Improved nature provision in urban environments offers great potential for achieving both biodiversity conservation and public health objectives. Yet there are few experimental studies that address links between specific natural environments and physiological and/or psychological changes that could contribute to the health and wellbeing co-benefits of urban nature. In addition, relative to green space, the salutogenic impact of aquatic environments are understudied. Here, we present a feasibility study examining the use of low-cost wearable technology to quantify the psychophysiological effects of short-term exposure to urban wetlands. The study took place at the WWT London Wetland Centre, which is characterized by its contrasting biodiverse wetland habitat and surrounding urban setting. Thirty-six healthy participants experienced counterbalanced exposures to an indoor space, a wetland, an urban site. We continuously recorded electroencephalographic (EEG) data and real-time physiological stress responses; with additional monitoring of post-exposure self-reported mood states. We found a significant effect of site on mean resting heart rate (HR), with increased HR in the urban setting, although this was only observed in participants with pre-existing high stress. We found no significant differences in other measures of physiological stress responses . The EEG data showed modulation of high beta band activity only in the wetland setting, potentially related to changes in attention. However, the EEG findings were confounded by low quality signals and artifacts caused by movement and environmental interference. Assessments of self-reported mood states demonstrated an increase in positive feelings in the wetland setting. A pronounced decrease in negative feelings in the wetland setting was observed in stressed individuals only. Our results suggest that pre-existing stress levels may be an important modulator of the salutogenic effect of blue-green space. We provide partial support for the hypothesis that exposure to blue-green space promotes stress recovery and for the use of low-cost psychophysiological measurements to quantify the potential stress-reducing effects of blue–green space exposure in urban dwellers. Further technological refinement is required for this approach to become a viable tool to support evidence-based decision-making for public health and green/blue space provision. It is estimated that by 2050 two-thirds of the global human population will live in urban centers . Future In recent years, numerous studies have reported on the role of exposure to natural environments in both disease prevention and salutogenesis, the latter referring to the promotion of HWB rather than simply the absence of disease f London . To explf London .n = 34) had never visited the wetland center before. Of the participants who gave responses, 83% (n = 29) were not WWT members or affiliates, while 68% percent (n = 25) were not members of other conservation charities. Overall, the majority of our sample was unfamiliar with wetland conservation prior to participating in the study. All participants were screened against exclusion criteria, i.e., the presence of neurological conditions or injuries, psychiatric disorders, cardiovascular disease, taking of prescription or over-the-counter medicines, and any departure from normal or corrected-to-normal eyesight or hearing. This resulted in exclusion of n = 2 participants. Written prior and informed consent was obtained from each participant.We recruited 36 participants for this study, the majority of which were contacted through a social corporate responsibility scheme which allows local professionals to volunteer for WWT during normal work hours. Just over half  and shoveler (Anas clypeata). This site was selected for this experiment because of its unique location , an area of living collection of wetland birds and mammals, and surrounding managed wetland habitats. The latter carries the Site of Special Scientific Interest (SSSI) designation due to its national importance for UK breeding birds in lowland open waters and for its assemblages of non-breeding gadwall (location  that enaA multipurpose function room at WWT London Wetland Centre (51°28′37.9′′N 0°14′09.2′′W) was used as a control setting for baseline measures of PPG and EEG . The rooThe control site is situated in the visitor center area (51°28′47.2′′N 0°14′08.5′′W), which includes the café, shop and education center of the WWT London Wetland Centre site. Adjacent is a captive animal area and beyond the captive area is ‘Wildside’: a managed ‘natural’ wetland area . We wereThe study area (WWT London Wetland Centre) was chosen to create maximally opposing sensory experiences and to test differential responses to these environments. With similar consideration to the wetland site, the urban site (51°28′31.3′′N 0°14′21.3′′W) was chosen because it was the first suitable urban bench outside the Centre. Suitability was judged by the number of features present at this site that are typical of busy urban settings e.g., a traffic intersection, traffic lights, a pedestrian crossing, passers-by, shops, and cafes.2). The level of other potentially confounding variables  was also recorded. Over the 6 week testing period no sessions were canceled due to adverse weather. Participants attended their allocated session individually, with scheduled start times at 9am, 12pm, or 2pm. Upon arrival participants were greeted by a researcher and shown to the visitor center. We first conducted brief, semi-structured interviews to explore participants’ motivations to take part, expectations about the experiment, anticipated benefits, pre-existing knowledge of the site, any formal or informal affiliation to WWT , support for other conservation charities, and their understanding of the aims and procedures. Participants were also asked how they felt in the moment, and whether that was a departure from their regular mental and/or physical state. Following the interview, participants were asked to complete a set of questionnaires to obtain demographic information , and an assessment of relevant psychological variables including life stress, recent levels of depression, anxiety and stress, and participants’ sense of connectedness with nature to provide an assessment of participants recent exposure and response to stressors. The in-depth interview data were intended to provide qualitative contextual information, but a report on this is beyond the scope of the current study.The study was conducted between the 14th July and 23rd August 2017. Prevailing weather conditions were recorded for each participant session ]. Fitting the measurement equipment took approximately 15 min. Following calibration, EEG and physiological data were continuously recorded for the full duration of the experiment. Participants remained seated in the indoor control room for 10 min to obtain stable baseline readings . For theThe study had a repeated measures design, with three exposure conditions/sites: indoor (control), urban and wetland. Condition order of the urban and wetland sites was counterbalanced between participants; the control condition was always administered first.The Holmes & Rahe Stress Inventory, also known as the Social Readjustment Rating Scale  compriseWe used the 21-item version of the Depression, Anxiety and Stress Scale DASS-21;  to measuThe Nature Relatedness Scale  is a 21-Cardiovascular and electrodermal activity metrics are commonly used to assess and monitor the relative activity of the sympathetic and parasympathetic branches of the autonomic nervous system, which are respectively associated with states of threat (fight or flight response) and calm (rest and digest). These metrics can therefore be interpreted as indicators of physiological and psychological stress . We usedpeak exercise to 2 min post-exercise  epochs: the root mean square of successive differences of the inter-beat intervals (RMSSD), the number of adjacent NN intervals that differ from each other by more than 50 ms (NN50) and the Triangular Interpolation of the NN Interval Histogram (TINN). Note that NN intervals refer to the intervals between ‘normal’ peaks, i.e., excluding abnormal beats due to measurement artifacts. For the frequency-domain, we employed the most common method to calculate HRV spectra, i.e., a Fourier transform, to derive the primary components: the low frequency  and high frequency  spectra. For further analysis, we opted to transform the data using the natural logarithm to yield an approximately normal distribution .The Empatica E4 sampled EDA at 4 Hz, and skin conductance (SC) was measured in micro-Siemens (μS). Due to variation in the length of EDA readings across participants, analysis windows were standardized by removing the first minute of data. The subsequent 8 min were used in the analysis.Continuous Decomposition Analysis (CDA) was performed on EDA data in Ledalab  to decom® EPOC+ EEG system to obtain brain electrical activity measurement non-invasively through the scalp. The EPOC+ has 14 channels in the international 10–20 position system , with P3 and P4 as reference electrodes. To prevent electrode dehydration we added 5% glycerol to the saline solution before wetting the electrodes. Electrodes were checked for contact quality, signals were sampled at 128 Hz per channel and sent via Bluetooth to a portable laptop computer. EEG data was pre-processed using EEGLAB  and examined Pearson correlations (assessing associations with age). We created separate general linear models for each of the continuous outcome measures, with Site  as the within-subjects factor, while controlling for Site Order, which was entered as a between-subjects factor. To account for individual differences that could impact physiological responses, we also controlled for current (self-reported) levels of stress. For this purpose, we classified participants into ‘self-reported low stress’ and ‘self-reported high-stress’ groups on the basis of their DASS-21 stress score relative to published UK population mean scores , and incSD = 10.28). Women were slightly over-represented in the sample (61.8%) compared with men (38.2%). Descriptive statistics for the baseline assessments are summarized in Participants were on average 41 years of age  fell within the 58–68th percentile for Depression, the 54–69th percentile for Anxiety and the 60–69th percentile for Stress . ParticiF = 3.703, p = 0.031, η2 = 0.117, and a significant interaction between Site and Stress Level, F = 4.848, p = 0.011, η2 = 0.148, but no interaction effect between Site and Site Order, F = 0.261, p = 0.771, η2 = 0.009. Neither the main effect of Site Order, nor Stress Level was significant, F = 0.846, p = 0.366, η2 = 0.029 and F = 0.215, p = 0.647, η2 = 0.008, respectively.There was a significant effect of Site on mean HR, F = 0.399, p = 0.675, η2 = 0.029, but a significant effect of Site in ‘self-reported high stress’ individuals, F = 6.045, p = 0.007, η2 = 0.309. Pairwise comparisons indicated increased HR in the urban setting compared with both the wetland (p = 0.030) and the control setting   .F = 0.711, p = 0.502, η2 = 0.061, and no effect of Site Order, F = 0.29, p = 0.867, η2 = 0.003, or Stress Level, F = 0.769, p = 0.399, η2 = 0.065. We observed no significant interaction effects between Site and Site Order, F = 1.285, p = 0.297, η2 = 0.105, or Site and Stress Level, and F = 1.781, p = 0.192, η2 = 0.139.We found no significant differences between the exposure conditions on any of the time-domain HRV metrics. There was no significant main effect of Site on RMSSD, F = 0.775, p = 0.481, η2 = 0.064. There was no significant main effect of either Site Order, F = 0.576, p = 0.464, η2 = 0.050, or Stress Level, F = 1.317, p = 0.275, η2 = 0.107. None of the interaction effects were significant, for Site and Site Order, F = 0.669, p = 0.522, η2 = 0.057; for Site and Stress Level, F = 1.544, p = 0.236, η2 = 0.123.Similarly, the main effect of Site on NN50 was not significant, F = 0.991, p = 0.387, η2 = 0.083. There was no significant main effect of either Site Order, F = 0.551, p = 0.474, η2 = 0.048, or Stress Level, F = 0.603, p = 0.454, η2 = 0.052. None of the interaction effects were significant, for Site and Site Order, F = 0.158, p = 0.782, η2 = 0.014; for Site and Stress Level, F = 0.459, p = 0.638, η2 = 0.040.The main effect of Site on TINN was not significant, F = 0.156, p = 0.857, η2 = 0.014, nor the High Frequency signal, F = 2.614, p = 0.096, η2 = 0.192. Site Order seemed to have a marginally significant effect on the Low Frequency signal, F = 4.206, p = 0.065, η2 = 0.277, but not on the High Frequency signal, F = 0.216, p = 0.651, η2 = 0.019. No significant effects were observed of Stress Level on the Low Frequency or the High Frequency signal, F = 0.529, p = 0.482, η2 = 0.046 and F = 0.656, p = 0.435, η2 = 0.056, respectively. The interaction effect between Site and Site Order was not significant for the low Frequency signal, F = 0.291, p = 0.750, η2 = 0.026, nor for the High Frequency signal, F = 0.255, p = 0.777, η2 = 0.023. Finally, no significant interaction was observed between Site and Stress Level for the Low Frequency signal, F = 0.385, p = 0.685, η2 = 0.034, nor for the High Frequency signal, F = 2.610, p = 0.096, η2 = 0.192.With respect to the frequency-domain, we did not find a significant main effect of Site on the Low Frequency signal, We found no significant effects on the phasic EDA activity , nor on the measure of tonic EDA activity.F = 0.414, p = 0.663, η2 = 0.014. There was no significant main effect of either Site Order, F = 0.638, p = 0.431, η2 = 0.022, or Stress Level, F = 0.821, p = 0.372, η2 = 0.028. The interaction effects were not significant, for Site and Site Order, F = 1.068, p = 0.350, η2 = 0.036; or for Site and Stress Level, F = 0.657, p = 0.522, η2 = 0.022.The main effect of Site on nSCR was not significant, W = 0.382, 2(2) = 26.96, p < 0.001]. There was no significant main effect of Site, F = 0.119, p = 0.785, η2 = 0.004. There was no significant main effect of either Site Order, F = 0.325, p = 0.573, η2 = 0.011, or Stress Level, F = 0.030, p = 0.865, η2 = 0.001. The interaction effects were not significant, for Site and Site Order, F = 0.421, p = 0.563, η2 = 0.014; for Site and Stress Level, F = 0.671, p = 0.449, η2 = 0.023.For the within-subjects effects on tonic EDA, we report Greenhouse–Geisser correct results, as Mauchley’s test of sphericity was significant . There was no significant main effect of Site on the RMS of the EEG signal, F = 0.656, p = 0.529, η2 = 0.056, and there were no systematic differences between the Frequency Bands, F = 0.418, p = 0.795, η2 = 0.037. Site Order also did not affect the EEG signal, F = 0.614, p = 0.450, η2 = 0.053 and neither did Stress Level, F = 1.667, p = 0.223, η2 = 0.132. However, of key interest is the interaction effect between Site and Frequency Band, which indicates differential environmental effects depending on the EEG band, F = 2.195, p = 0.035, η2 = 0.166. Simple effects analysis showed that EEG responses varied by site only in the High Beta band, F = 6.320, p = 0.017, η2 = 0.558. Significantly stronger signals were observed in the wetland versus the urban setting (p = 0.015), whereas they only marginally differed from the control setting (p = 0.076), and the urban and control setting also did not differ (p = 0.654)  = 2.482, p = 0.133, η2 = 0.332, alpha, F = 0.691, p = 0.524, η2 = 0.121, Theta, F = 0.264, p = 0.773, η2 = 0.050 and Delta, F = 0.349, p = 0.714, η2 = 0.065).For the effect of Frequency Band, we report Greenhouse–Geisser correct results, as Mauchley’s test of sphericity was significant [= 0.654) . None ofF = 2.729, p = 0.087, η2 = 0.199, Site by Stress Level, F = 0.30, p = 0.971, η2 = 0.00, Frequency Band by Order, F = 0.253, p = 0.805, η2 = 0.023, and Frequency Band by Stress Level, F = 1.309, p = 0.291, η2 = 0.106. The three way interaction between Site, Frequency Band and Order was not significant, F = 1.652, p = 0.122, η2 = 0.131, and neither was the interaction between Site, Frequency Band and Stress Level, F = 0.533, p = 0.829, η2 = 0.046.None of the other two-way interactions were significant: Site by Site Order, F = 3.698, p = 0.031, η2 = 0.113, demonstrating the strongest positive feelings were reported in the wetland setting , followed by the control , and then the urban . There was no significant main effect of either Site Order, F = 0.186, p = 0.669, η2 = 0.006, or Stress Level, F = 2.434, p = 0.130, η2 = 0.077. None of the interaction effects were significant, for Site and Site Order, F = 1.588, p = 0.213, η2 = 0.052; for Site and Stress Level, F = 0.083, p = 0.921, η2 = 0.003.For the positive subscale of the PANAS, we found a significant main effect of Site, F = 8.671, p = 0.001, η2 = 0.230. There was no significant main effect of Site Order, F = 0.209, p = 0.651, η2 = 0.007, but the main effect of Stress Level was significant, F = 5.352, p = 0.028, η2 = 0.156. Because we also found an interaction effect between Site and Stress Level, F = 0.083, p = 0.921, η2 = 0.003, we conducted a simple effects analysis, which demonstrated that for the subgroup classified as ‘self-reported low stress,’ PANAS negative scores did not differ between sites, F = 1.188, p = 0.320, η2 = 0.078, but those classified as more stressed reported significant differences, F = 8.585, p = 0.001, η2 = 0.380. Pairwise comparisons demonstrated significantly fewer negative feelings in the wetland setting compared with both the control (p = 0.006) and the urban setting   .in situ. Here, we examine acute physiological and psychological stress responses during short-term exposure to a typical urban setting and an urban wetland in London, providing insight into the feasibility of using low-cost wearable technology the monitor the HWB co-benefits of urban blue-green space.Mental health is negatively associated with urban living  and posiOf the various physiological measures we acquired, only the HR response was found to be significantly different between exposure conditions. While acute cardiovascular responses to nature have been noted in earlier studies e.g., , novel iIn addition to the physiological metrics, we also asked participants to report changes in mood states using a validated self-report instrument. Again, effects on mood state varied depending on participants’ pre-exposure stress levels. Those with self-reported elevated stress levels in the week prior also reported a reduction in negative feelings after 10 min in the wetland compared with both the urban and control settings. This suggest at least a temporary improvement in subjectively experienced mood for those experiencing high stress in everyday life. In a similar vein, in situ and across different environmental gradients, and results have been inconsistent. Previous studies using the Emotiv® EEG technology applied proprietary algorithms that automatically process and translate the EEG signals into mental state indicators. post hoc statistical artifact correction. Significant loss of signal quality restricted the quantity of data available for processing. In addition, the low spatial resolution of the 14-channel Emotiv® system precludes analysis of signal origins, further complicating the interpretation of the data ,  but see . More reWe experienced similar technical difficulties with the acquisition and processing of some of the other psychophysiological metrics, which may have produced the negative findings on the HRV measures and indicators of EDA. Despite promising results in laboratory settings using images and 3-D simulations e.g., , studiesContrary to our expectations, we also found no difference in EDA between the exposure conditions, despite the fact that skin conductance is less susceptible to the confounding effect of concurrent physical activity, and more closely linked to psychological arousal or stress. Evidence from other studies suggests that low-cost mobile sensors can provide data on par with research-grade systems . One expIn addition to the technological challenges outlined earlier, we recognize a number of limitations associated with our experimental design. Time constraints limited the environmental exposure to a single wetland site, comprising both blue and green elements, and an urban site. The environments vary on a great number of variables, across a range of perceptual domains. This precludes judgments on the specificity of the observed effects. It remains unclear whether exposure to other types of blue and/or green spaces could deliver similar benefits. The absence of a green environment control  means we are unable to determine the impact of urban wetland habitat as opposed to any other non-built environment. As such our conclusions are drawn using more generic terms around ‘blue–green space’ as opposed to ‘blue space.’ Future studies should incorporate a greater range of environmental stimuli to identify specific elements producing strong effects, what the role of biodiversity is  and whetDue to the age range and anticipated variation in physical condition within the participant sample, any psychophysiological measure would be subject to large inter-individual variation, which introduces measurement noise and impacts the detection of statistically significant differences in a relatively small sample. While our sample size compares favorably with similar quasi-experimental studies , we cautOur categorization of participants into self-reported stressed and non-stressed .The dataset generated for this study can be found on a dedicated page within the Open Science Framework domain, maintained by the Center for Open Science (COS) . All data was stored in accordance with the UK Data Protection Act of 1988 (which was in force when the project commenced) and the EU General Data Protection Regulation which came into force in 2018. An internal review process within the Department of Life Sciences was used to investigate the ethical dimensions of the proposed research. Further elevation to a full review by the Imperial College Research Ethics Committee (ICREC) was not required on the basis of this initial assessment. The above process followed standard procedures for the assessment of low-risk, non-medical human research studies, as approved by the Head of Department of Life Sciences and the Head of Regulatory Compliance (within the Joint Research Compliance Office) at Imperial College London.This research was conducted in accordance with the RCUK Policy and Guidelines on Governance of Good Research Conduct (updated in 2017) and the guidelines set out by Imperial College London (JR, AV, RC, and AK conceived the research and secured funding for the project. JR and AV developed the study design, oversaw the data collection and analysis, and drafted the initial version of the manuscript. JR, AV, and ES analyzed and interpreted the data. VH contributed to participant recruitment and data collection. CN contributed to the development of the study design and data analysis. All authors contributed to the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lake sediments reveal that parts of the High Arctic were ice free and vegetated during the culmination of the Last Ice Age. Lake sediments retrieved from the beds of former nonerosive ice sheets offer unique possibilities to constrain changes in the extent and style of past glaciation, and place them in an absolutely dated context. We present the first pre-Holocene lake sediments from Arctic Svalbard. Radiocarbon dating of terrestrial plant fossils reveals that the investigated catchment was unglaciated and vegetated between 30 and 20 ka B.P. during the global Last Glacial Maximum. The presence of volcanic ash from a contemporaneous Azorean eruption also provides evidence for ice-free conditions. Indicators of sediment compaction and a depositional hiatus suggest subsequent coverage by nonerosive ice until 11 ka B.P. Comparison with regional paleoclimate data indicates that sea ice variability controlled this pattern of ice sheet evolution by modulating moisture supply. Facing rapid regional sea ice losses, our findings have implications for the future response of the Arctic’s cryosphere, a major driver of global sea-level rise. We characterized the stratigraphy of unit 4 sediments using four parameters on a common 0.5-cm resolution. Sediment density [dry bulk density (DBD)] and water content, basic measures for compaction . For this purpose, we sieved sample material through a 63-μm mesh and separated clay from silt using gravity settling. Both fine fractions were then analyzed as randomly oriented dry powders on a Bruker D8 ADVANCE ECO X-ray diffractometer. Last, we visualized sedimentary structure in three dimensions (3-D) using a ProCon X-Ray ALPHA Computed Tomography (CT) scanner. Extensive sampling of HAP0212 for previous work (n = 5) were devoid of identifiable compounds following gas chromatography mass spectrometry and flame ionization detection.This study focuses on HAP0212, a 332-cm-long undisturbed core taken from the deepest part of the basin. Here, we reappraised bottom unit 4 (272 to 320 cm)— homogenous, dense, and minerogenic sediments that were excluded from previous work on the Holocene lake history by (3). To detect discrete horizons, we sampled and analyzed the entire length of targeted unit 4 sediments. First, we focused on contiguous 10-cm sediment slices. Then, we zoomed in on sections that contained glass using 1-cm interval samples. Last, shards were picked for analysis from the discrete tephra horizon presented here (278.5-cm core depth) with the help of a gas chromatography syringe [cf. (Volcanic glass (tephra) was extracted from the investigated sediments using the flotation procedure of  and LuS 10870 (317 cm), first reported by .14C dates are in stratigraphic order and separated by stable time increments, which suggests that the investigated sediments have neither been disturbed nor reworked (table S2). The CT orthoslices in The chronology shown in Complementing the presented chronological evidence, minimal organic content—reflected by stable low (1.5%) LOI values (fig. S2A) (14C age above the investigated late Weichselian sediments (unit 4) at 265 cm yields an Early Holocene age of 11,260 to 11,082 cal. yr B.P. (2σ) (3) (−5 SI) values, this depositional signature is notably different from the Holocene sediments in Lake Hajeren (fig. S2A) but characteristic for compression by ice in similar Arctic lakes where pre-Holocene sediments have been preserved  analyses allay concerns of carbonate contamination of radiocarbon ages by freshwater reservoir effects: unit 4 host sediments that predominantly consist of the phyllosilicates (illite and muscovite) and quartz minerals that dominate the bedrock of the surrounding catchment  (14). Th(2σ) (3) . Togethe3) suggests that ash was deposited from primary air fall  content ranging from 13.29 to 14% (table S6). To find a geochemical match, we compared our data to reference material of trachytic eruptions from volcanoes that were active around the time of deposition  concentrations (>4.3%)  setting . An Azor3 dense rock equivalent) during the LAI eruption, which has been dated to between ~20 and 23 14C ka B.P. (14C ages (n = 3) taken from the base of LAI deposits to constrain the onset of this phase (table S5)  , providiable S5)  has a di10Be) dates with a complex exposure (inheritance) history. The reported association of undisturbed and compacted sediments with an overlying hiatus in Lake Hajeren is indicative for coverage by nonerosive (cold-based) ice, as observed in similar settings elsewhere [e.g.,  ice. The inferred timing of this transition from ice-free to ice-covered around 20 cal. ka B.P. is consistent with the most regional evidence of the culmination of ice sheet expansion. Using amino acid ratios on dated shells, Mangerud and co-workers . HoweverFrom a paleoclimate perspective, regional marine geological records reveal that the advance of ice over Hajeren and across Svalbard around 20 cal. ka B.P. coincides with sea ice shifts of an unprecedented magnitude during the discussed ~30 to 11 cal. ka B.P. While perennial sea ice formed along the western Svalbard margin after ~22.5 cal. ka B.P., seasonally open conditions were established farther north on the eastern Yermak plateau  and 5 4. Convers11). Climatologically, deglaciation can be attributed to high radiative forcing and intensifying heat advection by the surface ocean . This estimate is in agreement with geochronological evidence from the surrounding Mitra peninsula   (11). ClWe report the first evidence of pre-Holocene lake sediments on the Arctic Svalbard archipelago. The presented chronostratigraphic data reveal that unglaciated areas existed on northwest Spitsbergen between 30 and 20 cal. ka B.P., coincident with the LGM. Bryophyte macrofossils, used for dating, indicate that these cryptic refugia were vegetated. In light of evidence for ice-free conditions on high (>300 m a.s.l.) plateaus since 80 cal. ka B.P. (


Humulus lupulus) and two derived prenylflavanones, isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) gained increasing attention as potential anti-diabetic and cancer preventive compounds. Two enzymes of the aldo-keto reductase (AKR) superfamily are notable pharmacological targets in cancer therapy (AKR1B10) and in the treatment of diabetic complications (AKR1B1). Our results show that XN, IX and 8-PN are potent uncompetitive, tight-binding inhibitors of human aldose reductase AKR1B1  and of human AKR1B10 . The activity of the related enzyme AKR1A1 was left unaffected by all three compounds. This is the first time these three substances have been tested on AKRs. The results of this study may provide a basis for further quantitative structure–activity relationship models and promising scaffolds for future anti-diabetic or carcinopreventive drugs.Xanthohumol (XN), a prenylated chalcone unique to hops ( This has raised the demand for selective inhibitors that preferentially bind to AKR1B1 and leave the activity of AKR1A1 unaffectedAdditionally, the AKR1B subclass contains the AKR1B1 homologue AKR1B10 (71% sequence identity to AKR1B1), a NADPH-dependent oxidoreductase that converts both endo- and exogenous carbonyl group containing compounds to their corresponding alcohols,These metabolites are involved in protein prenylation, a process suspected of being crucial for carcinogenesis,,,,,,,,,AKR1B10 is physiologically expressed in the small and large intestine and in the adrenal glandsThus, the finding of new potent and selective inhibitors for both AKR1B10 and AKR1B1 are current challenges in biomedical research, in order to prevent uncontrolled cell proliferation and to reduce microangiopathies, as found in diabetic complications. In this article, we investigated the inhibitory potential of three hop-derived substances on three members of the AKR superfamily . Our results provide evidence that XN, IX and 8-PN are potent inhibitors of AKR1B1 and AKR1B10, while not affecting the activity of the closely related enzyme AKR1A1.2.2.1.2.1.1.XN was a friendly gift from Dr. Klaus Kammhuber, Lfl Hop Research Center Huell . IX and 8-PN were purchased from Biomol . NADPH was obtained from Carl Roth GmbH+Co. . Glucose, glyceraldehyde and farnesal were purchased from Sigma-Aldrich .2.2.2.2.1.Escherichia coli expression system according to previously published methods: AKR1A1 was a gentle gift from Prof. Dr. Vladimir WsolE. coli BL21 (DE3) cells. For over-expression of 6 × His-tagged enzymes, a 400-ml culture  was grown to an optical density of 0.6 at 600 nm at 37 °C. Protein over-expression was induced by adding isopropyl-1-thio-galactopyranoside (IPTG) to the culture medium . After 3 h, cells were harvested by centrifugation  and re-suspended in 20 ml PBS-I buffer . Cell disruption was performed by ultrasonication with cooling on ice, to avoid heating. The sample was subsequently centrifuged at 100,000 g at 4 °C for 1 h. The obtained supernatants, containing the target protein were purified using Ni-affinity chromatography on an ÄKTA-Purifier System . Purification progress was monitored by SDS-PAGE of the obtained fractions (not shown). Enzyme concentrations were determined using a Qubit 2.0 fluorometric quantitation system  according to the manufacturer’s instructions.The recombinant enzymes AKR1A1, AKR1B1 and AKR1B10 were prepared in an 2.2.2.2PO4 buffer (pH 7.4) and an appropriate amount of enzyme in a total assay volume of 0.8 ml. Final enzyme concentrations in the assay ranged from 583 nM (AKR1B10) to 712 nM (AKR1B1). For inhibitor selectivity studies on AKR1A1, AKR1B1 and AKR1B10 stock solutions of the inhibitors XN, XI and 8-PN were prepared in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the assay was ≤0.5%. Activity measurements were started without pre-incubation by adding an appropriate amount of enzyme. When collecting data for dose–response curves, initial velocities of the glyceraldehyde reduction  in the presence of inhibitors were assayed as described above. The percentage of inhibition was calculated considering the activity in the absence of inhibitor to be 100%.Catalytic properties were determined by measuring the decrease in absorbance at 340 nm at 37 °C . A reaction mixture without inhibitor consisted of different concentrations of substrate  were determined for each inhibitor in the presence of each enzyme, using the shared substrate glyceraldehyde  to assess specificity among the structurally similar members of the AKR-superfamily. For IC50 determination, experimental data were normalised and fitted to a sigmoidal curve as implemented in GraphPad6 . Whenever tight-binding inhibition was observed, the inhibition constant Ki was determined by fitting inhibition data to the Morrison equation as implemented in GraphPad Prism6 Initially, the half maximal inhibitory concentrations (ICM = 32 mM) and AKR1B10  were used to determine inhibition parameters. Enzyme inhibition parameters were assayed as described above. The inhibition mechanism of each compound for the respective enzymes was analysed by plotting IC50 values at different substrate concentrations (at least five inhibitor and substrate concentrations),In order to verify the inhibitory potency, enzyme-specific physiological substrates for AKR1B1 . The IC50 for IX is 6 to 15 times lower than compared to XN (50 > 50 µM).Initially, dose–response curves for XN, IX and 8-PN with AKR1A1, AKR1B10 and AKR1B1, using glyceraldehyde, were calculated . 50- and Ki-values , ICbstrates  and 6.A closer examination of the binding mechanism of the three inhibitors was carried out by investigating their mode with respect to the co-factor binding site for NADPH. Interestingly, all the three substances behave as uncompetitive inhibitors of AKR1B10, while only IX displays the typical pattern of an uncompetitive inhibitor on AKR1B1, whereas XN and 8-PN inhibit in a non-competitive fashion  and AKR1B10 are involved in numerous pathologies and have therefore been proposed as suitable targets for drug development. Under hyperglycemic conditions, AKR1B1 promotes osmotic imbalance and modifies physiological ratios of the redox couples of both NAD50 and Ki values ranging at low µM or nM concentrations, using d,l-glyceraldehyde as a generic substrate .Our results show that XN, IX and 8-PN are potent inhibitors of the AKR1B’s subfamily members B1 and B10 with ICrate see . In the rate see . After aInterestingly, all three inhibitors exhibit a 4′-OH group and a 2-benzyl substituent in their structure  and its isomer 6-prenylnaringenin (6-PN), which is also a phytoestrogenic prenylflavanone occurring in hops  at the C-8 position of their flavonoid skeletons, playing important roles in aldose reductase inhibition,vice versa, in order to exclusively treat complications of diabetes or cancer. Yet, only a few selective inhibitors have been identified so far, including Androst-3β,5α,6β,19-tetrol (IC50 = 0.86 µM) or oleanolic acid (IC50 = 0.09 µM),We further demonstrated that the three substances have no inhibitory effect on the closely related AKR member AKR1A1. This is an important result since many candidate inhibitors failed in clinical trials due to their undesired property of inhibiting both enzymes, AKR1B1 and AKR1A150 = 0.57 μM for AKR1B1 and IC50 =  1.09 μM for AKR1B10) and 8-PN (IC50 = 0.81 μM for AKR1B1 and IC50 = 0.99 μM for AKR1B10) inhibit both enzymes with an efficacy 6 to 15 times greater than XN when applying GA as substrate (Our results show that IX (ICubstrate .50 =  0.88 μM for AKR1B1 and IC50 =  0.63 μM for AKR1B10) and 8-PN (IC50 =  1.87 μM for AKR1B1 and IC50 =  3.96 μM for AKR1B10) is 10–30 times greater than that of XN , thus circumventing competition with the substrate for the active site. For AKR1B1, this constitutes a promising mechanism of inhibition, since the affinity of uncompetitive inhibitors is greatest at saturating concentrations of the substrate, as observed under hyperglycemic conditions In order to closely examine the binding mechanism of the substances, further experiments were performed to investigate whether XN, IX and 8-PN compete with NADPH at the co-factor binding site. However, our work revealed no competition between the three tested substances and NADPH, neither for AKR1B1, nor for AKR1B10. All substances tested exhibited an uncompetitive mode of inhibition for AKR1B10 with respect to NADPH, while only IX showed an uncompetitive inhibition pattern for AKR1B1, with respect to the co-factor binding site. XN and 8-PN exhibited a non-competitive mode of inhibition on AKR1B1.M. mulatta).Interestingly, the abovementioned substances show an uncompetitive mode of action with respect to both, the substrate and the co-factor binding site. Hypothetically speaking, this would mean that the inhibitor does not bind to the enzyme–NADPH binary complex as expected, but, instead, to the subsequent and transient enzyme conformation after the product release but before the release of NADP+. This mechanism was previously hypothesised by Harris and Kozarich, 1997Hence, XN, 8-PN, IX for AKR1B10, and IX for AKR1B1 might act as uncompetitive inhibitors that bind to an enzyme species, following the formation of the initial ES complex. More challenging to explain is the behavior of 8-PN and XN, which both have been showing a non-competitive mode of inhibition with respect to AKR1B1 at the NADPH binding site.Since the models obtained from molecular docking experiments (data not shown) did not clarify the binding mechanism of the three compounds, further investigations by X-ray diffraction are required in future studies.Overall, we could show that the hop compounds tested have a selective inhibitory effect on the AKR1B subfamily, with respect to AKR1A1. All substances exhibit an uncompetitive mode of inhibition on both, AKR1B1 and AKR1B10. This fact might be promising when it comes to designing novel drugs and molecular therapies with high efficacy even at low concentrations. The results of this study may also provide a basis for further quantitative structure–activity relationship models and favorable scaffolds for new selective inhibitors of AKR1B1 or AKR1B10.


The development of high‐performance oxygen evolution reaction (OER) catalysts is crucial to achieve the clean production of hydrogen via water splitting. Recently, Co‐based oxides have been intensively investigated as some of the most efficient and cost‐effective OER catalysts. In particular, compositional tuning of Co‐based oxides via doping or substitution is shown to significantly affect their catalytic activity. Nevertheless, the origin of this enhanced catalytic activity and the reaction mechanism occurring at catalytic active sites remain controversial. Theoretical investigations are performed on the electrocatalytic properties of pristine and transition metal ‐substituted Co oxides using first‐principle calculations. A comprehensive evaluation of the doping effects is conducted by considering various oxygen local environments in the crystal structure, which helps elucidate the mechanism behind the doping‐induced enhancement of Co‐based catalysts. It is demonstrated that the local distortion induced by dopant cations remarkably facilitates the catalysis at a specific site by modulating the hydrogen bonding. In particular, the presence of Jahn–Teller‐active Fe(IV) is shown to result in a substantial reduction in the overpotential at the initially inactive catalysis site without compromising the activity of the pristine active sites, supporting previous experimental observations of exceptional OER performance for Fe‐containing Co oxides. The active sites and reaction mechanism are predicted from extensive examination of the oxygen sites near the dopants. The OER mechanisms are discussed in detail for each site, including the terminal/bridge oxygen sites neighboring the dopant or Co cations in the structure. Notably, it is demonstrated that the bridge oxygen site, which has generally been considered to be inactive for the OER, can be catalytically activated by Fe incorporation. We further unveil the structural properties of the relevant intermediates and propose that the modulation of the hydrogen bonding length induced by the Jahn–Teller‐active Fe(IV) cation leads to the activation of the bridge site. Finally, with a comprehensive view of the estimated OER thermodynamics, we discuss the origin of the improved OER performance of Fe‐containing Co‐oxide‐based catalysts. Our findings help establish theoretical design rules for Co‐based multimetal‐oxide‐based catalysts, and we propose a viable strategy to resuscitate the catalytic activity of inert sites via structural modulation using cation doping.2Figure2‐O) is the oxygen that bridges the doped metal and Co. The acid–base mechanism was considered to involve four elementary steps of the OER, where the intermediates OH*, O*, and OOH* are formed in sequence through the four proton‐coupled electron transfer (PCET) steps (see the Supporting Information for details).An oxide cluster model was adopted in our theoretical investigations on the catalytic mechanism for both the pristine and doped Co oxides, as shown in FigureB) to probe the electron transfer at each step. During the first two PCET steps, the holes required for O—O bond formation are accumulated in the terminal Co=O group. The initial extraction of one H+ and e− from the terminal Co—OH2 group oxidizes Co(III) to Co(IV), preceding the formation of the terminal Co—OH group. The removal of another H+ and e− further oxidizes the terminal Co—OH group, yielding a Co=O group with η‐oxo ligand. The two holes produced by the first two oxidation steps are accommodated in both the Co ion and η‐oxo ligand, as observed by the change in the spin moments of each species (from 0.00 to 1.39μB for Co and from 0.00 to 0.83μB for O) in Figure 2 molecule, completing the OER cycle with the restoration of the catalyst to its initial state.A schematic illustration of the OER on the cobalt terminal site (green circle) of the pristine cobalt oxide is displayed in The blue line in Figure Figure+ from the terminal Fe—OH2 group, which is accompanied by the oxidation of Fe(III) to Fe(IV). In the second step, while the terminal Fe—OH group is further deprotonated to form a η‐oxo ligand, one proton is transferred from the neighboring Co—OH2 to the other Fe terminal oxygen adjacent to the reaction site, as indicated by the black dotted arrow in Figure 2 molecule. For the Fe‐doped system, the O—O bond formation step was observed to be the potential‐determining step, requiring a theoretical η of 0.44 V, which is similar to that at the cobalt terminal site, 0.46 V, as shown in Figure 2* → OH*) only required a reaction free energy of 1.08 eV, suggesting that the Co oxide host provides an environment in which the higher valence state of Fe can be stabilized. This observation is also consistent with previous analysis on the structurally analogous β‐CoOOH, for which similar theoretical η values of 0.47 and 0.43 V were reported for the pristine and Fe‐doped structure, respectively.We further investigated the OER process occurring at the dopant  terminal sites, as shown in 2* state of the OER cycle, the doped Ni is in the 2+ valence state because of the charge transfer between Ni and Co —OH, Ni(III)—O•, and Ni(III)—OOH groups in turn during the OER cycle. Of the four PCET steps, the second step (OH* → O*) is estimated to be potential‐determining, delivering a theoretical η of 1.17 V , the introduction of Fe significantly reduced the energy for the equivalent step to 1.64 eV, indicating that the activation of the bridge site in the Fe‐doped oxide is likely to originate from the more stable OOH* adsorption compared with that of the undoped model. We observed that this stabilization is closely related to the structural distortion induced by the Jahn–Teller‐active Fe(IV). Figure  eV, the GOOH* − ΔGO* and ΔGOH* in FigureGOOH* = ΔGOH* + (3.34 ± 0.10) eV , which indicates only a small discrepancy in the intercept value with an approximate universal scaling relation of ΔGOOH* = ΔGOH* + (3.2 ± 0.20) eV estimated for rutile oxides and perovskites.t‐Co) and dopant terminal site (denoted as Fet‐Do) in the Fe‐doped model exhibited similar theoretical overpotentials as the terminal site of pristine Co oxide (denoted as Cot‐Co). However, the activity of the bridge site was remarkably enhanced in the Fe‐doped Co oxide compared with that in the pristine Co oxide. This finding suggests that the exceptional performance of Fe‐containing Co oxides originates from the provision of additional active sites represented by the bridge site (denoted as Feb) rather than from the further enhancement of the intrinsic activity of existing active sites. Unlike Fe doping, the doping with Ni or Mn is predicted to result in deterioration of the OER performance of Co oxides. Although the effects of Ni and Mn doping on the cobalt terminal sites were modest, the Ni and Mn terminal sites were estimated to be inactive toward the OER. In addition, the catalytic activation of the bridge site is not expected for Ni‐ and Mn‐doped systems. Therefore, the large overpotential of the dopant terminal sites would induce an overall reduction in the number of exposed active sites, resulting in degradation of the OER performance.The resulting overpotentials of all the reaction sites considered for the pristine and doped Co oxides are represented in the 2D map of Δ2), and it is well‐known that they typically crystalize in the plate‐like structures in the nature or in the conventional synthetic process.Co oxyhydroxides are one of the most representative layered structures along with their lithium analog ‐substituted Co oxides using first‐principle calculations. Regardless of the dopant type, the changes in η of the cobalt terminal site were less than 0.1 V. The Fe terminal site exhibited an η of 0.44 V, which was comparable to that of the terminal site of pristine Co oxide (η = 0.46 V), whereas the activity of the Ni and Mn terminal sites was severely limited by difficulties in forming O* intermediates. Notably, the presence of Fe significantly reduced the η at the bridge site from 0.71 to 0.46 V. This catalytic activation of the bridge site was attributed to the stabilization of OOH* formation, which benefits from the local distortion of the Fe cation and consequent dramatic change in the hydrogen bond strength. From these results, we propose that the key origin of the enhanced performance of Fe‐containing Co oxides is the supply of additional active sites represented by the bridge site rather than the improvement of the existing active site denoted as the terminal site. These findings help to clarify the OER performance of recently reported Co‐oxide‐based catalysts and provide theoretical guidelines for the rational design of multimetal‐based catalysts. Moreover, it is newly proposed that applying local strain to the native environment of reaction sites using cation doping could be a viable strategy to enhance the catalytic activity of materials.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Hendrick Avercamp: Master of the Ice Scene. Wintery weather predominated across most of Europe during the Little Ice Age, which started around 1300 and lasted for about 350 years. The atypically cold, harsh weather affected agriculture, health, commerce, emigration, and social stability, precipitating unrest, revolt, warfare, and migration. Throughout this time, epidemics of plague flared across Europe, and malaria persisted despite frigid conditions. “No other nation in the world looks forward to an icy season the way the Dutch do,” wrote Wim Pijbes and Earl A. Powell III in the preface to The coldest period of the Little Ice Age roughly overlapped the 90 years of the Dutch Golden Age. Unlike most of Europe, the Dutch Republic, which preceded the Netherlands of today, thrived during the icy 17th century. Despite its small size and limited natural resources, the country achieved stability and prosperity, chiefly because of its unmatched commercial fishing and trading fleets and its agricultural practices. That confluence of “political, economic, religious, and social circumstances created a unique and fruitful climate for the arts,” according to the National Gallery of Art, which also notes, “A remarkable number of pictures of extraordinary quality were produced during the Dutch Golden Age.” Perhaps less known than some of his contemporaries, Hendrick Avercamp is nonetheless considered to be among the great masters of that time. Avercamp painted vibrant, detailed winter landscapes during the first third of the 17th century. The United Sates National Gallery of Art states that he “did not invent the winter landscape, but he was the first artist to specialize in this quintessential Dutch subject.” Details of Avercamp’s life are sparse. He was born in Amsterdam in 1585, though the next year his family relocated to the smaller city of Kampen. His father, Barent, served as the town apothecary and later as its doctor until he died of the plague in 1602. His first art teacher and one of his brothers also died of the plague during that frigid 17th century. Apparently, Avercamp could not speak and was known as “the Mute of Kampen.” He came from a learned family: his father and brothers were physicians and apothecaries, and his grandfather and uncle were headmasters of a prestigious Latin school. Avercamp probably lived in the house that his mother Beatrix owned. Art historian Arthur K. Wheelock Jr. observed that “There, in relative isolation from the mainstreams of Dutch art, he devoted himself almost entirely to the painting of winter scenes and specifically to depictions of crowds of people engaging in a wide range of activities on frozen rivers.” In total, Avercamp produced about a hundred paintings of winter scenes.Winter Landscape with Ice Skaters, this month’s cover art, following the exceptionally frigid winter of 1607–1608. The painting, which is his largest known work, reveals an icy fog cloaking the winter sun and the edges of the village. A stark filigree of bare tree limbs claws toward the winter sky. The artist achieved an impression of depth by fading colors and blurring shapes in the distance. His placement of buildings and trees that flank the frozen canal also guides viewers toward the misty horizon. Pieter Roelofs, a curator at Rijksmuseum, Amsterdam, observes that Avercamp’s use of vantage points and perspective served to “create a more natural space so that the viewer is involved more directly in the events on and around the ice.” Avercamp completed The Rijksmuseum offers additional details: “The high vantage point of this painting turns it into a sampler of human―and animal―activity during a harsh winter. Hundreds of people are out on the ice, most of them for pleasure, others working out of dire necessity.” A crowd of busy figures is scattered across the canvas. Among the working class, a beggar seeks bread, an angler balances a long trident and grips the day’s catch, and a brewery worker fetches water from a hole cut into the ice. Knots of people conversing, couples skating, and sportsmen playing kolf on the ice (a hybrid golf and hockey) reveal that for many others this is a day for recreation and socializing. Animals scattered throughout the painting include several horses pulling sledges; birds hovering in the silvery air or clinging to bare branches or sloping rooftops; domesticated fowl pecking for food; and a dog scampering over the ice. Roelofs notes, “Avercamp did not concentrate solely on winter fun and games and the sunny side of the cold season.” For example, he placed crows and a dog feasting on a horse carcass in the foreground of this painting. Winter Landscape with Ice Skaters provides a glimpse of village life during Europe’s Little Ice Age and may stimulate viewers to consider the interrelationship of climate and human, animal, and environmental health. Environmental history professor Dagomar Degroot comments on the Little Ice Age: “Changing weather patterns altered the range of insects that carried pathogens, bringing new and deadly ailments to the previously unexposed. Because malnourished bodies have weak immune systems, farmers and their livestock soon fell sick. Refugees from the famine-stricken countryside spread disease to cities, where epidemic outbreaks often inflicted a fearsome toll.”


Administration of endogenous mediators or exogenous chemicals in migraine patients provoke early headaches and delayed migraine-like attacks. Although migraine provoking substances are normally vasodilators, dilation of arterial vessels does not seem to be the sole contributing factor, and the underlying mechanisms of the delayed migraine pain are mostly unknown. Sustained mechanical allodynia is a common response associated with the local administration of various proalgesic substances in experimental animals and humans. Here, we investigated the ability of a series of endogenous mediators which provoke or do not provoke migraine in patients, to cause or not cause mechanical allodynia upon their injection in the mouse periorbital area.Mechanical allodynia was assessed with the von Frey filament assay. Stimuli were given by subcutaneous injection in the periorbital area of C57BL/6J mice; antagonists were administered by local and systemic injections.2 (PGE2) and prostacyclin (PGI2), but not PGF2α, evoked a dose-dependent periorbital mechanical allodynia. The painful responses were attenuated by systemic or local  administration of antagonists for CGRP (CLR/RAMP1), PACAP (PAC-1), histamine H1, PGE2 (EP4), and PGI2 (IP) receptors, respectively.Calcitonin gene related peptide (CGRP), but not adrenomedullin and amylin, pituitary adenylyl cyclase activating peptide (PACAP), but not vasoactive intestinal polypeptide (VIP), histamine, prostaglandin E2, PGI2), or do not provoke (VIP and PGF2α), migraine-like attacks in patients and periorbital allodynia in mice suggests that the study of allodynia in mice may provide information on the proalgesic mechanisms of migraine-provoking agents in humans. Results underline the ability of migraine-provoking substances to initiate mechanical allodynia by acting on peripheral terminals of trigeminal afferents.The correspondence between substances that provoke (CGRP; PACAP, histamine, PGE Migraine is a pain disorder that affects about 15% of the adult population worldwide. Thus, the burden of migraine is enormous in terms of suffering, disability, healthcare, and social and economic costs . AlthougA prototypical example of a migraine-provoking agent is glyceryl trinitrate (GTN). Occupational exposure to, or treatment with, organic nitrates has long been known to provoke headaches –10. TypiIn the last three decades, rigorous studies with randomized, double blind and crossover designs have been undertaken, resulting in a systematic investigation of the ability of a series of endogenous mediators or exogenous chemicals to provoke early headaches and delayed migraine-like attacks . Vasodil2 (PGE2), prostacyclin (PGI2) and prostaglandin F2α (PGF2α). The receptor type implicated in the PMA evoked by each mediator was also studied. A close correspondence was found between agents that provoke/not provoke delayed migraine in patients and PMA in mice. Thus, the study of PMA in mice may provide information on the proalgesic mechanisms that, in humans, result in the development of migraine-like attacks provoked by endogenous mediators and exogenous chemicals.Here, we have investigated whether a series of endogenous mediators, which have been found to provoke or not provoke migraine-like attacks in patients, elicit or do not elicit delayed and prolonged PMA after their injection in the periorbital skin of mice. Provocation tests in humans are usually performed by systemic administration of the stimulus . Howevervivarium, maintained on a 12-hour dark/light cycle (light off from 7.00 PM to 7.00 AM), and with free access to food and water. Animal studies were reported in compliance with the ARRIVE guidelines ) and the inactive immunoglobulin  were from Abcam CGRP, amylin, PACAP, VIP, PGF2, PGI2 and PGF2α  or their vehicles (0.9% NaCl). The subcutaneous injection was performed unilaterally on the right side of the periorbital area. The mouse was restrained by the double handed manual restraint method [C57BL/6J mice were injected subcutaneously in the periorbital area  with CGRP, adrenomedullin, amylin, PACAP, VIP, histamine, PGEt method . BrieflySome mice were pre-treated (30 minutes before the stimuli) with intraperitoneal  olcegepant (1 μmol/kg corresponding to 0.869 mg/kg), astemizole (4 μmol/kg corresponding to 1.8 mg/kg), ER819762 (60 μmol/kg corresponding to 29.4 mg/kg) or their vehicle  and intravenous  PACAP6-38 (12 nmol/kg corresponding to 48 μg/kg) and Ro1138452 (30 μmol/kg corresponding to 10.4 mg/kg), or their vehicle (0.9% NaCl). Other mice received locally  olcegepant (4 nmol/site), astemizole (10 nmol/site), ER819762 (10 nmol/site), or their vehicle (4% DMSO and 4% tween 80 in 0.9% NaCl) and PACAP6-38 (240 pmol/site) and Ro1138452 (10 nmol/site), or their vehicle (0.9% NaCl), or a mouse monoclonal anti-CGRP antibody or, as a control, a mouse monoclonal IgG2a . In another set of experiments, C57BL/6J mice received intraplantar  injections of olcegepant (4 nmol/site), astemizole (10 nmol/site), ER819762 (10 nmol/site), or their vehicle (4% DMSO and 4% tween 80 in 0.9% NaCl), or PACAP6-38 (240 pmol/site) and Ro1138452 (10 nmol/site), or their vehicle (0.9% NaCl).Immediately after the p.orb. injections, mice were placed inside a plexiglass chamber and spontaneous nociception was assessed by measuring the time (seconds) that the animal spent face rubbing the injected area with its paws  over thei.e., the area of the periorbital region facing the sphenoidal rostrum) of the mice [The measurement of PMA was performed by using the up-and-down paradigm as described previously , 28 in tthe mice  Fig 1a)ai.e., thi.e. lighter) filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated from these scores by using a δ value of 0.205, previously determined.The day of the experiment, after 20 minutes of adaptation inside the chamber, a series of 7 Von Frey filaments in logarithmic increments of force  were applied to the periorbital area perpendicular to the skin, with sufficient force to cause slight buckling, and held for approximately 5 seconds to elicit a positive response. The response was considered positive by the following criteria: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking. The stimulation initiated with the 0.16 g filament. Absence of response after 5 seconds led to the use of a filament with increased weight, whereas a positive response led to the use of a weaker , as well as IBM SPSS . P<0.05 was considered statistically significant.CGRP, amylin and adrenomedullin belong to the larger calcitonin family of peptides, which activate, with different potencies, a series of receptors resulting from the multiple combinations of the 3 forms of the calcitonin b subtypes) receptor and the CT receptor-like receptor (CLR) with the 3 forms of receptor-activity-modifying proteins (RAMPs) . Althoug-47b subtLocal p.orb., 10 μl) administration of adrenomedullin or amylin at the same pro-allodynic dose of CGRP (1.5 or 15 nmol/site), was unable to produce any measurable acute nociceptive response, even at the highest dose. Adrenomedullin or amylin also failed to produce PMA over the entire period of observation (6 hours) , is a mainstay of the acute migraine attack, thus implicating prostaglandins in migraine pain [2 administration in the mouse paw is known to evoke spontaneous acute nociception [2 , but not PGI2  or PGF2α (1.5-15 nmol/site) injection into the mouse periorbital skin elicited a marked spontaneous nociceptive response  PMA  antagonist, ER819762, prevented PGE2-induced spontaneous nociception and PMA  did not affect PMA evoked by PGI2 , and amylin, which is equipotent to CGRP on the receptor combinations formed by the three CT subtypes with RAMP1, RAMP2 or RAMP3, failed to evoke allodynia. A possible effect of amylin and adrenomedullin on the RAMP1/CLR, or of CGRP on the different combinations of receptors for amylin and adrenomedullin has been claimed to contribute to the pro-migraine action of CGRP or its receptor [The members of the calcitonin family of peptides activate a variety of receptors deriving from the dimerization of CLR or CL with RAMP proteins. Adrenomedullin, which stimulates the combinations of the CLR with RAMP2 or RAMP3 with a potency higher than CGRP (AMreceptor . As prevreceptor  and perireceptor , we confreceptor , whereasreceptor , strengt2 and PGI2, two prostaglandins that induce headaches and migraine-like attacks in humans [2α, a prostaglandin, which was unable to evoke migraine-like attack in patients [4 and IP receptors showed that PGE2 and PGI2 caused allodynia exclusively by activating the respective preferred receptor. This conclusion suggests that in humans PGE2 and PGI2 elicit migraine-like attacks by acting on EP4 and IP receptors, respectively. As reported previously, in the mouse hindpaw [2 was the sole compound among all the presently investigated substances that evoked an early spontaneous nociceptive response, which, similarly to allodynia, was abated by EP4 receptor antagonism. However, given that only one of the migraine-provoking substances elicited spontaneous nociception, the significance of such early non-evoked pain-like responses for migraine pain mechanism remains unclear.Clinical trials with anti CGRP or anti RAMP1/CLR monoclonal antibodies, while showing excellent efficacy and safety profile, also indicate that a subset of migraine patients either do not respond or have a partial benefit , 43, 44.n humans –48, elicpatients , failed  hindpaw , PGE2 wa1 receptor subtype, histamine evokes PMA in mice and provides indirect support to the contribution of the H1 receptor, rather than H2 receptors [via H1 receptor activation, the H1-antagonists were not always effective in reducing migraine [Histamine, a key proinflammatory and allergic mediator with a proalgesic role provokes migraine-like attacks in patients –52. Furteceptors , in proveceptors . It shoumigraine .per se a major factor contributing to allodynia in mice and migraine pain in humans. These findings are in line with previous observations that PACAP, but not VIP, causes delayed activation and sensitization of central trigeminovascular neurons via activation of the PAC1 receptor [via a hitherto uncharacterized receptor, degranulates mast cells [VIP and PACAP, which belong to the glucagon/secretin family of regulatory peptides, stimulate three distinct receptors: the PAC-1, selectively activated by PACAP, and the VPAC-1 and VPCA-2, which are equipotently activated by both PACAP and VIP. The observation that PACAP, but not VIP, elicited allodynia, suggests that the PACAP/PAC-1 is the sole pathway implicated in generating pain-like responses. PACAP and VIP are both vasodilator substances , 56. Thereceptor . The impreceptor , and PACst cells . The preThe underlying mechanism that promotes migraine attacks is unclear. Clinical investigation with small molecule CGRP receptor antagonists underscores the key role of CGRP in the genesis of migraine pain , 61. HowDifferences may exist between the trigeminal fibres of the skin and those innervating meningeal blood vessels , 63 thatper se sufficient to elicit pain. Cutaneous allodynia is frequently reported by migraine patients during the attack [The major finding of the present study is the strict correspondence between mediators that provoke migraine in patients and evoke periorbital allodynia in mice. The same correspondence was observed between mediators that do not provoke migraine in patients and do not evoke allodynia in mice. An additional relevant finding is that, although most of the pro-allodynic substances tested in the present study are vasodilators, two robust vasodilators, VIP and adrenomedullin, did not evoke allodynia, thus indicating that vascular activity is not e attack , 69. How


In the present study, the potent inducers of phase II detoxifying and antioxidant stress responsive to icariside II was investigated. First, a dose of 0–10 µM icariside II showed no significantly cytotoxicity on HepG2 cells by MTT assays and icariside II could enhance cellular GSH levels by ELISA assay. Then, the potential roles of ERK, Akt and JNK in the regulation of icariside II-induced Nrf2-dependent ARE transcriptional activity as well as ARE-mediated endogenous HO-1 and glutathione GST protein expression in HepG2 cells were estimated. Icariside II activated the nuclear translocation of Nrf2 and the up-regulated expression of Nrf2-related antioxidant protein OH-1 and GST were evaluated by Western blotting. Then the phosphorylation levels of ERK1/2, Akt and JNK1/2 were further examined by Western blotting assays. Results showed that icariside II significantly increased the phosphorylation levels of ERK1/2, Akt and JNK1/2. Furthermore, icariside II-induced ARE transcriptional activity was attenuated by the inhibition of ERK, Akt and JNK signaling using biochemical inhibitors. These results suggest that the Nrf2/ARE pathway plays an important role in the regulation of icariside-mediated antioxidant effects in HepG2 cells. GSHglutathioneHO-1heme oxygenase-1GSTglutathione S-transferaseAREantioxidant responsive elementKeap1Kelch-like ECH-associated protein 1GCLg-glutamyl-cysteine ligasePKCProtein kinase CERendoplasmic reticulumVEGFvascular endothelial growth factorGlut4glucose transporter 4There are many natural and synthetic compounds that can induce phase II detoxifying and antioxidant stress responsive genes. The induction of those genes is a highly effective strategy for protection against antioxidant stress ,2,3. TheEpimedium koreanum Nakai is a perennial herb that grows in the northeast part of China, north part of North Korea and Japan  × 100%.Inhibition of cell proliferation by icariside II was measured by the MTT assay . Brieﬂy,For measurement of intracellular GSH levels, ELISA analysis was performed as previously described . HepG2 c6) were seeded in a 6 cm plate and incubated at 37 °C for 24 h. Cells were treated with various concentrations of icariside II for 0–6 h and then washed twice with ice-cold PBS, and lysed using cell lysis buffer . The lysates were collected by scraping from the plates and then centrifuged at 10,000 rpm at 4 °C for 5 min. The protein samples (20 g) were loaded on a 12% of SDS-polyacrylamide gel for electrophoresis, and transferred onto PVDF transfer membranes  at 0.8 mA/cm2 for 2 h. Membranes were blocked at room temperature for 2 h with blocking solution (1% BSA in PBS plus 0.05% Tween-20). Membranes were incubated overnight at 4 °C with primary anti-bodies at a 1:1000 dilution  in blocking solution. After thrice washings in TBST for each 5 min, membranes were incubated for 1 h at room temperature with an alkaline phosphatase peroxidase-conjugated anti-mouse secondary antibody at a dilution of 1:500 in blocking solution. Detection was performed by the BCIP/NBT Alkaline Phosphatase Color Development Kit  according to the manufacturer’s instructions. Bands were recorded by a digital camera .For isolation of protein fractions, HepG2 cells (1 × 10p < 0.05.Data are presented as means ± SEM. Differences were evaluated using unpaired Student’s t-tests. The level of statistical signiﬁcance was set at In several diseases, such as ischemia-reperfusion injury, major burns and pyaemia caused by oxidative stress in cardiovascular and transplant surgery, free radical-mediated oxidative stress play an important clinical role, which causes the depletion of the body’s antioxidant defense system. Antioxidant supplements enhance the body’s antioxidant defense capacity and reduce the resulting tissue damage. The present study indicates that icariside II expresses antioxidant effects in HepG2 as a result of inducing antioxidant enzymes via the Nrf2/ARE pathway. The study provides evidence of a protective role for icariside II against oxidative injury and further indicates the multiple effects of icariside II, which could potentially contribute to antioxidant protection.


This study shows the feasibility of the Electrospinning method as a process step for advanced and fast production Li ion cells. Lamination is a key technology for Lithium-ion battery production. It bares different advantages, such as a fast production line speed by fixing the separator to the electrodes. Unfortunately, this technology is inapplicable for separator and electrode formulations not based on thermoplastic binders. Using Electrospinning, this disadvantage can be overcome. In our study, beaded PVDF polymer nanofibres were spun onto a fibre-reinforced, inorganic-filled separator. This modified separator was then laminated onto a NMC111-cathode using a temperature profile of 110/110/120°C within the laminator. After Lamination, the separator was pulled-off again and placed in a SEM to see the adhesive behaviour of the applied polymer. The information gathered with SEM clearly shows a successful lamination of the separator to the electrode. With the rise of portable consumer electronics over the last decades, lithium-ion battery technology has become one of the most used storage technologies. Beginning in the 1970s, after the commercialization by Sanyo –4, elect® 5130, SOLVAY, Italy) using N-methyl-pyrrolidone  as a solvent. The resulting slurry is then single-side coated using a doctor-blade coater in a roll-to-roll process. The electrodes are dried during the coating process in a two-step in-line drying tunnel at a temperature range of 135°C to 150°C. The fibre-reinforced, inorganic-filled separators have been place on to the rotating drum collector of the Inovenso NE300-XP Electrospinning set-up. The set-up used a common rail three-nozzle emitter and a spinning distance of 12 cm at a voltage of 25kV. To ensure a homogeneous coating of fibres, the collector stage was moved continuously parallel to the drum collector axis 60 mm to each side, with a speed of 5 mm/s. The separator was placed on a drum collector, which rotated with 100 rpm during the spinning process. The humidity inside the spinning chamber was controlled to be 30% relative at 25°C. The spinning solution held a polymer concentration of 10 wt % THV 221 (3M) solved in tetrahydrofuran (THF) using a magnetic stirring plate. The solution is stirred at 75°C for 1 h under reflux and afterwards cooled down to room temperature over a period of 1.5 h. The spinning process itself is conducted for only 3 min for each separator sheet. The electrodes are punched into sheets with an area of 5 x 8 cm2. The modified separators are cut to rectangular shape with an area of 5.5 x 8.5 cm2. Afterwards, the separators are placed with the electrospun side onto the active material of the electrode and inserted in a roller lamination press . Our modified lamination technique is tested for NMC-cathodes, using a roll speed of 1.38 m/min, a line force of 157 N/cm in a temperature profile of 110/110/120°C in the pre-heating unit of this. The thus prepared cathode-separator pair is then ripped apart to investigate the interfaces after lamination. To control the results and to have further information on the distribution of the polymer on the separator, samples are prepared by electrospinning and lamination without a cathode. To further investigate the lamination, a peeling force test using a manual peeling test stand  is administered. The measurable force with this set-up is between 0 and 5 N with a resolution of 0.001 N. The samples are therefore taped to the machine using double-sided tape. The electrode is pulled of directly with a self-adhesive tape from the separator. The samples are prepared for SEM by punching out disks of 12 mm diameter. These disks are attached to sample holders using graphite tape. The samples are not sputtered, therefore only a low acceleration voltage is used for taking the pictures.NMC  is mixed in a planetary mixer  with Super C65 carbon , KS6L graphite  and PVDF binder Click here for additional data file.


We extended these studies to the desmethylated form of ovothiol, 5-thiohistidine, and confirmed that this ovothiol derivative also acts as a non-competitive-like GGT inhibitor, with a potency comparable to ovothiol. We also found that both 5-thiohistidine derivatives act as reversible GGT inhibitors compared to the irreversible DON. Finally, we probed the interactions of 5-thiohistidines with GGT by docking analysis and compared them with the 2-thiohistidine ergothioneine, the physiological substrate glutathione, and the DON inhibitor. Overall, our results provide new insight for further development of 5-thiohistidine derivatives as therapeutics for GGT-positive tumors.Gamma-glutamyl transpeptidase (GGT) is a cell surface enzyme involved in glutathione metabolism and maintenance of redox homeostasis. High expression of GGT on tumor cells is associated with an increase of cell proliferation and resistance against chemotherapy. GGT inhibitors that have been evaluated in clinical trials are too toxic for human use. We have previously identified ovothiols, 5(Nπ)-methyl-thiohistidines of marine origin, as non-competitive-like inhibitors of GGT that are more potent than the known GGT inhibitor, 6-diazo-5-oxo- To date, the organisms known to be able to synthesize l-ergothioneine are bacteria, more specifically mycobacteria and cyanobacteria acetic acid), DON (6-diazo-5-oxo-l-norleucine), and Azaserine (O-diazoacetyl-l-serine)  concentration of the inhibitor. The values of the apparent Ki obtained at each inhibitor concentration were averaged to obtain a unique value of apparent Ki. The apparent inhibition constant (t-Student test).Kinetic parameters and their corresponding standard errors were evaluated using a simple weighting method (The reversibility of the inhibition was assessed using a dilution method described previously . Briefly3 cell/well) by a pipetting robot  and treated with 5-thio, at the indicated concentrations for 24–48 h. Cytotoxicity was assessed by resazurin-based assays  according to the manufacturer’s recommendations using a Spark® multimode microplate reader (TECAN). Experiments were performed in triplicate. Data were expressed as units of fluorescence 560Ex/590Em nm.For cytotoxicity assays, HEK 293 cells were plated in 384-multiwell plates (2 × 10https://sybyl-x.software.informer.com/2.1/). The crystal structure of human GGT in complex with the GGsTop inhibitor (4ZBK.pdb) was chosen for the study and was derived from the Protein Data Bank (https://www.ebi.ac.uk/pdbe/). Before the docking, water molecules and the inhibitor GGsTop were removed from the protein structure. The hits were docked into the active site of GGT by the use of Glide within Schrödinger-Maestro  [The 2D/3D structures of thiohistidines, including ovo, 5-thio and erg, and the ones of the natural substrate of the GGT enzyme, i.e., glutathione, and of the commercial GGT inhibitor, DON, were constructed using PubChem Sketcher V2.4 and SYBYL-X 2.1 software  ,46,47 an