  in PBS.The tissue was deparaffinized in xylene (2 × 15 min). Tissue was rehydrated at decreasing concentrations of ethanol . Sections were washed 3 times for 5 minutes each in phosphate buffered saline  (2 L of 10x PBS (0.1 M): 160 g NaCl, 4 g KCl, 28.8 g Na2HPO4, 4.8 g KH2PO4, pH 7.4). Antigen retrieval was carried out via the pressure cooker  method using a citrate buffer (dHSections were incubated overnight at 4 °C in primary antibodies (1:200). These included mouse anti-ionized calcium-binding adapter molecule 1  as a marker of microglia/macrophages; mouse anti-myelin basic protein  as a marker of myelination, mouse anti-2′,3′-Cyclic-nucleotide 3′-phosphodiesterase  as a marker of immature/mature oligodendrocytes; mouse anti-APC antibody  as marker of mature oligodendrocytes; mouse anti-oligodendrocyte lineage transcription factor 2  as a marker of total oligodendrocytes and rabbit anti-glial-fibrillary-acidic protein  as a marker of astrocytes in 3% NGS.After washing with PBS (3 × 5 min each), sections were incubated at room temperature for 3 hours in 1:200 biotinylated goat anti-mouse  or anti-rabbit  secondary antibody in 3% NGS. Incubation in 1:200 ExtrAvidin horseradish peroxidase  was conducted for 2 hours at room temperature. 3,3 diaminobenzidine  was used to visualize positive staining. The tissue was visualized by microscopy to limit background staining. Staining was stopped by immersion in PBS solution. Slides were then dehydrated in increasing alcohol concentrations  and xylene (2 × 15 min) before being mounted  with coverslips.Two sections per animal were imaged using the Nikon eclipse 80i light microscope  for each antibody. Iba-1, Olig-2 and CC-1 stained sections were imaged at 20x magnification, whilst MBP, GFAP and CNPase at 40x magnification using the NIS elements Basic Research software . Images were taken from the intragyral white matter tract of the first parasagittal gyrus (IGWM1) and second parasagittal gyrus (IGWM2) and the periventricular white matter (PVWM) from both hemispheres. A total of 4 fields of view were obtained for each region from each animal.Immunohistochemistry images were quantified using Image J software . Iba-1 + ve microglia/macrophages, GFAP + ve astrocytes, Olig-2 + ve oligodendrocytes and CC-1 + ve mature oligodendrocytes were quantified by manual counting of each positively labelled cell. Both resting (ramified) and active (amoeboid) microglia were included. Quantification of the area fraction present with staining of MBP, CNPase and GFAP was performed using the Auto (MBP) and Huang (CNPase) and Mean (GFAP) auto-threshold settings on Image J .The effects of cerebral ischemia and hypothermia on physiological and histological parameters were assessed with a mixed-design ANOVA, using time or region as a repeated measure . When statistical significance was found, post-hoc comparisons were performed with univariate ANOVA and Tukey correction. Non-parametric data were assessed using the Kruskal–Wallis test. Statistical significance was reported when P < 0.05, and data are presented as mean ± standard error of the mean (SEM).There were no differences in sex or fetal body or organ weights or proportion of singletons between groups at post-mortem Table . Ischemi11. Esophageal temperature decreased to 37 ± 1.0 °C during hypothermia (P < 0.05). In the ischemia-48h hypothermia and ischemia-72h hypothermia groups, the end of cooling was marked by a rapid increase in brain temperature at 48 and 72 hours, respectively. In contrast, in the ischemia-slow rewarming group, a gradual increase in brain temperature (0.2 °C/hour) was observed from 48 to 72 hours after ischemia.Hypothermia was associated with a significant decrease in brain temperature to 31.3 ± 0.2 °C in the ischemia-72 h hypothermia group and 32.3 ± 0.3 °C in the ischemia-48 h hypothermia and ischemia-slow rewarming groups, compared with 39.5 ± 0.1 °C in the ischemia-normothermia group (p < 0.05)st (IGWM1) and 2nd (IGWM2) parasagittal gyri and periventricular white matter (PVWM) compared to sham control (P < 0.05), which was partially attenuated by all hypothermia protocols  but a trend towards reduced total area fraction P = 0.068) in all white matter regions compared to sham control  are very common after HIE and both are highly associated with subsequent neurodevelopmental impairment19. However, this was based on limited evidence from case studies suggesting that fast rewarming might lead to hypotension or rebound seizures21, and empirical concerns arising from preclinical studies that it might reverse the depression of potentially injurious processes23. There is only limited evidence from animal studies that speed of rewarming after prolonged hypothermia can materially affect neurological outcomes. In neonatal piglets exposed to hypoxia-ischemia, 18 hours of hypothermia followed by rewarming at 0.5 °C/h was associated with less caspase-3 activation in the cerebral cortex and white matter tracts compared to rewarming at 4 °C/h25. However, 18 hours is a very short, highly suboptimal duration of hypothermia. There is now considerable preclinical evidence from both adult and neonatal studies that following delayed initiation of treatment, hypothermia must be continued for approximately 72 hours to achieve maximal neuroprotection26. Thus, as these previous studies did not control for total duration of cooling25, it is likely that the apparent reduction in white matter injury after slow rewarming was mediated by the increased total duration of therapeutic hypothermia.The optimal speed of rewarming for infants with HIE after therapeutic hypothermia to reduce white matter injury is unknown. The randomized clinical trials of therapeutic cooling for HIE typically rewarmed neonates at no more than 0.5 °C per hour11. In the present study, by contrast, the partial protection of total numbers of oligodendrocytes seen after 48 hours of hypothermia, was not affected by either the rate of rewarming or by a longer duration of hypothermia. There was a small improvement in the survival of mature oligodendrocytes with 72 hours of hypothermia but not with slow rewarming after 48 hours of hypothermia. Given that there was no improvement in the total number of oligodendrocytes, this suggests that the longer duration of hypothermia may have been associated with improved oligodendrocyte maturation rather than greater survival.We have recently shown that 48 hours of hypothermia after ischemia in near-term fetal sheep was associated with partial neuroprotection in the cortex and hippocampus that was not improved with slow rewarming, whereas neuronal survival was restored to near sham control levels when the duration of hypothermia was increased to 72 hours27. Extending the duration of hypothermia from 48 to 72 or 120 hours did not affect total numbers of oligodendrocytes but significantly modulated neuronal survival28.Interestingly, oligodendrocyte survival appears to be more sensitive than neuronal survival to greater delay in starting hypothermia rather than to the duration or rate of rewarming after hypothermia. For example, we have previously shown that hypothermia started at 90 minutes after ischemia was highly protective for oligodendrocytes and neurons but when the start of hypothermia was delayed until 5.5 hours there was loss of oligodendrocyte protection despite significant, albeit partial, neuronal protectionIn the present study, there was no effect of the rate of rewarming on the expression of the myelin proteins MBP or CNPase. Interestingly, 72 hours of hypothermia was associated with a small decrease in the area fraction of CNPase expression compared to the ischemia-48 hour hypothermia and ischemia-slow rewarming groups. It is not clear why CNPase expression was decreased in the ischemia-72 hour hypothermia group given the increase in mature myelinating oligodendrocytes in this group. Further, the implications of this small reduction in CNPase on the structure and function of the myelin are not known.9. All hypothermia protocols significantly reduced numbers of microglia. However, 72 hours of hypothermia with rapid rewarming was associated with a greater reduction in microglial number compared to 48 hours of hypothermia. The slow rewarming group showed an intermediate number of microglia that was not significantly different to 72 or 48 hours of hypothermia. This suggests that any benefit seen in the slow rewarming group is likely attributable to the greater duration of hypothermia rather than a true effect of the rate of rewarming per se.Consistent with many previous studies, cerebral ischemia was associated with a marked increase in the number of Iba-1 positive microglia in all white matter regions, e.g.29. These data suggest that in the current study ischemia was associated with an increase in the number of astrocytes but loss of these fine processes. The slow rewarming protocol best restored both astrocyte number and area fraction of GFAP labelling, suggesting improved astrocyte morphology. Given that the slow rewarming protocol was more effective than either 48 hours or 72 hours of hypothermia with rapid rewarming, this suggests a true benefit of slow rewarming on astrocytosis, rather than an effect of prolonging the duration of hypothermia.Ischemia was associated with a significant increase in the number of GFAP positive astrocytes but an apparent trend to reduced area fraction of labelling (P = 0.068), consistent with a change in the morphology of astrocytes. Only ischemia-slow rewarming was associated with a significant reduction in numbers of astrocytes compared to ischemia-normothermia, while both the ischemia-48 h and ischemia-72 h hypothermia protocols had no effect on astrocyte number. Both the ischemia-48 h hypothermia and ischemia-slow rewarming groups showed a significantly greater area fraction of GFAP labelling compared to ischemia-normothermia, whereas the ischemia-72 hour hypothermia group showed an intermediate effect that was not significantly different to ischemia-normothermia or sham control. Structural remodeling of astrocytes has previously been shown 72 hours after hypoxia-ischemia in the neonatal pig brain, whereby injured astrocytes showed loss of the complex arbor of fine processes that normally extend out from the cell body of a healthy astrocyte, leading to reduced area fraction of labelling11. Thus, we speculate that secondary astrocytosis may contribute to electrophysiological recovery. Astrocytes play a critical role in maintaining homeostasis in the brain, including uptake and recycling of extracellular glutamate, buffering of potassium ions and modulation of synaptic transmission via their contribution to the “tripartite synapse” as reviewed30. Further, in the present study, we only assessed changes in white matter astrocytes. However, similar astrocytic structural remodeling after hypoxia-ischemia has been shown in grey matter astrocytes in the neonatal pig brain31. It is plausible that if the slow rewarming protocol showed superior protection of grey matter astrocytes, this may also contribute to the improved recovery of EEG power seen after slow rewarming11.We have previously shown that the slow rewarming protocol was associated with a significant improvement in the recovery of EEG power compared to 48 hours of hypothermia with rapid rewarming, despite slow rewarming having no effect on neuronal survivalper se. Moreover, the duration of hypothermia and rate of rewarming after normothermia had no effect on total numbers of oligodendrocytes or preservation of MBP. Finally, hypothermia for 72 hours with rapid rewarming was associated with the greatest recovery of mature oligodendrocytes albeit with a small reduction in recovery of the area fraction of CNPase labelling. Overall, pragmatically, these data suggest that the net duration of hypothermia has a more important impact on hypoxic-ischemic white matter injury than speed of rewarming after therapeutic hypothermia.The effect of rate of rewarming after hypothermia and duration of hypothermia in white matter recovery from global cerebral ischemia are complex and cell type specific. Very slow rewarming at a rate of 0.2 °C per hour after 48 hours of hypothermia was associated with modestly improved normalization of astrocyte number and area fraction compared to either 48 or 72 hours of hypothermia with rapid rewarming. However, there was intermediate improvement in Iba-1 positive microglia with slow rewarming after 48 hours of hypothermia compared with rapid rewarming after 48 hours and 72 hours of hypothermia, consistent with an effect of prolongation of the duration of hypothermia rather than the rate of rewarming


Bulk polytetrafluoroethylene (PTFE) possesses excellent chemical stability and dielectric properties. Indeed, thin films with these same characteristics would be ideal for electret applications. Previously, the electret properties of PTFE-like thin films produced by rf sputtering or plasma enhanced chemical vapor deposition were found to deteriorate due to structural changes and surface oxidation. In this article, the technique of initiated chemical vapor deposition (iCVD) is evaluated for electret applications for the first time. The iCVD method is known for its solvent-free deposition of conformal, pinhole-free polymer thin films in mild process conditions. It is shown that PTFE thin films prepared in this way, show excellent agreement to commercial bulk PTFE with regard to chemical properties and dielectric dissipation factors. After ion irradiation in a corona discharge the iCVD PTFE thin films exhibit stable electret properties, which can be tailored by the process parameters. Due to the mild deposition conditions, the iCVD technique is suitable for deposition on flexible organic substrates for the next-generation electret devices. It is also compatible with state-of-the-art microelectronic processing lines due to the characteristics of conformal growth and easy scaling up to larger size substrates. An electret is a dielectric material with a quasipermanent dipole polarization or surface charge, the latter one usually generated by ion irradiation in a corona discharge or electron beam irradiation3. It can deliver a built-in potential in portable electronic devices to power it without external bias voltage. The best known application is probably the millionfold produced electret condenser microphone for mobile electronic devices, like mobile phones and hearing aids5. Various other applications take advantage of electrets ranging from electrostatic air filters to organic transistor memory and electret memristors8. Most recently the increasing interest in electret generators13 demands furthermore high performance electrets, often on flexible substrates13. For next-generation electret devices, a fluoropolymer thin film deposition technique is highly demanded to enable device miniaturization, precise fluoropolymer thin film control, as well as the integration into state-of-the-art industrial microelectronic processing lines. Particularly for PTFE electrets, high quality PTFE thin films are needed, that preserve the original nature of PTFE. Some promising approaches have been reported16. Nevertheless corona-charged PTFE thin films deposited, e.g. by radio frequency (rf) sputtering show significant differences in charge storage and electrical properties compared to commercial PTFE14, due to deviations from the original PTFE structure. Also much higher dissipation factors are reported for rf sputtered PTFE thin films17. PTFE thin films prepared by plasma polymerization suffer often from C=O group formation due to oxidation which increases also the dissipation factor of the film18 and consequently impairs the electret properties15. Promising PTFE film fabrication approaches with pulsed laser deposition (PLD)16 are not suitable for large-area deposition. High temperatures are furthermore involved in most of these approaches and are thus not suitable for sensitive flexible polymer substrates used, e.g. in next generation flexible organic electronic devices like wearable electret generators13. In this approach, the mentioned problems are solved by using PTFE thin films synthesized via initiated chemical vapor deposition (iCVD). This technique is known to preserve the original polymer functionality and to maintain a low substrate temperature, due to its mild deposition conditions20, allowing thus the deposition on flexible organic susbtrates. The iCVD process is already well established for PTFE thin film deposition23. The technique does not involve any organic solvents, because the thin film is directly polymerized by a free radical polymerization on a substrate, which is cooled to room temperature. An initiator provides CF3 end groups, preventing polar group formation at dangling bonds, and increases thus the dielectric performance regarding, e.g. dissipation factors. It furthermore increases the deposition rate significantly. Rates of several µm h−1 are reported for iCVD PTFE23, so that the demanded film thickness of typically 10 µm for stable polymer electrets is easily obtained in this way. The CVD-typical conformal growth enables furthermore smooth film surfaces and easy scale-up of the process to larger dimension substrates, making it interesting for industrial applications23. The PTFE thin film electrets in this study were fabricated in a custom-made, hot filament, radial-flow-type reactor with ring-inlet, as illustrated in Fig. Besides its excellent chemical stability and dielectric properties polytetrafluoroethylene (PTFE) is well known for its outstanding performance as a real charge electret materialThe self-initiated monomer gas hexafluoropropylene oxide (HFPO) is used together with perfluorobutanesulfonyl flouride (PFBSF) to polymerize PTFE thin films on the cooled substrate. The chemical structures of these two molecules are shown in Fig. σs) after the ion irradiation generates an internal electric field (Ein) and an external electric field (Eex) in the vicinity of a counter electrode. It is related to the surface potential (VS) of the electret by:x1 represents the film thickness and ε0 and εr denote the vacuum permittivity and relative permittivity, respectively. The initial value of σs is limited by the corona parameters and dielectric strength of the material. Non charged iCVD PTFE films deposited on C-Si are used for the chemical characterization. The similarity of iCVD PTFE films to commercial PTFE is reported by many authors24. Also the X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy conducted for this study confirm the similarity of iCVD PTFE thin films to commercial bulk PTFE, as shown in Fig. The characteristic parameters of an electret are illustrated in Fig. 25. The peak around 690 eV is identified as the primary F 1 s peak for the fluorine atoms, bound to the carbon backbone, while the peaks in the region between 800 eV and 900 eV can be associated with the Auger peak for fluorine (F KLL)25. Considering the sensitivity factor 5.1 for F 1 s, a concentration of 66.9% for F 1 s and 32.5% for C 1 s can be estimated implying the characteristic F/C ≈ 2 ratio for PTFE. Further information on the high resolution C 1s peak can be found in the supplementary information . In the setup used in this study, no difference between iCVD PTFE and commercial PTFE was found. This confirms that iCVD PTFE does not only resemble commercial PTFE with regard to chemical properties, but further on with regard to the dielectric properties. Due to the CF3 end-groups, provided by the PFBSF initiator, the dissipation factor is much lower as typically observed in, e.g. plasma polymerized PTFE thin films18, because polar C=O group formation can be excluded. The corona charged iCVD PTFE electrets are investigated with regard to their charge stability with an electrostatic voltmeter  to a pseudohexagonal disordered structure at 30 °C35. The thermally stimulated current dn/dt released from the iCVD PTFE electrets at heating rate β can be calculated from VS by36:E be the trap energy level. The function f0(E) represents the initial occupancy of the traps and N(E) the energy distribution of the trap levels, the product f0(E)N(E) thus the initial number density38. With P as a shape-factor, the carrier current can be expressed, according to Simmons et al., by40:P in this connection is given by:e is the release probability including the attempt-to-escape frequency ω and can be described by:P can be approximated by a δ-function considering the associated area D (see39 for more details) by:dP/dE = 0, Simmons et al. suggested the solution−4, b = 3.2 × 10−4 and c = 1.5 × 10−2. From this equation, ω can be estimated by measuring the temperature of the current peak at two different heat rates (β1 = 3.75 °C min−1 and β2 = 6 °C min−1). The determined value of 3.6 × 1013 Hz for iCVD PTFE shows excellent agreement to reported values for commercial PTFE (7 × 1013 Hz)34. The activation energy of the traps is estimated to Ea = 1.163 eV. The iCVD technique enables furthermore to tune these properties by a change in the deposition parameters. They have a direct influence on the underlying free radical polymerization and hence the resulting structural properties, like molecular weight and crystallinity of the PTFE thin film. Fig. 8 or similar applications. The presented results show that the iCVD technique is well applicable for the production of PTFE thin films for electret applications thanks to their excellent resemblance to commercial PTFE with regard to chemical and dielectric properties. The mild deposition conditions enable furthermore the deposition on temperature sensitive substrates like flexible organic substrates for next generation organic electret devices. The conformal growth and easy scale-up allows furthermore an easy integration into state-of-the-art microelectronic processing lines and can be considered for next generation fluoropolymer thin film electret fabrication.The relative permittivity of the iCVD PTFE is determined from the complex impedance ter Fig. . Thermalion Fig. . Let E bPTFE thin films were grown on aluminium and crystalline silicon  substrates by iCVD. The system was operated in continuous flow mode. Perfluorobutanesulfonyl flouride  was mixed with hexafluoropropylene oxide  at a ratio of 1/5. The HFPO flow was controlled by a mass flow controller (Alicat Scientific MC series). PFBSF was kept in a glass jar at room temperature and the PFBSF vapor was delivered through a leak valve (Varian) to the reactor. The overall flow was 1.2 sccm. The filament power of 50 W was applied during the deposition by a power supply (Knürr-Heinzinger), using a Nickel Chromium  filament array. The sample stage was cooled to 20 °C by a circulating thermostat (Huber CC-K6). The process pressure of 50 Pa inside the reactor was controlled by a downstream butterfly valve (VAT Series 615), coupled to a capacitance manometer (MKS Baratron), which was attachted to the reactor. Additional sample sets of identical film thickness were produced using different process pressures (125 Pa and 25 Pa) to study the influence of pressure on the charge storage behavior of the polymer film.−1 with spectral resolution 5 cm−1. The same measurements are performed for commercial bulk PTFE (Goodfellow). Further measurements were performed with a variable-angle spectroscopic ellipsometry  in reflection mode at four angles . The optical data has been recorded from 250 nm to 1700 nm. The resulting data was processed with the CompleteEASE software. The system was modeled with one layer for the c-Si substrate and a Cauchy layer for the polymer film.The film thickness for the iCVD PTFE samples was obtained by profilometer measurements (Bruker Dektak XT). X-ray photoelectron spectroscopy (XPS) were performed using a UHV system equipped with a hemispherical electron analyser and a twin anode–x-ray source . An Al Kα X-ray source at a power of 240 W is used in the system. CasaXPS software was used to perform a quantitative analyses and detailed peak deconvolution. Raman spectroscopy is performed with a confocal Raman spectroscopy microscope (WITec Alpha300 RA). The laser excitation line is 532 nm, using a power of 5 mW in the range of 80–2000 cmVtip = −6.5 kV tip potential and used to irradiate the samples with ions in order to fabricate electrets. The discharge is performed in ambient air for 60 s. The tip-to-sample distance is 5 mm. In this way iCVD PTFE thin film electrets, spin coated Teflon AF (Du Pont) electrets and bulk PMMA (Goodfellow) electrets are fabricated.13 µm iCVD PTFE films were grown on 0.5 mm aluminum. A custom-made point-to-plane DC corona discharge setup is operated at −1 and 6 °C min−1 were used. The computation of all presented results were performed in Matlab R2016a.The phase angle and absolute value of the impedance of the non-charged iCVD PTFE samples were measured from 100 Hz to 1 MHz in a custom-made parallel plate setup containing a programmable LCR meter (Fluke PM6306). The surface potential of the charged electret samples was determined by an electrostatic voltmeter (Trek 347). The probe was mounted over a grounded, heatable stainless steel plate. Thermally stimulated potential decay (TSPD) measurements were performed by recording temperature and surface potential. Heating rates of 3.75 °C minSupplementary Information


In this study, we set out to identify the growth media component(s) responsible for ‘cut’ phenotype suppression in Δcbf11 and cut6–621 cells. We show that mitotic defects occur rapidly in Δcbf11 cells upon shift from the minimal EMM medium (‘cut’ suppressing) to the complex YES medium (‘cut’ promoting). By growing cells in YES medium supplemented with individual EMM components, we identified ammonium chloride, an efficiently utilized nitrogen source, as a specific and potent suppressor of the ‘cut’ phenotype in both Δcbf11 and cut6–621. Furthermore, we found that ammonium chloride boosts lipid droplet formation in wild-type cells. Our findings suggest a possible involvement of nutrient-responsive signaling in ‘cut’ suppression.Fission yeast ‘cut’ mutants show defects in temporal coordination of nuclear division with cytokinesis, resulting in aberrant mitosis and lethality. Among other causes, the ‘cut’ phenotype can be triggered by genetic or chemical perturbation of lipid metabolism, supposedly resulting in shortage of membrane phospholipids and insufficient nuclear envelope expansion during anaphase. Interestingly, penetrance of the ‘cut’ phenotype in mutants of the transcription factor  Ammonium chloride, a nitrogen source used in fission yeast growth media, was found to rescue cell division defects in mutants with perturbed lipid metabolism. Schizosaccharomyces pombe features very short G1 and S phases, a long G2 phase (∼70% of the cycle) and a rapid mitosis, during which the nuclear envelope does not break down. Thus, when grown in standard complex medium , the daughter nuclei have already completed S phase by the time cytokinesis has finished  family  catalytic subunit and general stress-response MAP kinase, respectively. Furthermore, the ‘cut’ phenotype of Δcbf11 and cut6–621 cells is largely diminished when cells are grown in the minimal defined EMM medium  subunits, cause loss of viability at increased temperature when cells are grown in complex, yeast extract-based YPD medium. Notably, the lethality can be suppressed by inactivating the PKA pathway in the cut4–533 and cut9–665 mutants, or by growing the cells in EMM medium in the case of cut4–533  affecting this coordination is currently unknown. In this study, we set out to identify the EMM medium component(s) responsible for ‘cut’ phenotype suppression in the Schizosaccharomyces pombe strains used in this study were JB32 (+h), MP44 (+ Δcbf11::kanRh)    and YES variants supplemented with EMM-contained chemical compounds at concentrations listed in Table S. pombe cells cultured in EMM were collected by centrifugation , resuspended in the same volume of fresh YES and incubated at 32°C. In all other experiments, cultures were grown in the indicated media for the whole duration of the experiment.S. pombe cells were first grown exponentially in YES. Culture volumes corresponding to ∼1.2 × 106 cells were collected and centrifuged . Supernatants were removed and cell pellets were washed with the appropriate media. The resulting cell suspensions were then centrifuged again , supernatants were discarded, and cell pellets were resuspended in 1.5 mL of appropriate media. Aliquots of 1.4 mL of resulting cell suspensions were loaded into 12-well plates and introduced into the VarioSkan Flash plate reader (Thermo Scientific). Plates were incubated at 32°C with background shaking . Optical densities were measured at 10 min intervals at λ = 595 nm. Doubling times (DT) were calculated according to the formula DT = 1/k, where k represents the slope of logarithmic phase of growth. Microsoft Excel 2007 was used for data processing and determination of k-value.For growth rate measurements, S. pombe cells were collected by centrifugation  and fixed by resuspending in 70% ethanol. Ethanol-fixed cells were centrifuged again  and resuspended in deionized H2O. Cells were stained in suspension with 1 μg mL−1 4΄,6-diamidine-2΄-phenylindole dihydrochloride (DAPI). Cell images were taken using the Olympus Cell R and Leica AF 6000LX microscopic systems. Frequency of ‘cut’ phenotype occurrence was determined by manual counting of ‘cut’ cells using the ImageJ software, version 1.51j8  and briefly mixed by vortexing. No washes or sample dilution/concentration steps were performed to avoid stressing the cells or affecting their metabolism. Cells were centrifuged  and promptly imaged on soybean lectin-coated slides using the Olympus Cell R microscope. For imaging Ptl2-GFP, cells were fixed with 10% formaldehyde for 15 min, and washed three times with PBS, followed by microscopy. Fluorescent images were acquired as 16-bit Z-stacks  in the green channel and were processed using the ImageJ software, version 1.51n . By contrast, the growth rate of WT cells did not decrease after the shift to YES  present in EMM but absent or limiting in YES are responsible for the observed mitotic defects, as opposed to a situation in which YES would contain a ‘poison’ component  responsible for suppressing defects in the YES Fig. , even thΔcbf11 cultures . We observed strong ‘cut’ suppression in Δcbf11 cells when ammonium chloride (NH4Cl), which serves as the nitrogen source in EMM, was added to YES .To directly test the impact of EMM components on ‘cut’ phenotype occurrence, we performed experiments with supplemented YES variants and analyzed the percentage of ‘cut’ cells by microscopy. We found that the ‘minerals’ component of EMM was able to suppress ‘cut’ incidence in res Fig. . To idenYES Fig. . Both WT4Cl, we analyzed the cut6–621 temperature-sensitive mutant at the semi-permissive temperature of 30°C. Expression of the cut6 acetyl-coenzyme A carboxylase gene is regulated directly by Cbf11, and the cut6–621 mutant also shows high frequency of the ‘cut’ phenotype when grown in YES at semi-permissive temperature  and EMM at 30°C. Lipid droplets are storage bodies composed of neutral triacylglycerols and sterol esters. Their formation shows cell-cycle and growth-phase dynamics . Δcbf11 cells have decreased lipid droplet abundance when grown in YES and many cells do not contain any detectable lipid droplets at all. Both these phenotypes, low lipid droplet abundance and cell-to-cell heterogeneity, were largely rescued by growing Δcbf11 cells in EMM .First, we stained live, exponentially growing cells with BODIPY 493/503 to visualize neutral lipids. BODIPY dyes are insensitive to pH , a major component of EMM and an efficiently utilized nitrogen source, as the compound specifically responsible for the rescue. Furthermore, we have shown that addition of NH4Cl increases lipid droplet content in WT cells, suggesting that NH4Cl affects fatty acid and/or neutral lipid metabolism.Fission yeast mutants in the genes S. pombe shows exquisite sensitivity to both nitrogen source quantity and quality  compared to the pronounced lipid droplet accumulation in stationary S. pombe cells. Therefore, these might be two independent phenomena with distinct regulation, each taking place at a different stage of culture growth.We found that NHlls Fig. . CuriousHe et al.. Importaet al.et al.4Cl rescues the ‘cut’ mitotic defects? One possibility is that NH4Cl increases membrane phospholipid production, thereby providing mutants with compromised lipid metabolism with the required nuclear envelope components. While we found association between the ‘cut’ phenotype and decreased lipid droplet content in the Δcbf11 strain, we did not see any significant corrective effect of NH4Cl on lipid droplet amount and distribution in either Δcbf11 or cut6–621 mutant cells  and this dynamics is important for successful cell division  responsible for the suppression were not analyzed  and other nitrogen sources, in general, including suboptimal ones. The potential involvement of TOR signaling, another major nitrogen-responsive regulator, in the suppression of the ‘cut’ phenotype should also be addressed.In future, it will be interesting to determine how fission yeast cells (and particularly ‘cut’ mutants related to lipid metabolism) respond to different doses of NHSupplement Table 1Click here for additional data file.


During 120 days exposure to 0.5 M Cl− solution, the rebars in cracked MSCC keep showing open circuit potentials above the critical value of −250 mV and they lose less than 2% of the total rebar material which corresponds to half the material loss in cracked plain mortar. Overall, the obtained rebar protection performance is comparable with that of uncracked mortar and mortar containing chemical inhibitor, hence the microbe-based system becomes an alternative to the traditional methods.Chemicals and synthetic coatings are widely used to protect steel against corrosion. Bio-based corrosion inhibition strategies can be an alternative in the arising bioeconomy era. To maintain the good state of steel reinforcement in cracked concrete, microbe-based self-healing cementitious composites (MSCC) have been developed. Yet, proposed strategies involve reasonably slow crack filling by biomineralization and thus risk the possible rebar corrosion during crack healing. Here we upgrade the rebar protection to a higher level by combining MSCC with microbial induced corrosion inhibition. Presented NO Hence, the steel passivation may quickly be lost and rebar corrosion starts which results in further durability issues and ultimately structural failure. Conventionally used coatings and corrosion inhibitors are often expensive and they may contain toxic volatile organic solvents, causing severe environmental concerns3. The construction sector needs effective, cost-efficient, sustainable and green corrosion control methods to deal with durability issues.Microcracks in reinforced concrete structures, especially in marine environment, facilitate the ingress of substances such as Cl10. So far, regardless of the proposed metabolic pathway, MSCC perform better than conventional mortars in terms of water tightness regain10. Such regain in functionality is desired to hinder the ingress of aforementioned aggressive substances and to protect the good state of the rebar13. In previous microbe-based self-healing studies, it was claimed that biomineralization in microcracks is an effective method for protection of the steel reinforcement14. Yet, available data lacks solid evidence on the protective effect of MSCC against rebar corrosion. Especially the corrosion behaviour of steel reinforcement in cracked and uncracked MSCC is unknown.Microbial induced calcite precipitation Eqs  and 2) e e2)) ena5. We speculate that rebars are still susceptible to attack by aggressive ions during the reported time period of biochemical crack healing due to the fact that the water tightness could be regained effectively only after complete healing of the crack. Therefore, especially for applications of MSCC in marine environment, complementary mechanisms that provide corrosion inhibition are necessary to effectively protect rebars from corrosion during microbe-mediated healing of cracks.The biochemical crack healing process requires a time span of 28 to 70 days for immersed mortar specimens3− reducing microorganisms simultaneously produce NO2−, a commercially used anodic corrosion inhibitor, as an intermediate product15, and induce bio-mineralization through the following reactions.Among the reported microbial pathways, microbial nitrate reduction can be more advantageous on this pursuit compared to the others (urea hydrolysis and aerobic respiration) as NO2− in steel corrosion inhibition is to interfere with the migration of the ferrous ions from the anodic zones of the rebar. Ferrous hydroxide formed in anodic reactions is rapidly stabilized by the NO2− ions in the form of ferric oxide . Theref3− reducing microbial culture named as ‘activated compact denitrifying core’ (ACDC), which reveals satisfactory distribution and stability in mortar and effectively promotes self-healing of cracks18. Using the same culture, our initial attempts to explore the influence of microbially produced NO2− on steel plates revealed the possibility of microbial induced corrosion inhibition in weak salt solutions (0.05 M Cl−) (ref.17). The findings revealed that the microbial produced NO2− started to be effective when the [NO2−]:[Cl−] ratio exceeded 0.50. Yet, the investigated test conditions provided limited information which was not enough to demonstrate the corrosion behaviour of reinforced MSCC containing ACDC in marine environment. First of all, in marine environment, typical Cl− concentration is around 0.50 M which is 10 times higher than the concentration tested in our previous study and the ionic activity (µ) of the seawater is around 0.70 (refs20). Either of these variables may adversely affect the activity of microorganisms21 and thus their influence on the activity of in-house developed ACDC granules should be investigated. Secondly, the effect of the mortar matrix was not included in the reported corrosion tests where steel plates were directly tested in a corrosive nutrient solution. Since the alkalinity of mortar can passivate the steel with a thin oxide layer, the corrosion behaviour of a rebar can be better understood only if the steel is tested while embedded in mortar. Finally, the corrosion was induced by polarization experiments rather than the natural disruption of the passive film and further corrosion of the steel in long-term. Therefore, the influence of biomineralization on corrosion behaviour of the rebar was yet unknown. Due to the limitations in the experimental design of the preliminary study, the actual rebar corrosion inhibition performance of ACDC in a cementitious composite exposed to marine conditions still remains unclear. Therefore, our main objective was to evaluate the rebar protection performance of ACDC containing MSCC in corrosive marine conditions during and following the crack healing period. The study was conducted in two consecutive steps; (i) pre-assessment of the ACDC culture for salt tolerance by testing resuscitation and microbial activity of ACDC culture under a variety of ionic strengths (µ), (ii) investigation of the corrosion behaviour of rebars embedded in MSCC in simulated marine conditions.Our team previously developed a non-axenic granulated NOµ ~ 0.7 and [Cl−] ~ 0.5 M) and gradual accumulation of non-metabolised ions that leach from the mortar  are expected in situ. Therefore, the effects of the most relevant ions (Cl− and NO3−) on resuscitation and microbial activity of NO3− reducing ACDC culture were tested  of the solution rather than a change in specific ion concentration. Among the investigated concentration couples at similar ionic strengths NO3− reduction and NO2− accumulation rates were similar. For instance, in two cases where concentrations of the individual ions were different but the ionic activity of the solution was identical (µ = 0.8), the NO3− reduction and NO2− accumulation activity of ACDC was also similar and recorded as 1 mmol/g−1.d−1 . The negative influence of an increasing ionic strength was more pronounced between µ = 0.7 and µ = 0.9 when compared to the changes at higher ionic strengths , the NO3− reduction and NO2− accumulation rates were recorded as 2.70 mmol.g−1.d−1 and 2.30 mmol.g−1.d−1, respectively. These first results revealed that ACDC culture was adequate for the further tests in a corrosive chloride solution .The increase in either NOes Table . When di− solution and measuring the open circuit potential (OCP) values for 120 days. The initial crack widths of the cracked mortar specimens were similar. The average crack widths were recorded as 310 ± 30 µm, 307 ± 41 µm, 348 ± 41 µm, 312 ± 23 µm and 300 ± 30 µm, for plain mortar, the positive control, the abiotic control, the self-healing control and the microbe-based test mortars, respectively.The corrosion behaviour of rebars embedded in cracked MSCC was investigated by exposing the specimens to a 0.5 M Cl− solution showed minor variations and was recorded as 9.1 ± 0.1 over the course of the experiments   while the rebar embedded in positive control specimens containing NO2− always showed OCP values higher than −250 mV throughout the 120-days experimental period   at the end of 120 days exposure to salt solution  Fig. . For the05) Fig. . When coion Fig. .Figure 4Scanning electron microscope (SEM) micrographs of the rebar surfaces were acquired and energy dispersive spectroscopy (EDS) analyses were conducted to clearly distinguish between the surface degradation of rebars taken from different mortars after 120 days Fig. . A largenEDS = 9) which indicated that the surface was mostly composed of iron oxides  weight ratio, Fe content in particular, decreased in the corroded rebar surface as the corrosion became more severe, and other metals of the rebar alloy such as copper, chromium and aluminium appeared owing to the degraded passive layer  while in negative controls it was 10% (nEDS = 13)  Fig. .3− in aquatic environments with an ionic strength up to 1.2, which are essential metabolic activities to induce calcium carbonate precipitation and corrosion inhibition. At the ionic strength of a typical seawater (µ ~ 0.7 M), the specific NO3− reduction and NO2− accumulation rates of ACDC were found to be 2.7 mmol.g−1.d−1 and 2.3 mmol.g−1.d−1, respectively. Previous studies aiming at wastewater treatment reported NO3− reduction rates as high as 86 mmol.g−1.d−1 (at µ = 0.8) after acclimating the nitrate reducing culture to high Cl− and NO3− concentrations22. It should be noted that the bacteria in those studies were already active, the composition was rich in nutrients (in terms of micro- and macro-nutrients) and considerable amount of time was spent for acclimation at high ionic strengths. In this study, only an electron donor (HCOO−) was available as macro-nutrient and the solution was deficient in terms of phosphorus and micro-nutrients. Moreover, the activity was recorded immediately after resuscitation of the ACDC culture without any acclimation. Therefore, the observed difference with the previously reported performances could be attributed to aforementioned harsh conditions in our test setup. The main objective of this first set of experiments was to clarify whether ACDC can resuscitate in marine conditions, reduce NO3− and accumulate NO2− by only relying on a readily degradable electron donor (HCOO−). It can be said that there is still room for improvement of the resuscitation performance and activity by making necessary modification on the cultivation process of ACDC. Perhaps such improvements can positively influence the corrosion inhibition performance as well, and thus should be noted for a further study.In this study, we revealed that ACDC can be useful for concrete applications in marine environment. The culture can resuscitate and reduce NO3−, which is believed to have some inhibitory effect on the corrosion of steel23. However, the passivation due to NO3− was not as effective as NO2− 2 is economically more feasible than the use of Ca(NO2)2 (refs3), ACDC containing MSCC not only offers a bio-based alternative for corrosion prevention but it can also decrease the cost of rebar protection in marine environment.The corrosion equally progressed in all cracked mortars without corrosion inhibitors Fig. . The delO2− Fig.  which wa14. Corrosion of the steel reinforcement in self-healing control continued even after the noted complete crack closure on day 28. Although the crack closure performances of the two different types of MSCC tested in this study were similar here and reported to be similar earlier7, when they were compared in terms of steel protection, the one containing NO3− reducing ACDC culture became more prominent owing to its remarkable corrosion prevention performance. The quality of the steel protection in ACDC containing MSCC was equivalent to that in the cracked positive control and that in uncracked mortar. Additionally, the closing of the crack in 28 days improved the aesthetic and functional properties of the ACDC containing specimen as compared to those of the positive control.The self-healing control tested in this study was significant to reveal the sole effect of microbial induced crack healing on the corrosion behaviour of the steel. Obtained results revealed that only biomineralization is not an effective method for protection of the steel reinforcement as opposed to the claims in previous microbe-based self-healing studies3− reducing ACDC culture, we developed an advanced MSCC that can protect the rebar in cracked mortar up to 4 months while healing a 300 µm wide crack in the first 28 days. Corrosion inhibition achieved through microbial nitrate reduction was as effective as that achieved by using commercially used chemical inhibitors such as Ca(NO2)2. The material performance is promising to effectively protect the rebar against corrosion in marine environment and increase the service life of structures. We expect such composites to be applicable in a broad range of environments including underwater tunnels and nuclear repositories. Our advanced approach may also contribute to other concepts in microbial induced corrosion inhibition such as bio-competitive exclusion. Overall, these represent a departure from certain existing chemical based corrosion inhibitors which are not welcomed in the scope of a more sustainable world.Taken together, these findings unveiled the necessity of a corrosion inhibition mechanism together with the self-healing functionality to delay rebar corrosion in cracked cementitious composites and to limit its development in the post-healing period. By using NO3− reduction was investigated by using a non-axenic self-protected NO3− reducing culture called ACDC. A cylindrical sequencing batch reactor (SBR) was operated under feast/famine conditions for four months (4 cycles/day) by slightly modifying a previously described operational procedure7 and in total 35 g of ACDC granules were cultivated. Different from the previous setup, height to diameter ratio of the SBR was 10 . Minimal nutrient solution (COD: N—5:1) was used as feed (4 times/day) and the solution composition is given in Table th week of the operation. Further time points for harvests were arbitrarily chosen after achieving the stable granular biomass (94% of the biomass) with an active denitrifying core. The harvested ACDC granules were dried for 36 h in a drying tunnel at 60 °C with ventilation and stored at room temperature until their incorporation into the mortar specimens. The VSS content (which represents the amount of bacteria and organic matter inside the granular culture) was 70% of the ‘total suspended solids’ (TSS) content  of the granulated culture.Microbial NO24. The CERUP used in this study was cultivated in Avecom, NV, Belgium based on the previously described thermo-cycle based procedure that induce sporulation (at 70 °C) twice a day6. The VSS content of the obtained CERUP culture was 50% of the TSS content. The feed composition used in CERUP production is given in Table In corrosion experiments, as a control culture, we used a self-protected non-axenic ureolytic culture, so called CERUP, so its effect on mortar properties is similar to ACDC and it can induce crack healing through urea hydrolysis2 and Ca(NO3)2 were used as biomineralization precursors (details are given for each batch in the following section) for the ACDC culture, and CO(NH2)2 was used as a biomineralization precursor for the CERUP culture, in the mortar.Admixtures of Ca(HCOO)− and NO3− concentrations. The test conditions are given in Table − and NO3− concentrations. We chose a Cl− concentration range from 0.3 M (which has no effect)25 to 0.5 M , in 0.1 M increments. The tested NO3− concentrations were determined on the basis of the Cl− concentrations. The NO3− concentrations we chose ensured a [NO2−]:[Cl−] ratio of at least 0.34:1.00 when NO3− was completely reduced to NO2−. This ratio was previously found to be the minimum ratio for effective steel corrosion inhibition in concrete environment15. The initial pH inside the batch reactors was set to 9.5 by using NaOH (1 M). The initial VSS concentration in the reactors was 2.5 g/L. Abiotic controls were also tested for each mixture.ACDC culture was tested at varying ionic strengths by changing the initial Cl2−, four control series were investigated in addition to the microbial test mortar containing ACDC granules. The control series were (i) a plain mortar, (ii) a positive control , (iii) an abiotic control (the effect of nutrients) and (iv) a microbe-based self-healing control . The plain mortar series contained DIN EN 196-1 standard sand (1350 g), CEM I 52.5 N cement (450 g) and tap water (225 g), with 3.0:1.0:0.5 wt/wt ratios according to the EN 196-1 standard. Microbial test mortar contained ACDC-VSS (0.5% wt/wt cement), Ca(NO3)2 (3% wt/wt cement) and Ca(HCOO)2 (2% wt/wt cement) in addition to the components present in plain mortar. The positive control mortar contained NO2− (1.6% wt/wt cement) as a corrosion inhibitor in the form of NaNO2 (2.4% wt/wt cement) and Ca(HCOO)2 (2% wt/wt cement). Abiotic control mortar contained the nutrients of the microbe-based test batch, Ca(NO3)2 (3% wt/wt cement) and Ca(HCOO)2 (2% wt/wt cement). The microbial self-healing control mortar contained CERUP-VSS (0.5% wt/wt cement) and CO(NH2)2 (5% wt/wt cement). The amount of nutrients in microbe-based mortars were determined based on the previous studies where successful crack healing performances were achieved by means of ACDC and CERUP7.In the experimental design, in order to distinguish the anodic corrosion inhibition occurring due to microbially produced NOncracked = 6, nuncracked = 6). Half of each series was monitored for 28 days and the other half was monitored for 120 days. We defined the first 28 days of the Cl− exposure experiment as pre-healing period on the basis of the previous self-healing reports on MSCC7. The results obtained for the 28-days-tested specimens  were analysed to determine the corrosion behaviour of rebars embedded in different mortars with 300 µm wide cracks and the influence of microbe-mediated self-healing on corrosion behaviour. The other half of the mortar specimens  were monitored further in Cl− solution, until day 120. Results obtained throughout 120 days exposure were interpreted to evaluate the corrosion behaviour of a microbial self-healing reinforced mortar in an extended time period subsequent to crack healing .Each series was tested under ‘uncracked’ and ‘artificially cracked’ conditions. In each series 12 specimens were tested  with a centrally positioned embedded smooth steel rebar  were prepared.2)2 which was dissolved in the mixing water prior to addition. Bacterial admixtures, ACDC and CERUP, were 0.5–2 mm in size.During mortar preparation all of the admixtures and bacteria were added in powdered form to the mixture, except CO. The plates were positioned with their notch partially surrounding the steel rebar inside the moulds, and thus, it was assured that the crack reached and partially surrounded  the steel rebar. After arranging the plate positions to obtain three cracked and three uncracked specimens from each moulded prism, the mortar was poured. After curing the specimens for 24 hours at a temperature of 20 °C and a relative humidity (RH) of >90%, the plates were carefully removed. This resulted in the formation of artificial cracks with a depth of 20 mm and a width of ~300 µm over the complete 40-mm width of the mortar samples.Artificial cracks were created for testing, because the creation of realistic cracks would affect the reproducibility of the results and complicate the comparison of different series. The cracks were formed according to the procedure described previouslyAt the end of the 28-days curing period at a temperature of 20 °C and a relative humidity (RH) of >90%, the trowelled surface of the mortar prisms was polished (removing a 2 mm thick layer) to create a uniform flat test surface. Subsequently, six artificially cracked and six uncracked pieces (38 mm × 40 mm × 15 mm) were sawn off from the prisms via wet sawing.The widths of the artificially created cracks were confirmed with a stereomicroscope (Leica s8 APO with a DFC 295 camera) . At the end of the 28-days and 120-days experimental periods, the crack widths were measured again to quantify self-healing performance.− solution and kept in contact with the solution for 28 and 120 days. The exposed surfaces were standardized (40 mm × 15 mm) by partially covering the side surface area of the mortar samples with aluminium butyl tape. Capillary sorption occurred only through the pores and cracks on the cracked surface that was in contact with the solution. Throughout the experiments, the dissolved oxygen concentration in the bulk solutions was 7.3 ± 0.2 mg/L and the pH was 9.1 ± 0.1.Capillary sorption was used to create the conditions for corrosion. The cracked surfaces of the specimens were soaked with 0.5 M Cl− solution (0.5 M Cl−) by using a Gamry Interface 1000 potentiostat . The setup parameters for the weekly OCP measurements were 500 sec, 0.5 sec and 0 mV/sec for the total measuring time, data sampling period and stability, respectively. All of the obtained data (versus reference SCE) were converted to values versus the standard hydrogen electrode (SHE), on the basis of the given equilibrium potentials27, to be able to interpret them using the Pourbaix diagram of the iron species. Obtained data was compared with the limit OCP value (−250 mV vs SHE) defined based on the findings in Moreno et al.28  versus a reference saturated calomel electrode (SCE) for 120 days. Different batches were separated into different containers, and specimens were placed at similar distances from the reference SCE. Data were recorded every 6 min. For the first 28 days, the validity of the automated system was checked every week through additional separate OCP measurements in equimolar ClPieces of steel rebar of varying lengths (from 14 to 18 mm) were sawn off from a smooth unexposed steel rebar , and their average density values were calculated. The lengths of the tested steel rebars (which were embedded in the mortar specimens) were accurately measured, using an electronic calliper, before the corrosion experiments. Therefore, the initial volume of each embedded rebar could be calculated. The initial mass was then calculated by using the average density of the steel rebar and the initial volume. At the end of the experiments, the steel rebars were taken out of the tested specimens by breaking the mortar, and all corrosion products were removed by gentle cleaning under running water. After drying with compressed air they were weighed. The difference between the calculated initial mass and the measured final mass is presented as the gravimetric mass loss.− solution for 120 days, further analyses were performed with a scanning electron microscope (SEM)  coupled with energy-dispersive X-ray spectroscopy (EDS). SEM micrographs were taken at accelerating voltage of 20 kV at a working distance of 17 mm. EDS analysis was conducted on the surface of the unexposed steel rebar to determine the actual surface composition of the steel rebar in terms of FexOy and Fe (wt%). Afterwards, the surface compositions of the exposed steel rebars were analysed. Reductions in FexOy content (wt%) were used to distinguish the corrosion degree of rebars taken from different batches.The degree of corrosion at the position where the crack reached the steel rebar, as well as the general state of the steel rebar, was observed under a stereomicroscope using Leica S8 Apo apochromatic optics . The resulting micrographs were used to differentiate between the corroded specimens of different batches. For the series that had been exposed to a Cl30. Throughout the experiments, the ion concentrations  were measured via compact Metrohm 930 ion chromatography (IC) .TSS and VSS analyses of the grown ACDC culture and obtained CERUP culture were conducted according to standard methodsp < 0.05).Unless stated otherwise, all of the experiments were conducted in triplicate and the relevant data are presented as mean ± standard deviation. Mean values of OCP measurements are presented without giving the standard deviations as they negatively affect the visibility of the presented data which was recorded every 6 min for 120 days. Sigma Plot v12.0  was used to compare the significant differences in values by means of a one-way analysis of variance (ANOVA) test (Supplementary Materials


For this purpose the growth parameters  already calculated in the related article “Modelling microbial growth in Modified-Atmosphere-Packed hake (Merluccius merluccius) fillets stored at different temperatures” [1] were used. The data include the fit and goodness of the fit parameters calculated as well as the comparison between fitted and observed data.This article presents the results obtained after applying the Ratkowsky model for developing secondary models describing the influence of storage temperature on microbial growth in hake fillets packaged under a modified atmosphere (MAP) rich in CO The influence of storage temperature on the μmax (2) and λ (3) values calculated for each bacterial group  is shown in b, Tmin) together with its standard errors are included in vsμmax) and vs λ). The secondary models developed are included in 2 and RMSE values calculated for each microbial group.Growth curves in hake fillets stored under MAP 50% CO/50% N2 ots model  in the rratures” . In this22.12/50% N2) and stored at four different temperatures  were modelized using the Ratwosky [The growth parameters  previμmax. This model is defined by the following equation:The Ratkowsky model  was usedb is the slope of the regression line, T is temperature, and T0 is a conceptual minimum temperature for microbial growth, where T and T0 are given in °C.Where λ) was described with the inverse Ratkowsky model [Three influence of storage temperature on lag time (ky model :(2)1λ=b.GraphPad PRISM software  was used for curve fitting, and Microsoft Excel software  was used to calculate the goodness of the fit parameters (R