P < 0.001) in women subsequently experiencing PTB or cervical shortening. Hsa-miR-150-5p had the strongest ability to predict PTB (AUC = 0.8725) and cervical shortening (AUC = 0.8514). Plasma miRNAs in the first trimester can predict PTB and cervical shortening in women at risk of preterm delivery. This is a key period in pregnancy when early identification of PTB risk allows time to deliver outcome-modifying interventions.Preterm birth (PTB) is the leading cause of infant death and disability worldwide. The onset of preterm uterine contractions is preceded by asymptomatic cervical remodelling and ripening, which can be seen on trans-vaginal ultrasound as cervical shortening. This study aimed to identify plasma miRNA biomarkers that predict preterm birth and/or cervical shortening. We collected serial plasma samples from pregnant women prospectively from 12 to 22 weeks gestation. The nCounter miRNA assay was used to identify differentially expressed miRNAs associated with spontaneous PTB and/or cervical shortening . Predictive values of the miRNA biomarkers were confirmed in an independent validation cohort consisting of 96 women who delivered at term, 14 preterm and 21 early cervical shortening at <20 weeks gestation. Nine miRNAs  were differentially expressed ( It is the leading cause of mortality in children under five years-old worldwide, accounting for more than 1 million deaths per year2. The onset of labour, both term and preterm, is diagnosed clinically by uterine contractions, but is preceded by cervical remodelling which occurs over many weeks3. Cervical ripening is associated with an inflammatory signature, including up-regulation of prostaglandins, chemokines and cytokines, inflammatory cell infiltration and increased matrix metalloproteinase activity4.Preterm birth (PTB) before 37 weeks of gestation is a complex clinical syndrome with multiple aetiologies5. It has been reported that ultrasound measurement of CL at 19–24 weeks detects most PTL <28 weeks and 50% of PTL <37 weeks gestation5. This is clinically useful in populations already identified as being at high risk of PTL, since interventions  can reduce the risk of PTB. However, CL measurements using TVUS is not routinely offered to low-risk pregnancies where 60% of all low-risk pregnancies with short cervix at 22–24 weeks deliver <28 weeks of gestation and 90% <32 weeks6. It has been suggested that universal CL measurement should be introduced into maternity care for all women for the prediction of PTL8. However, this would have substantial cost implications, and would require expertise in ultrasound, and multiple appointments. An easily measured circulating biomarker which, when measured early in pregnancy, predicts cervical shortening and PTB, would have significant practical and economic advantages. Early risk identification would also allow earlier intervention, which may be more effective at prolonging pregnancy and improving neonatal outcome9.Cervical remodelling can be observed on trans-vaginal ultrasound (TVUS) as shortening of the cervix. Shortened cervical length (CL) is a risk factor for spontaneous preterm labour (PTL) in both low- and high-risk pregnancies11. Individual tissues, cells and body fluids have their own unique miRNA expression profiles and these change in pathological processes and disease states. MiRNAs are shed into the circulation where they remain stable and easily measurable, and have thus emerged as potential biomarkers in a variety of diseases including malignancy, infection and inflammatory conditions13.MicroRNAs (miRNAs) are small, single-stranded, non-coding, 19–25 nucleotide molecules regulating mRNA stability and transcription. They are important regulators of gene expression in almost all eukaryotes; up to 80% of all human genes are thought to be regulated by miRNAs15. However, sampling from the cervix is impractical for obstetric population screening. We hypothesised that, in women at risk of PTB, miRNAs may be shed from reproductive tissues, or other body sites, into the peripheral circulation where they may be used as peripherally available biomarkers of cervical ripening and PTB. To identify miRNA biomarkers of PTB and cervical shortening, we compared circulating miRNA profiles between women at high risk of PTB who delivered preterm with or without cervical shortening in the late first and second trimester of pregnancy with the profiles of women who had no cervical shortening and who delivered at term.Recent studies suggest that specific miRNA expression profiles measured in the cervix or in cells taken from the cervix have the potential to differentiate women destined to deliver pretermA total of 511 women were recruited and the study was completed in two phases. Firstly, a ‘discovery’ cohort was selected from the first 280 women recruited. Secondly, a ‘validation’ cohort was identified from the subsequent 231 women recruited. In the discovery cohort, the PTB rate before 34 weeks’ gestation was 5.2% (11.9% <37 weeks) and in the validation cohort was 5.3% (12% <37 weeks). In the discovery cohort 13 women delivered preterm (<34 weeks) with complete serial datasets (Table +6 weeks) and C (18–21+6 weeks). Of these women 96 did not demonstrate cervical shortening and delivered at term (OUTCOME 1/TERM), 14 women delivered preterm (<34 weeks) (OUTCOME 2/PRETERM) and 21 women demonstrated cervical shortening (OUTCOME 3/SHORT). In this cohort, there were 7 women who delivered preterm but did not demonstrate early cervical shortening. Of the 21 women with cervical shortening, 11 women delivered at term and 10 delivered preterm (<34 weeks gestation). Clinical characteristics of the discovery and validation cohort patients are presented in Table From the validation cohort we selected 124 pregnant women with samples available at time points A , all of which significantly differed in at least one of the three time points  did not further improve the diagnostic performance . However, a combination of the seven best performing miRNAs produced a ROC curve with an AUC of 0.91 for PTB  in those women who demonstrated cervical shortening. In these cohorts the intervention was cervical cerclage, not progesterone. Since the intervention may have affected the rate of PTB, we also defined premature cervical shortening as an outcome, irrespective of gestational age at delivery. Nevertheless, we have identified a panel of miRNAs, which are highly predictive of both PTL and premature cervical shortening independent of cervical cerclage as an intervention.+6 weeks gestation). Test performance was also undertaken at time point C, which coincides with routine visits to health care providers during pregnancy for diagnostic testing.Although the nCounter platform enabled plasma profiling of more than 800 miRNAs simultaneously, cost and practicality considerations limited the sample size of the discovery cohort. However, subsequent technical validation and confirmation of our main findings in an independent patient cohort using a different miRNA quantification platform (RT-qPCR) represents a strength of our study. Independent validation was performed using a ratio of controls to pathological cases (7:1) to better represent the rate of preterm birth in this population. In the discovery cohort, prediction of both PTB and premature cervical shortening was equally robust at all three time points. Considering prediction of PTB or premature cervical shortening is clinically desirable as early in pregnancy as possible, biological validation was performed in samples collected at time point A , just as physiological ripening occurs gradually at term. It is thus reasonable to hypothesize that the underlying mechanism of pathological cervical ripening is active many weeks prior to clinical diagnosis. In support of this concept, Elovitz16 used the nCounter miRNA profiling array to examine maternal plasma miRNAs at 20 weeks’ gestation in extremely small number of samples (7 preterm and 9 term). They found miR-223 increased in the preterm group and miR-302b, miR-1253, miR-548a, miR-548aa, miR-548ak and miR-548n reduced in the plasma of preterm women. Analysis of our discovery cohort for these targets gave us much lower nCounter values for all the miRNAs apart from miR-223 which had comparable counts to our study  were differentially expressed at time point A (12–14+6 weeks) and are shown in Supplementary Fig. 19. Elovitz et al used microarray to examine serum derived miRNAs and could not differentiate women who delivered early from those whose symptoms settled and delivered at term18, whereas Fallen et al used a small RNA library construction protocol in circulating plasma from women with symptoms of preterm labour and identified a spectrum of miRNA changes19. These studies examined a very different obstetric population which had already developed uterine contractions at later gestational ages (24–29 weeks gestation) for a variety of reasons. The onset of contractions would also likely have a significant effect upon release of miRNAs from within the cervix or uterus into the circulation.A number of recent pilot studies have been undertaken in an attempt to examine the capacity of circulating miRNAs to predict subsequent preterm birth. However, these studies have been severely limited by a lack of power, failed to undertake validation or included poorly defined clinical outcome groups that limit the application of proposed biomarkers. Recent pilot study21. Known targets of hsa-miR-150-5p include members of the membrane-type-1 matrix metalloproteinases involved in cervical ripening23. Consistent with a role in the pathophysiology of PTL, we have found that its plasma concentrations rise from 12–14+6 through 15–18+6 to 19–21+6 weeks gestation  in women who deliver preterm and  in women who demonstrate early cervical shortening. The observed increased detection of plasma hsa-miR-150-5p in women with later cervical shortening/weakness and PTB could be secondary to increased active shedding from the reproductive tissues or alternatively it could be indicative of underlying increased expression. There also exists a class of miRNAs that are preferentially sorted and packaged into exosomes for excretion into the circulation, and hsa-miR-150-5p is an example of this group24.The best performing miRNA detected in our study, hsa-miR-150-5p, is a biologically plausible biomarker of cervical shortening and PTB. It is a conserved miRNA, located on chromosome 19, expressed in the cervix and placenta and regulated by the inflammation associated transcription factor NFkappaB, which we have shown to be pivotal to labour onsetth percentile for the general population25. Therefore, these subjects likely fall within an aetiological group characterised by cervical weakness and increased risk of delivery <34 weeks, and represent those women at highest risk of associated maternal and neonatal infection and/or inflammation. Early gestational age at delivery and infection/inflammation are the major risk factors for prematurity associated cerebral palsy and adverse neurodevelopmental outcomes26. This is also a group with high PTB recurrence rates, and increased utilisation of medical resources. Currently, women at increased risk of PTB are likely to be offered more intensive antenatal care and CL surveillance to identify those likely to benefit from interventions; commonly progesterone and/or cervical cerclage. In most clinical settings, women in their first pregnancy are not routinely offered CL measurement, for cost and practicality reasons. Screening in early pregnancy using circulating miRNAs could therefore offer an effective triage of women at risk of PTL allowing intervention at an earlier time in pregnancy and more effective targeting of resources.The assessment of circulating miRNAs in a cohort of women at high risk of PTL could be considered both a strength and a weakness of our study. The recruitment of women at high-risk of PTL permitted enrichment of those women most likely to subsequently deliver preterm and/or demonstrated cervical shortening. It also further focused our study towards women with a more defined underlying PTB aetiology; women with abnormal cervical function or vaginal host immune interactions characterised by early cervical shortening. Although our primary outcome was preterm delivery before 34 weeks, it was not considered ethical to withhold intervention  in those women who demonstrated cervical shortening. We therefore consider that comparing two pathology outcomes  with the control group  represent more appropriately this prospective sample and metadata collection study with outcomes defined after delivery. All the women that delivered preterm in our discovery cohort demonstrated preterm cervical shortening but 11 women who demonstrated cervical shortening delivered at term after receiving an USS indicated cervical cerclage. In our validation cohort, seven women delivered preterm without a shortened cervix <25 mm and therefore did not receive intervention but their mean Time Point C cervical length was 27 mm (range 26–29 mm), only slightly above the clinical cut-off value for intervention and close to the 10The value of miRNA screening for early PTB will be to triage women already defined at increased risk, and to identify women at risk in their first pregnancy. This will require a test with a high sensitivity. Any woman defined through screening at high risk could then undergo TVUS surveillance which although expensive and invasive, is widely accepted and clinically used. Our data suggest that hsa-miR-150-5p provides a ‘cut-off’ or ‘screen-positive’ value that gives 100% sensitivity for both PTL and cervical shortening with 60% specificity meaning that the ratio of ‘screen-positive’ to ‘true positive’ will be less than 2 to 1. Refinement of risk at the end of the first trimester, in women already identified of being at increased risk of cervical shortening and PTL will allow improved targeted surveillance and delivery of interventions at an earlier stage in pregnancy, which may improve effectiveness. We further anticipate that miRNA screening could also be used to identify women at risk of early PTL in otherwise low-risk general populations, although this will require further study.+6 (time-point A), 15–17+6 (time-point B) and 18–21+6 (time-point C) weeks gestation were collected from women with singleton pregnancies attending preterm birth prevention clinics at Imperial College Healthcare NHS Trust hospitals in London, UK. Women were defined as being at increased risk of PTB because of previous mid-trimester loss and/or PTB, or previous excisional treatment for cervical intraepithelial neoplasia (CIN). At each sampling, whole blood was collected and placed immediately on ice before centrifugation at 1300 × g for 10 min at 4 °C within 30 min of collection. Isolated plasma was stored in 1000 μl aliquots in RNase-free microtubes at −80 °C. Samples demonstrating macroscopic haemolysis were discarded.Ethical approval was granted by the Hertfordshire Research Ethics Committee (22/02/2011-REC reference number 11/H0311/6) and informed written consent was received from all participants. All research was performed in accordance with the relevant guidelines and regulations. Prospective serial maternal plasma samples and CL measurements at three time-points in mid-pregnancy: 12–14Women who developed obstetric complications  were excluded from the study. Following delivery, subjects were categorised into three different outcome groups. Outcome 1/TERM comprised women with uncomplicated pregnancies and no cervical shortening who delivered at term. Outcome 2/PRETERM comprised women who spontaneously delivered preterm (<34 weeks) and outcome 3/SHORT comprised women who demonstrated second trimester cervical shortening. Cervical shortening was defined as a CL measurement of <25 mm at time-points B or C when measurement at time-point A was >25 mm. All CL measurements were obtained using TVUS. All women who developed cervical shortening underwent cervical cerclage as per local guidelines. Women included in our study had not received progesterone before or after cervical shortening.g for 10 min at 4 °C to minimise cellular and platelet RNA contamination27. The upper 750 μl was removed for processing and the remaining plasma was discarded. RNA was extracted using the ‘Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit (Slurry-Format)’ . A spike-in of 5000 attomoles of synthetic cel-254  was added following the addition of lysis and denaturing buffers to allow downstream normalisation of any technical variation to the extraction process. Eluted RNA was further purified and concentrated using Amicon Ultra YM-3 columns  to a final volume of 25 μl.Plasma aliquots were thawed on ice and centrifuged at 800 × +6 (time point A), 15–17+6 (time point B) and 18–21+6 (time point C) weeks gestation. The nCounterTM plasma miRNA cassette  permits target miRNA expression levels to be directly assessed, without cDNA synthesis or enzymatic reactions, via two sequence-specific probes28.nCounter miRNA plasma profiling was used to compare plasma miRNA expression across gestational time points in a ‘discovery cohort’ consisting of maternal plasma samples collected serially at three time-points in mid-pregnancy: 12–14TM Universal RT miRNA cDNA synthesis kit II , following the addition of 0.625 μl synthetic miRNA UniSp6 (108 copies/μl)  to allow downstream normalisation of any technical variation to the reaction. A constant initial volume of 750 μl plasma was used for each extraction. To minimise the impact of endogenous inhibitors, the reverse transcription (RT) reaction for plasma miRNAs was optimised by comparing Ct values derived from multiple volumes of RNA in-put  into 10 μl total RT reaction. 1 μl RNA/10 μl RT reaction was found to be optimal. RT-qPCR was performed on ABI StepOnePlus  using custom ‘pick and mix’ PCR panels containing LNATM (locked nucleic acid) primers for the nine miRNA targets, according to the manufacturer’s instructions . Optimised and experimentally validated miRCURY LNA™ Universal RT microRNA PCR system primer sets were purchased from Exiqon. LNATM primers were specifically designed to have maximum sensitivity with at least 95% efficiency, which enables multiple miRNA targets to be assayed under the same experimental conditions. Melt curve analysis was used to confirm presence of a single PCR product. Duplicate ‘no template’ control reactions were performed with all assays, confirming the absence of RNA contamination. A calibrator primer was included in all plates which showed that the intra-assay variation (coefficient of variance) of Ct values was <2%.RT-qPCR was then used to confirm the data derived from the nCounter assay and to measure miRNA expression in an independent validation population cohort. For this, RNA was reverse transcribed to cDNA using miRCURY LNA29.The background signal of the nCounter assay was defined as two standard deviations above the mean of negative control probes and subtracted from the raw miRNA molecule counts. Expression counts were normalised to the mean expression of the top 100 expressed miRNAs. Raw values of nCounter expression counts are included as Supplementary Data. MiRNA probes without expression above background in more than half of the samples from any clinical group were removed from further analysis. Samples with very high expression of haemolysis-associated miRNAs, hsa-miR-16-5p, hsa-miR-25-3p and hsa-miR-93-5p were removed from analysis (n = 2)P < 0.05 and a false discovery rate (FDR) <0.05. Datasets were assessed using the D’Agostino and Pearson omnibus normality test and longitudinal data were interrogated using either the repeated measures ANOVA with the Greenhouse-Geisser correction followed by a post test for linear trend in either direction, or a Freidman test followed by Dunn’s multiple comparison test, if data were found to have a Gaussian or non-Gaussian distribution, respectively. MiRNAs with gestation dependent expression were defined as P < 0.05.We initially compared log-transformed geometric means of expression counts  between gestational groups, using nSolver v2.0 software . MiRNAs with significant differences in mean expression counts between time points were defined as k), where Sk represents the standard deviation value of the variable to ensure that all variables in the model retained equal importance. Goodness-of-fit (R2Y) and predictive ability (Q2Y) parameters were calculated, the latter by a seven-round internal cross-validation of R2Y, and used to assess model fit31.nCounter assay data were subjected to unsupervised principal component analysis (PCA) and supervised partial least squares-discriminatory analysis (PLS-DA) using SIMCA-P . Data were scaled to unit variance by dividing each variable by 1/ values for each miRNA were calculated using StepOne v2.3 software . All qPCR reactions were performed with a technical replicate. All assays demonstrated no amplification in ‘no template’ control reactions. Ct values were median normalised firstly to an inter-plate calibrator and then to the extraction and reverse transcription spiked-in controlsDetection rate (DR) (or sensitivity) and false positive rate (FPR) (or 1-specificity) for both PTB and cervical shortening were calculated using data from time point A combining both the discovery and validation cohorts. ROC curves were constructed and the AUC for prediction of PTB or cervical shortening was calculated.Supplementary InformationSupplementary dataset


During the Holocene , northern peatlands accumulated significant C stocks over millennia. However, virtually nothing is known about peatlands that are no longer in the landscape, including ones formed prior to the Holocene: Where were they, when did they form, and why did they disappear? We used records of peatlands buried by mineral sediments for a reconstruction of peat-forming wetlands for the past 130,000 y. Northern peatlands expanded across high latitudes during warm periods and were buried during periods of glacial advance in northern latitudes. Thus, peat accumulation and burial represent a key long-term C storage mechanism in the Earth system. 2 and methane in polar ice cores have been attributed, in part, to changes in global wetland extent, but the wetland distribution before the Last Glacial Maximum  remains virtually unknown. We present a study of global peatland extent and carbon (C) stocks through the last glacial cycle (130 ka to present) using a newly compiled database of 1,063 detailed stratigraphic records of peat deposits buried by mineral sediments, as well as a global peatland model. Quantitative agreement between modeling and observations shows extensive peat accumulation before the LGM in northern latitudes (>40°N), particularly during warmer periods including the last interglacial  and the interstadial . During cooling periods of glacial advance and permafrost formation, the burial of northern peatlands by glaciers and mineral sediments decreased active peatland extent, thickness, and modeled C stocks by 70 to 90% from warmer times. Tropical peatland extent and C stocks show little temporal variation throughout the study period. While the increased burial of northern peats was correlated with cooling periods, the burial of tropical peat was predominately driven by changes in sea level and regional hydrology. Peat burial by mineral sediments represents a mechanism for long-term terrestrial C storage in the Earth system. These results show that northern peatlands accumulate significant C stocks during warmer times, indicating their potential for C sequestration during the warming Anthropocene.Glacial−interglacial variations in CO However, the mechanisms behind these observations are still unknown; hypotheses include both marine . As the largest natural source of methane (CH4) to the atmosphere . In addition to location, we use several attributes of buried peat deposits in our analysis: (i) the timing of active peat accumulation when sites were actively accumulating peat (determined from sediment dating methods) as opposed to being buried or otherwise inactive; (ii) the number of sites that were actively accumulating peat at the same time , which is a proxy for peatland extent; and (iii) the thickness of these buried peat deposits, which is a proxy for the total C stock of the peat deposits that accumulated during the period of active deposition . We model peatland C stocks from 126 ka to the preindustrial (1850 CE) using an Earth System Model of Intermediate Complexity coupled to a higher resolution Dynamic Global Vegetation Model  and model global peatland C stocks for the past 130,000 y (130 ka) to test the response of peatland C stocks to the highly variable climate conditions before the Holocene. We create a synthesis dataset of buried peat deposits that includes 1,063 profiles globally , includion Model  . During a period of cooling during MIS 4 . As temperatures increased during MIS 3 (57 ka to 29 ka), the number of northern buried peat records increased sixfold, particularly between 57 ka and 45 ka .There is substantial evidence for widespread northern peatlands from more than 40 sites during the last interglacial , when continental ice sheets were largely absent in the Northern Hemisphere  as peats not covered by glacial sediments were buried by aeolian deposits (27%), coastal sediments (30%), and permafrost-associated deposits (16%). The number of active northern peat deposits reached a minimum during the LGM .After 29 ka, the number of active northern peat deposits decreased by >80% , coincid the LGM D as temp the LGM B and ice the LGM A. EightySI Appendix, Fig. S2). The rapid establishment of northern peatlands occurred during the first half of the Holocene . During MIS 5e, the maximum modeled active northern peatland C stocks were 340 Pg at 120 ka, corresponding to the largest number of northern sites with active peat deposition before the LGM (Modeled northern peatland C stocks agreed well with observations of active peat accumulation in now-buried peat deposits before the LGM ( the LGM D. During the LGM . During  the LGM . As glac the LGM .r = 0.99). A significant number (33%) of present-day peatlands were formed after 5 ka . The first evidence of peatland establishment in equatorial and southern Africa dates to 50 ka to 45 ka ; the majority of sites from that time persist to the present day . The number of actively accumulating peats in the tropics increased after 45 ka, then decreased during MIS 2 through the LGM as peats were buried by fluvial and coastal processes . However, the formation of new peatlands resulted in little apparent change in tropical peatland distribution .The first known buried peat deposit from the tropics formed between 164 ka and 122 ka . The number of active tropical peat records decreased and peat initiation slowed between the Bølling−Allerød and early Holocene . As the sea level stabilized  as peatlands expanded across Indonesia and Malaysia . While the areas of active tropical peatland formation shifted in space and time , the total modeled active tropical peatland C stocks remained relatively constant throughout the interglacial at an estimated 145 Pg C .As global temperatures increased after the LGM and into the Bølling−Allerød, the rate of peatland initiation increased after ∼15 ka for both buried and present-day tropical peatlands  as peatsHolocene  as contiabilized , the numPeat accumulation occurs when vegetation productivity exceeds decomposition losses and is facilitated by anoxic conditions due to poor drainage in wetlands. Understanding the drivers of peat accumulation and loss under a broad range of climatic conditions can ultimately improve projections of the response of peatland C stocks to future climatic changes  through SI Appendix, Table S2). Regardless, net peat accumulation will likely continue in topographically favorable areas with warming as long as disturbances such as wildfire, drainage, or flooding are not significant  corresponded to a higher occurrence of northern peat deposition and greater northern peatland C stocks, evidenced by the observed number of sites, observed peat thickness, and modeled C stocks . Whethernificant .SI Appendix, Fig. S1). Colder periods may not have directly resulted in the loss of peat , but instead favored processes  that resulted in rapid mineral deposition and subsequent peat burial while limiting new peatland development or recovery of peat accumulation due to dry or continental conditions . Instead, tropical peat formation responded mainly to changing hydrologic conditions. For example, approximately one-third of the tropical buried peats that formed during the Holocene were buried as sea level rose (15/46 sites), while others were formed as rising sea level altered regional hydrology in coastal regions (During warm periods (the Holocene) and in warm locations (the tropics), peat burial was related to factors other than temperature. Tropical peatland deposition was relatively insensitive to global temperature fluctuations, as evidenced by the persistent presence of tropical peatlands on the landscape after 50 ka during a range of climatic conditions in both data and model results . While decomposition of buried peat may occur, this may be limited in deep soils and subglacial sediments by anoxia resulting from slow rates of oxygen diffusion or saturation . We identified profiles based on author knowledge, solicitations through existing research networks, and literature searches on Web of Knowledge and Google Scholar using the terms “buried peat,” “buried peat deposits,” “histic paleosols,” “organic paleosols,” “interglacial peat,” “MIS 5 and peat,” and “MIS 3 and peat.” We defined peat broadly as organic-rich sediment derived from wetland or limnic environments deposited in situ or within the local catchment. We extracted information on the profile location, depth of the organic-rich sediments, the timing of deposition (when available), the depositional environment of the organic-rich sediment , the type and origin of the overlying sediments, and other site descriptors. The dataset is publicly available via the PANGAEA data archive  and was based mainly on calibrated radiocarbon dates for 786 profiles younger than 50 ka . For profiles older than 50 ka, chronologies were based on tephrochronology (14 profiles), optically or thermally stimulated luminescence dating (six profiles), stratigraphic position relative to tills and other sediment types of known depositional age , pollen (11 profiles), or Uranium−Thorium dating (eight profiles). Multiple dating proxies were used at 41 profiles. The use of radiocarbon dating imposes some important considerations. Notably, the apparent increase in the number of buried peat sites after 50 ka D and F iDataset S2). The peatland initiation dataset is archived and publically available  to place the development of the buried peats in the context of the development of present-day peatlands. The peatland initiation dataset consisted of 3,942 basal ages and was based on a compilation of several existing basal age datasets for northern peatlands , 12, 36 vailable .SI Appendix. Profiles without chronological control  remain in the database . Briefly, CLIMBER2-LPJ consists of the Dynamic Global Vegetation Model LPJ (2 concentrations derived from ice core data, and ice sheet extent determined using an ice sheet-enabled version of the CLIMBER2 model (SI Appendix.We performed a transient model experiment using a climate−carbon cycle model, an updated version of the peatland-enabled CLIMBER2-LPJ model , to deteodel LPJ , coupledodel LPJ . LPJ is odel LPJ , 42. Iceodel LPJ . The gloodel LPJ , as oppoodel LPJ . This alR2 model . The modSupplementary FileSupplementary FileSupplementary File


In contrast to mammals, adult fish display a remarkable ability to fully regenerate central nervous system (CNS) axons, enabling functional recovery from CNS injury. Both fish and mammals normally undergo a developmental downregulation of axon growth activity as neurons mature. Fish are able to undergo damage-induced “reprogramming” through re-expression of genes necessary for axon growth and guidance, however, the gene regulatory mechanisms remain unknown. Here we present the first comprehensive analysis of gene regulatory reprogramming in zebrafish retinal ganglion cells at specific time points along the axon regeneration continuum from early growth to target re-innervation. Our analyses reveal a regeneration program characterized by sequential activation of stage-specific pathways, regulated by a temporally changing cast of transcription factors that bind to stably accessible DNA regulatory regions. Strikingly, we also find a discrete set of regulatory regions that change in accessibility, consistent with higher-order changes in chromatin organization that mark (1) the beginning of regenerative axon growth in the optic nerve, and (2) the re-establishment of synaptic connections in the brain. Together, these data provide valuable insight into the regulatory logic driving successful vertebrate CNS axon regeneration, revealing key gene regulatory candidates for therapeutic development. However, even under these growth-enhanced conditions, regeneration mostly occurs only in a subset of RGCs2, with the majority of axons rarely growing beyond a few millimeters. Furthermore, the regenerating axons frequently grow in an undirected manner, resulting in the “regenerated” axons terminating growth far from their appropriate brain targets9. As such, there remains a gap in our understanding of the genetic programs that drive RGC axon regeneration culminating in target re-innervation and recovery of visual function.Damage to nerves in the CNS as a result of disease or injury most often results in a permanent loss of function in humans. The loss of function stems from the failure of adult mammalian CNS neurons to support regenerative axon growth. In adult mammalian retinal ganglion cell (RGC) neurons, genetic and pharmacologic manipulations of neuron-intrinsic pathways have shown promise in activating a regenerative state after optic nerve injury11, ultimately leading to functional recovery12. It is well known that proteins regulating RGC axon growth and guidance in the developing visual system are highly conserved across vertebrate species, and are transcriptionally down-regulated once the mature circuitry has been established14. In addition, like mammals, optic nerve injury in adult zebrafish induces the expression of axon growth attenuators such as socs3 in RGCs15, suggesting that expression of such negative regulators of axon growth is a normal part of the regenerative program. One major difference in response to CNS axon injury between mammals and fish is the re-expression of a genetic program that promotes axon growth and guidance17. However, we and others have found that the regulation of axon growth-associated genes differs between development and regeneration20. Thus, the difference between mammals and vertebrate species capable of optic nerve regeneration is their ability to reprogram adult RGCs for axon growth in response to optic nerve injury.In contrast to mammals, zebrafish display a remarkable ability to spontaneously regenerate RGC axons. As opposed to regenerating axons in the growth-enhanced mouse models, regenerating axons in the zebrafish optic nerve successfully navigate across the chiasm and re-innervate target neurons in the optic tectum21. What is less clear are the genome-wide changes in expression within the RGCs over time as they first grow toward their intermediate target of the optic chiasm and then navigate toward their principal brain target, the optic tectum. Here we present the first comprehensive analysis of gene expression changes (RNA-seq) and DNA regulatory element accessibility (ATAC-seq) in zebrafish RGCs at specific time points along the axon regeneration continuum from early growth to target re-innervation. Our analysis reveals that successful CNS axon regeneration is regulated by stage-specific gene regulatory modules, and punctuated by regeneration-associated changes in chromatin accessibility at stages corresponding to axonogenesis and synaptogenesis. Together, these data suggest candidates for gene regulatory targets for promoting successful vertebrate CNS axon regeneration.It has been established that specific regeneration-associated gene expression changes in the adult zebrafish retina begins within the first day after optic nerve crush and persist through the re-innervation of the optic tectum22. To achieve a comprehensive picture of the genetic programming driving successful vertebrate CNS axon regeneration, we used RNA-seq to identify changes in gene expression that accompanied axon growth in regenerating RGCs at critical time points after optic nerve injury. We specifically examined how transcript expression in naïve retinas compared with that in retinas dissected from fish at 2, 4, 7, and 12 days post-injury (dpi). Based on the previously established regeneration chronologies, our chosen time-points  > 4), were detected for six out of the seven temporal clusters . Enriched pathways, based on high stringency criteria .In order to understand the regulatory system that guides the post-injury transcriptional program, we identified putative regulatory DNA elements  in RGCs at different stages of regeneration. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq)26.We detected over 40,000 peaklets representing consensus regions of accessible chromatin in RGCs. The majority of peaks were located in intergenic regions, upstream or downstream from annotated genes Fig. . Of the Surprisingly, overall chromatin accessibility changed very little in response to optic nerve injury. In fact, only 233 consensus regions of accessible chromatin (0.5%) were differentially accessible in injured RGCs compared to control RGCs Fig. . All but27 and used a global differential  to compile a list of transcription factor-encoding transcripts associated with regeneration. We discovered 265 definitive transcription factor encoding genes associated with 339 transcripts that were differentially expressed at one or more post-injury time points . Each of these four families of transcription factors included representatives with peak expression early, middle, and late in the regenerative process Fig. , as well30. For each cluster of temporally expressed transcripts we identified potential regulatory elements in the form of accessible chromatin peaklets that were proximal (peaklet center within ± 1 kb from transcription start site) or distal  to the associated gene. Proximal and distal peaklet sequences were then queried for enrichment of binding motifs of transcription factors with similar temporal expression profiles (Table growth toward the midline cluster), we found that motifs for 17 out of the 31 transcription factors queried were enriched in the surrounding regions of accessible chromatin . By comparison, binding motifs for FOSL1 and JUNB displayed a high frequency of co-occurrence with binding sites for 6–7 other transcription factors . A similar trend was observed among proximal peaklets, although the differences were less marked . These differences suggest the possibility that binding site composition and potential transcription factor interactions may be distinguishing characteristics of regeneration-associated promoters and enhancers.Promoters and enhancers rarely function in response to binding of single transcription factors. This is due both to the propensity of transcription factors within the same family to dimerize, and the existence of multiple transcription factor binding sites within a given promoter or enhancer. To identify potential interactions between regeneration-associated transcription factors, we quantified the frequency with which binding sites for differentially expressed transcription factors co-occurred within putative regulatory elements. For example, although KLF6 and WT1 were the most prevalent binding sites within both proximal and distal peaklets, the co-occurrence rate with binding sites with other transcription factors present in this time window was among the lowest Fig. . Within We further quantified the frequency of specific co-occurring binding sites. The co-occurrence of members of the same family was expected due to similarities in recognition sequence specificity. This was observed most clearly in the frequency of shared enriched peaklets between KLF6 and WT1 binding sites, and between the bZIP factors Fig. . HoweverAlthough chromatin accessibility remains mostly stable in RGCs in response to optic nerve injury, we detected a small number of DNA elements in which chromatin accessibility changed in regenerating neurons compared to controls. Intriguingly, the few differentially accessible chromatin peaklets were not evenly distributed across the time points. Instead, most of the elements that became more accessible in response to injury did so at the earliest time point (2 dpi). This led us to postulate a role for these elements in triggering the regenerative growth program. We hypothesized that transcription factors regulated by such elements would not only regulate biological processes necessary for initiation of axon growth, but would also contribute to the regulation of downstream transcription factors that in turn regulate subsequent phases of regeneration.jun was the only regeneration-associated transcription factor encoding gene that was located within at least 100 kb of a differentially accessible chromatin region. In fact, the jun gene is flanked by three peaklets that are differentially open at 2 dpi with respect to controls  The elements may be responsible for initiating the expression of key regeneration-associated transcription factors, which would subsequently regulate other factors in the hierarchy; and (ii) The elements may serve to shift the higher order chromatin structure to reposition enhancers and promoters for the transitions necessary in adult RGCs to reinitiate programs for axonogenesis (2 dpi), and synaptogenesis (12 dpi).41. Supporting our second hypothesis, we find that roughly half the differentially-accessible regions at both early and late times are enriched in CTCF binding sites. CTCF is a transcription factor that has recently been implicated in mediating chromatin looping and marking the boundaries of topologically associating domains42. A logical next step would be to functionally validate interactions between the predicted jun promoter and enhancers, as well as additional long-range regulatory interactions between stably and differentially accessible elements using chromatin capture and genome editing technologies.Our data contain evidence supporting both hypotheses. Supporting the first hypothesis, we find that the gene encoding transcription factor JUN is flanked by promoter and distal enhancer elements that increase in accessibility during axon regeneration. In fact, JUN involvement in such epigenetic activation of pro-regenerative genes in response to axon injury was suggested in a recent review17. In addition, our temporal approach using RNA-seq has greatly expanded detection of regeneration-associated changes in gene expression over previous studies. We anticipate that functional testing of the networks predicted by this model will substantially expand our understanding of the molecular mechanisms governing successful optic nerve regeneration.The combination of gene expression and genomic accessibility over time provides a powerful model of axon regeneration-associated gene regulatory networks. As with any modeling approach, empirical testing and improved technology are expected to refine and strengthen the predictive power of the model. For example, current methods for associating enhancers with target genes will improve with the functional testing of these types of interactions as described above. Another potential caveat of these studies is that the transcriptomic analysis was carried out on whole retinas, rather than FACS-sorted RGCs that were utilized in examining chromatin accessibility. Because RGCs are the only cells directly injured by optic nerve crush, a comparison of gene expression between injured and uninjured retina should primarily reflect changes in RGCs. Yet, it is possible that some of the transcriptomic changes detected may be occurring in other cells within the injured retina, such as infiltrating microglia or retinal neurons connected to RGCs. However, we find good concordance between our transcriptomic results and microarray studies based on RNA extracted from RGCs isolated by laser capture microdissection43. However, to our knowledge, this is the first gene regulatory network analysis of optic nerve regeneration that couples temporal analysis of gene expression with the identification of putative regulatory interactions based on chromatin accessibility and transcription factor expression. We expect that these findings may be applicable to neurons in other regions of the CNS undergoing regenerative axon growth, such as the spinal cord and brain. In order to facilitate comparisons between our data and those derived from other regenerative models, we have created an interactive web application 44. This app enables users to upload a gene list of interest from any Ensembl-supported species and determine how those genes are expressed and potentially regulated in the context of optic nerve regeneration. Such cross-species and cross-system analyses will facilitate identification of novel pathways specifically associated with successful CNS regeneration.In summary, these data provide a roadmap for the identification of key combinations of transcription factors necessary to reprogram adult RGCs for optic nerve regeneration. Similar approaches have been employed to discover transcription factors necessary for direct reprogramming of somatic cells to produce motor neuronsDetailed methods can be found in supplementary information.Institutional Animal Care and Use Committee (IACUC) and carried out in accordance with the relevant guidelines and regulations thereof. The Tg (Tru.gap43:egfp) mil1 (aka fgap43:egfp) transgenic line and Ekkwill wild type strain were maintained as previously described25. Optic nerve crush (ONC) lesions were performed on adult zebrafish, 7–9 months of age, as previously described21.Zebrafish husbandry and all experimental procedures were approved by the University of Wisconsin – Milwaukee RNA was extracted and purified from retinas dissected from naïve (0 dpi) and regenerating adult fish at 2, 4, 7, and 12 dpi. RNA was extracted using the RNeasy Micro kit (Qiagen) and concentrated using the RNA Clean & Concentrator kit (Zymo). Three replicates were obtained for each time point. Whole retinas were used for transcriptomic studies because the quality of the RNA was much higher from whole retina. cDNA libraries were generated for each RNA sample using Tru-Seq Stranded Total & mRNA Sample Prep Kits, (Illumina 20020595). Each cDNA library was indexed for multiplexing and subsequently sequenced on four lanes of the Illumina Hiseq2000. Libraries were sequenced at 50 bp, 30–40 million paired-end reads/sample.45.After merging technical replicates of RNA-seq samples across lanes, adapter sequences were trimmed , sequence quality was validated , and transcript abundance was estimated using 500 bootstrap samples 46, and purified using DNA Clean & Concentrator kit .Chromatin was extracted from nuclei isolated from purified populations of RGCs. Retinas were dissected from naïve and regenerating fgap43:egfp fish as described above, and enzymatically dissociated for FACS. Cell suspensions were pooled from multiple retinas at each time point , and sorted for RGCs expressing GFP using a Becton Dickinson FACSAria™ III sorter. Although levels of endogenous Gap43 are low in the naïve adult retina, the GFP transgene tends to be long lived and is detectable by FACS at levels that are above background but below the level of visual detection. This enabled us to sort RGCs from naïve retinas since new RGCs continue to be added throughout the life of the animal and low-levels of GFP persist in RGCs even after these new RGCs have established connections in the tectum. However, we required twice the number of naïve retinas compared with injured retinas to accumulate the 50,000 RGCs required for constructing the ATAC-seq libraries. Three replicates of pooled cells were collected for each time point. ATAC-seq libraries were prepared using the Tn5 transposase system  as previously describedPrior to running the full sequence, sequencing depth was estimated and one sample was below the cut-off criteria and therefore omitted. The remaining fourteen samples were indexed for multiplexing and subsequently sequenced on four lanes of the Illumina Hiseq2000. Libraries were sequenced at 50 bp reads to obtain approximately 25 million paired-end reads/sample.47). Duplicate and multiple mapped reads were removed using samtools (v1.6)48. After concatenating aligned reads across all replicates and time points, MACS249 (v2.1.1.20160309) was used to call peaks from aligned reads. Only peaks with a p-value < 10−10 were retained for subsequent analyses. For each remaining subpeak summit, a 500 bp “peaklet” interval was defined using GenomicRanges (v1.30.3)50. We refer to these peaklets as consensus regions of accessible chromatin. Open chromatin in each replicate of each time point was then quantified using DiffBind  by counting the number of overlapping reads for each retained peaklet.After merging technical replicates of ATAC-seq samples across lanes, adapter sequences were trimmed  and sequence quality was validated . Reads were aligned to GRCz10 as well as the transgene sequence (BWA-MEM (v0.7.9a-r786)51.We used motif analysis to determine potential binding sites of differentially expressed transcription factors within regions of accessible chromatin identified by ATAC-seq. Motif enrichment and discovery was carried out within the MEME Suite of motif-based sequence analysis tools (version 5.0.3)52, either comparing each time point post injury  to the initial time point (0 dpi) using a Wald test statistic or using a likelihood ratio test (LRT) to compare the full model with a time factor versus the null model, controlling the false discovery rate (FDR) at 5% or 1%53. Expression heatmaps  were produced using ComplexHeatmap54 (v1.17.1), where transcript clusters were identified using the K-means algorithm, and hierarchical clustering  was used to cluster rows. The Ingenuity Pathway Analysis tool  was used to analyze enrichment of molecular and functional gene networks within the differentially expressed gene sets (FDR < 0.05) at each time point after injury  compared with the initial time point (0 dpi).Following pseudoalignment and quantification of transcripts, differentially expressed transcripts were identified using Sleuth (v0.29.0)55 was used to identify differentially accessible peaklets in an analogous manner to the RNA-seq analysis described above. Peaklets with FDR-controlled p-values < 0.05 in one of the four comparisons were considered to be differentially accessible. ChIPpeakAnno (v3.12.7)56, the danRer10.refGene UCSC annotation package (v3.4.2), and AnnotationHub (v2.10.1) were used to annotate peaklets with genes. Specifically, non-exonic  peaklets overlapping a transcription start site (TSS) or within 1 kb of a TSS were considered to represent proximal peaks, whereas those greater than 1 kb but less than 100 kb of a TSS were considered distal peaks. All statistical analyses were performed in R (v3.4.3). Integrative Genome Viewer (IGV) was used to visualize RNA-seq and ATAC-seq alignments57.After quantifying peaklet accessibility, DESeq2 (v1.18.1)Supplementary Information


N = 10) and two highly prevalent neurological conditions (N = 10 patients with Parkinson’s Disease and N = 10 stroke patients). We successfully implemented three AR games to evaluate these key aspects of motor function. As expected, PD patients moved slower than controls and needed more time for task completion. No differences were observed between stroke patients and controls, perhaps because motor impairments in this patient group were relatively mild. Importantly, usability of our AR system was good and considerably improved compared to our previous study due to more natural and patient-tailored interaction. Although our findings testify to the potential of AR for assessing motor impairments in patients with neurological conditions and provide starting points for further improvement, there are still many steps to be taken towards application in clinical practice.In clinical practice, upper extremity motor impairments are commonly assessed with disease-specific, subjectively scored and low-resolution rating scales that often do not consider the variations in tasks and environment that are essential aspects of daily life. Augmented reality (AR) systems with contactless tracking of the hand and upper body offer opportunities for objective quantification of motor (dys)function in a challenging, engaging and patient-tailored environment. In this study, we explore the potential of AR for evaluating 1) speed and goal-directedness of movements within the individually determined interaction space, 2) adaptation of hand opening to objects of different sizes, and 3) obstacle avoidance in healthy individuals (The online version of this article (10.1007/s10916-018-1100-9) contains supplementary material, which is available to authorized users. Objective assessment of upper extremity motor dysfunction is important for treatment selection and evaluation as well as monitoring disease progression in various neurological conditions (e.g. Parkinson’s Disease (PD), stroke). Motor impairments are commonly assessed with disease-specific, subjectively scored and low-resolution rating scales, or occasionally with cumbersome marker-based motion capture. Assessments are often limited to simple tasks, without considering variations in tasks and environment that are essential in daily life. Against this background, augmented reality (AR) systems with contactless tracking of the hand and upper body offer opportunities for objective quantification of motor (dys)function in a challenging, engaging and patient-tailored environment. AR gives clinicians full control over specific parameters  and allows for adjustment to individual capabilities. Importantly, it may result in more realistic behaviour than virtual reality , where iAR systems have successfully been developed for rehabilitation of upper extremity motor function, using ‘exergames’ in virtual worlds to motivate  and engaTen PD patients, ten stroke patients (>12 weeks post-stroke) and ten age-matched healthy controls participated in this study  and engVirtual content was visualized using an AIRO II head-mounted display (HMD) . The virtual world was aligned to the real world using the Vuforia tracking library . The Leap Motion Orion Beta software development kit (SDK) provided 3D-coordinates of hand ‘joints’ . The Kinect for Windows SDK (version 2.0) provided 3D-coordinates of body points . The following sections provide a brief description of each game. Technical details and illustrative movies are provided in Online Resources The ‘Balloons’ game  or non-dominant arm (N = 5). In all games, visual feedback was provided to facilitate interaction with the virtual content: ellipsoids represented the tips of the virtual index finger and thumb %) and PD patients  or stroke patients . Also Tballoon, movement speed, and relative path length were not significantly different between groups  than in PD patients  > 5.79, P < .001). Quality of object manipulation also depended on cube size: the smaller the cube, the higher Tcube  or stroke patients . Overall, success rate was lower in scenario VO (100 [94–100]%) than in scenario NO . The presence of an obstacle affected all outcome measures (Table Success rate in scenario VO did not differ between controls (100 [100–100]%) and PD patients . No group differences were observed for task load , engagement (3.8 ± 0.5 out of 5), presence (4.4 ± 0.7 out of 7), or usability (69.3 ± 13.7 out of 100). 53% of all participants scored above the threshold value (68) for good usability [Task load and engagement did not differ between games , adjustment of hand opening to virtual objects of different sizes (game 2) and obstacle avoidance (game 3) were comparable between patients and controls. As expected, PD patients moved slightly slower than controls , but onlIndividual assessment of interaction space (first part of game 1) allowed for patient-tailored positioning of virtual objects in all other games . Still, the so obtained MRD cannot be considered a proxy for the 3D upper extremity reachable workspace . This happened also in some NO trials, thereby reducing differences between NO and obstacle scenarios.As expected, the presence of an obstacle in game 3 led to increased relative path length, slower movement, and longer task completion time. Obstacles visible from the beginning (VO) were successfully avoided in almost 100% of trials. Unfortunately, implementation of the ‘surprise obstacle’ (SO) was not successful due to between-trial variations of movement speed in combination with the relatively short movement distance (limited by a minimum distance from HMD and maximum reaching distance), which made it almost impossible to present the obstacle In addition to game-specific considerations that have been addressed in previous sections, some general limitations and directions for future work need to be taken into account. Firstly, the small sample size fits the explorative purpose of our study. However, follow-up studies with larger groups are required to improve sensitivity to between-group differences, obtain insight into relations with clinical ratings of motor/cognitive impairments and evaluate test-retest reliability. Secondly, our findings are based on relatively mildly affected patients . Inspection of presence questions taught us that participants were not distracted or hindered by technical issues, although ease and realism of object manipulation could be improved.The patient-tailored and more natural interaction as well as the larger interaction space contributed to a good system usability that was considerably higher than in our previous study . InspectIn conclusion, our study testifies to the potential of patient-tailored AR games for assessing motor impairments in patients with neurological conditions and provides starting points for further improvement. We envision that rapid technical developments will lead to higher accuracy of contactless hand tracking and to improvements in price, aesthetics  and functionality  of HMDs. However, many steps are still to be taken towards application in clinical practice .ESM 1Online Resource 1 Presence questionnaire (PDF 79.1 kb)ESM 2Online Resource 2 Technical details on experimental setup and AR games (PDF 262 kb)ESM 3Online Resource 3 Illustrative video of game 1 “Balloons”. Two images are displayed on the laptop and in the screen captures: the left image is presented to the left eye and the right image is presented to the right eye to generate depth perception. The dark grey background appears almost transparent in the HMD so that the participant’s real hand and real environment (including the set of markers) are visible together with the virtual content (MP4 24.6 MB)ESM 4Online Resource 4 Illustrative video of game 2 “Melody Cubes”. Two images are displayed on the laptop and in the screen captures: the left image is presented to the left eye and the right image is presented to the right eye to generate depth perception. The dark grey background appears almost transparent in the HMD so that the participant’s real hand and real environment (including the set of markers) are visible together with the virtual content (MP4 27.2 MB)ESM 5Online Resource 5 Illustrative video of game 3 “Hungry Squirrel”. Two images are displayed on the laptop and in the screen captures: the left image is presented to the left eye and the right image is presented to the right eye to generate depth perception. The dark grey background appears almost transparent in the HMD so that the participant’s real hand and real environment (including the set of markers) are visible together with the virtual content (MP4 24.6 MB)ESM 6Online Resource 6 Reachable workspace assessment (PDF 319 kb)ESM 7Online Resource 7 Factors contributing to user experience (PDF 78.4 kb)


AbstractMesoniscusgraniger was the sole species closely associated with deeper parts of the depth profile and was present in most of the sites studied. Another ten species were represented by a small number of individuals and their occurrence deeper in the scree slope profile was rather accidental. A comparison between winter and summer periods indicates apparent differences in seasonal activities of isopods. Ethylene glycol seems to be more appropriate fixative solution for trapping isopods than formaldehyde.The shallow underground of forested scree slopes represents a little-studied subterranean biotope. In this paper, species diversity and depth distribution of terrestrial isopod communities studied in the depth profile (5–95 cm from the surface) of eight forested scree slopes in the Western Carpathians (Slovakia) is discussed. The southern edge of the Western Carpathians where the study sites are located represents the northernmost limit of distribution of obligate subterranean fauna in Europe. The sites differ from each other in type of bedrock, forest composition, slope aspect, and altitude. To sample isopods, a set of three subterranean pitfall traps consisting of a plastic cylinder (Ø 110 mm) and ten plastic cups was buried in each studied scree slope. In total, 252 isopods belonging to eleven species were sampled (1–5 species per site). Of the species found, the blind and depigmented  An extensive labyrinth of air-filled dark voids among the rocky fragments found inside the forested scree slopes represents a peculiar type of shallow subterranean habitat . The mesCommunities of terrestrial isopods dwelling shallowly underground in forested scree slopes have been explored minutely so far only in the Czech Republic , RomaniaPrevious research in subterranean biology in the Western Carpathians has favored caves . Mock etPageBreak(Slovakia). The studied scree slopes were predominately formed during the Pleistocene to Holocene by frost weathering and they differ in the type of bedrock . This study site lies on acidic granitoid bedrock but in its immediate vicinity, there is a karst area (Borinka Karst) characterized by several karst formations such as caves and sinkholes. The scree slope profile has four distinct layers: litter and humus (0–5 cm), organo-mineral layer (5–20 cm), a layer consisting of mixture of mineralized soil and rocks (20–75 cm) and scree partially clogged with soil (75–110 cm).1 Scree slope in the Strmina Natural Reserve  overgrown with beech forest (Querco-Carpinetum) and its profile consists of three different layers: litter and humus (0–5cm), organo-mineral layer (5–30 cm) and scree with spaces filled substantially with mineralized soils (30–110).2 Scree slope on basalt bedrock in the Belinské skaly National Nature Monument belonging to the Cerová vrchovina Highlands, an area known for the presence of a number of shallow pseudokarst caves. The scree slope profile is covered with a xerophilous oak-hornbeam forest (Fago-Carpinetum) and in the scree slope profile, there are four distinct layers: leaf litter and humus (0–5 cm), organo-mineral layer (5–25 cm), mixture of rocks and soil (25–70 cm) and scree partly filled with soil (70–110 cm).3 Limestone scree slope in the Drienok Valley (Revúcka vrchovina Highlands) situated a few meters below the entrance to the Špaňopoľská Cave. The site is surrounded by beech-hornbeam forest .5 Scree slope within the collapse above the Veľká drienčanská Cave. The slope profile without evident stratification consists predominately of rocky fragments originating from the previously collapsed cave ceiling. The spaces among the large limestone boulders are slightly filled with the mixture of soil and humus. Both sites (4 and 5) are located approximately 10 km west of the Drienok Valley (site 3) and are overgrown with beech hornbeam forest (Corno-Carpinetum). On the scree slope surface, there are several large boulders, most of them covered by bryophytes. Three clearly separated layers are recognizable inside the scree slope: a layer consisting of litter and humus (0–15 cm) is followed by organo-mineral layer with admixtures of tiny rocks (15–75 cm) and scree formed by large rock fragments (75–110 cm).6 Limestone scree slope in the Slovak Karst National Park situated about 30 m west of the entrance to the Ardovská Cave. The slope is overgrown with dogwood-maple forest (Tilio-Aceretum with Carpinusbetulus) trees and the scree PageBreakslope profile is divided into three layers: leaf litter and humus (0–5 cm), thick organo-mineral layer (5–30 cm) and scree (30–110 cm).7 Limestone scree slope along the doline near the entrance to the ice cave Silická ľadnica situated in the Slovak Karst National Park. The site is forested by linden-hornbeam, and maple (Tilio-Aceretum) is composed of the following layers: leaf litter and humus (0–15cm), organo-mineral layer (15–45 cm) clearly separated from scree (45–110).8 Limestone scree slope in the Sivec National Reserve (Čierna Hora Mountains) including a massive limestone cliff with several shallow caves. The vertical profile of the scree slope covered with linden-maple forest  was calculated using the formula C=100*pA/P where pA is the number of study sites on which, the specific species were sampled and P is the total number of study sites. Then after calculations, species were subdivided into the categories reflecting their dominance: subrecedent (D < 1%), recedent (D = 1–2%), subdominant (D = 2–5%), dominant (D = 5–10%), eudominant (D > 10%) and constancy: rare (C < 25%), widespread (C = 25–50%), constant (C = 50–75%), common (C > 75%). Shannon’s diversity index (H’) was calculated by the formula H = -∑(Pi*lnPi), where iP is the fraction of the entire population made up of species i . Pielou’s evenness index (J’) was computed by the formula J’ = H’/H’max = H’/lnS where S is the number of species encountered  was calculated by the formula D = 100*n/N where ountered .H’=1.35 and J’=0.32) was recorded on the scree slope near the entrance to the Ardovská Cave. Among the captured Isopoda, Mesoniscusgraniger was characterized as common, because of its highest constancy ; one species was classified as constant, three other species as widespread, and six species as rare. At the same time, M.graniger represented the only eudominant isopod species (D = 54.4%). The remaining species were classified as dominant (2 spp.), subdominant (3 spp.), subrecedent (3 spp.), and recedent (2 spp.) . Only three species were distributed deeper in higher numbers, nt Table . The indm Figure .M.graniger sampled on two pairs of sites with the same duration of samplings: the first pair is represented by the scree slopes in the Belinské skaly and Drienok Valley  while the second pair by the scree slopes next to the Ardovská Cave and ice cave Silická ľadnica . On the sites 2 and 6, warm/cold (= May-October/November-April) season ratio was almost 50:50 but on the sites 3 and 7, markedly more specimens were collected during the warm sampling period. Taking into account all 4 study sites together, 70% of specimens were collected during the warmer sampling periods . As regards the comparison of effectiveness of two types of fixative solutions used in this study, ethylene glycol appears to be significantly more attractive (or less repellent) for isopods than formaldehyde . Alkaline study sites are more diverse with average of four species on one study site, against two species on both acidic study sites. Total number of species on alkaline study sites is ten, on acidic study sites it is only four.Regarding pH, more study sites are located on limestone, thus soil pH is alkaline. Only two study sites have acidic soil pH. In comparison of abundance, on alkaline study sites 193 specimens were sampled, but on acidic sites only 18 (soil pH was not Mesoniscusgraniger (Mesoniscidae) and an undescribed blind species from the family Trichoniscidae , characterized by a stable microclimate and much higher organic matter content than in deep caves, are almost exclusively inhabited by Mesoniscusgraniger – an eyeless and depigmented eutroglophile often found in subterranean habitats of the Carpathian and Dinaric Mountains  to be the seasons with higher activity of M.graniger. Seasonal activity of other species was not assessed, due to their low abundance. This indirectly suggests that there is no massive seasonal vertical migration of the isopods into deeper layers of the forest scree slopes. Migration of terrestrial isopods into the deep soil is not necessary, since they can spend winter periods at shallow depths. Fallen leaves and snow cover provide a sufficient isolating layer to prevent lower lethal temperatures to isopods from being reached. Avoiding the uppermost layer of substrate, where the temperature has fallen below freezing point for several winter months, as overwintering isopod strategy could be minimized to move to a depth of a few centimeters under appropriate conditions, when the surface of soil is covered by fallen leaves and snow  are more abundant and diverse. Soil pH is not the sole reason of this phenomenon. We assume that it is a combination of soil pH, temperature, humidity and other externalities.It is not clear from our research, how pH is affecting terrestrial isopods assemblages, since our species and specimens counts are very low. If we take a look at the dominant  species . Terrest species . LimestoMesoniscusgraniger). The interior of forested scree slope can be considered as a part of subterranean environment inhabited in the long term by fauna specialized to live in permanent darkness. Eleven species were sampled in total, which is more than one third of the autochthonous isopod fauna in Slovakia. Well-preserved status of scree habitats is supported by the presence of the rare species, Mesoniscusgraniger, Orthometoponplanum, and minute Carpathian endemic Trichoniscuscarpaticus. Subterranean pitfall traps with ethylene glycol proved themselves as suitable apparatuses to collect macrofauna from this environment.The shallow underground of forested scree slopes in Slovakia is not inhabited by unique terrestrial isopods, but is uncommonly visited by surface (forest) species or by the sole subterranean species sampled (


Acute kidney injury (AKI) corrupts the outcome of about 50% of all critically ill patients. We investigated the possible contribution of the pathology acidemia on the development of AKI. Pigs were exposed to acidemia, acidemia plus hypoxemia or a normal acid-base balance in an experimental setup, which included mechanical ventilation and renal replacement therapy to facilitate biotrauma caused by extracorporeal therapies. Interestingly, extensive histomorphological changes like a tubular loss of cell barriers occurred in the kidneys after just 5 hours exposure to acidemia. The additional exposure to hypoxemia aggravated these findings. These ‘early’ microscopic pathologies opposed intra vitam data of kidney function. They did not mirror cellular or systemic patterns of proinflammatory molecules (like TNF-α or IL 18) nor were they detectable by new, sensitive markers of AKI like Neutrophil gelatinase-associated lipocalin. Instead, the data suggest that the increased renal proton excretion during acidemia could be an ‘early’ first hit in the multifactorial pathogenesis of AKI. Acute kidney injury (AKI) complicates the course of disease of up to 50% of all patients admitted to an intensive care unit –3 and co2 through the lungs and elimination of nonvolatile acids through the kidneys are the main regulators of acid-base balance. The kidneys have to increase acid elimination and retention of bicarbonate to protect pH homeostasis if the respiratory control of CO2 is impaired. The renal excretion of hydrogen ions is an active, energy consuming process that depends H+-ATPases, which exist ubiquitously in the nephrons with the highest concentration in intercalated cells [2-removal through the lungs, contributes to AKI. Furthermore, a reduction of mainly oxygen-dependent ATP-generation–as it can occur in ALI–might aggravate AKI further. Therefore, the hypothesis, that a short period of acidemia as well as a short period of combined acidemia and hypoxemia is an independent risk factor for the development of AKI, was tested. The investigations were performed in an experimental porcine model that enabled ‘standardized’ acidemic/hypoxemic conditions for several hours and a fast correction of the induced pathology within one hour before sacrificing the animals for kidney biopsies. The model accounted for the acute biocompatibility reactions of critically ill patients to ‘common’ organ support therapies like mechanical ventilation and renal replacement therapy.Elimination of a COed cells , 16. StiThe animals were held under standard conditions at the Department of Experimental Medicine (facilities are certified by ISO 9001:2000) according the guidelines for laboratory animal care of the European and German Societies of Laboratory Animal Sciences. The study protocol was approved by the Charité veterinarian and the University Animal Wellfare Officer as well as the federal authorities for animal research in Berlin , Germany.All experiments were performed in male pigs . Pigs were assigned to either of 4 groups with 6 animals per group (n = 6): mixed acidemia with continuous veno-venous hemofiltration (CVVH) or without (control) as well as mixed acidemia plus hypoxemia with CVVH and without (control). A full description of all used materials and methods is included in the supplementary materials. Two further groups with CVVH (n = 6) and without control, n = 5) were exposed to normoxemia and a physiological acid-base balance. Due to ethical aspects and strict local animal protection laws the normoxemic control group with normal acid-base balance is a control group of a former study . Thus, 2 (etCO2) target of 35–40 mmHg and a saturation of peripheral oxygen (SpO2) above 95%. After intubation the animals were placed in a supine position and cannulated with an arterial line, central venous line, shaldon catheter for CVVH and pulmonary arterial catheter (PAK). The urinary bladder was directly catheterized with a balloon catheter.General anaesthesia was induced in pigs after premedication, followed by orotracheal intubation and continuous intravenous anaesthesia . InitialaCO2 of 80–85 mmHg). Hypoxemia was induced by reduction of FIO2 until PaO2 was below 70 mmHg and SvO2 below 65%. The acid infusion rate had to be adjusted individually in the hypoxemia-groups to reach the target range, since hypoxemia induced a metabolic acidosis itself  and immediately processed. Blood samples were analysed at the local Institute for Clinical Chemistry, Charité—Universitätsmedizin Berlin using laboratory standard methods except for blood gas samples . Serum and urine levels of IL-18, IL-6 and TNF-α were determined by commercially available enzyme linked immunoabsorbant assay (ELISA)  .2) and oxygen consumption (VO2) were calculated using standard equations [Cardiac output (CO) was measured with the thermodilution technique and pulmonary artery occlusion pressure (PAOP) as determinant for left ventricular preload was measured at all main time points of the experiment using the PAK. Furthermore, oxygen delivery , thiopental (1 g) and potassium chloride , the kidneys were immediately removed and small corticomedular tissue samples were fixated in a 4 vol% buffered formalin solution. The no-flow time of the kidneys was about ten minutes. The tissue samples were embedded in paraffin; slides of 4 μm thickness were cut and stained with standard hematoxylin and eosin (HE)-staining. Kidney cell damage was analysed with a semiquantitative scoring system by adjusting existing scores –21 to ouIHC staining was performed for IL-18, IL-6 and TNF-α with an ABC-staining kit in concordance with the producer’s standard protocol  was performed using the Mann-Whitney Rank Sum Test. Post hoc analysis (pairwise comparison) was done using either Tukey’s test for equal sample size or Dunn's method for an unequal sample size. Friedman Repeated Measures Analysis of Variance on Ranks was performed for intra-group comparisons. Statistical significance was assumed at p <0.05.Plasma IL-6 levels increased in tendency in all groups during the experiment, except in the normoventilated control-group, which was exposed to the highest inspiratory pressures  values decreased during the induction of acidemia, but remained above 65 mmHg. A moderate PEEP of 5 mmHg did most likely not result in a compromised kidney perfusion, since diuresis remained above clinically accepted cut offs for oliguria (0.5 ml/kg/hr) , Fig 3. Slices of kidneys tissue samples were scored using a semiquantitative score in HE staining , despiteImmunohistochemical (IHC) staining of kidney slices for IL-18, IL-6 and TNF-α  was incoHigh diuresis rates were observed in all animals during acidemia/hypoxemia (3 ml/kg/hr) . SubgrouAlthough, urine neutrophil gelatin-associated lipocalin (NGAL) concentrations increased in the acidemic/hypoxemic CVVH-group, an irregular pattern was observed with respect to all groups as well as urine and serum NGAL concentrations after exposure to acidemia for five hours , Fig 9. Clinically, urinary output and laboratory measurements like serum creatinine are used to evaluate kidney function. Even immunological mediator levels, advanced hemodynamic monitoring and medical imaging can only give an indirect reflection of kidney perfusion, morphology and function. The hardest criterion of kidney cell damage is a biopsy and histopathological examination, which is almost never achievable in the critically ill patient. Thus, experimental investigations are necessary to identify conditions that result in cell damage and in turn AKI. To our knowledge, this was the first time that the impact of mixed acidemia on kidney function and histomorphology was investigated in large animals under clinical conditions.Unexpectedly, we found a severe impact of acidemia itself on the kidneys’ morphological integrity after only three hours of exposure to arterial pH values of 7.2 (about five hours with induction of the pathology). The cell damages were observed despite normal to high diuresis rates and without a relevant increase of novel biomarkers of AKI. A causal relationship between kidney damage and ventilation is unlikely, since the observed kidney cell damage was less in the groups which were exposed to normal acid-base balance and simultaneously the highest inspiratory pressures and tidal volumes as compared to the acidemic groups with low tidal volume ventilation. Furthermore, Tsuno et al. ventilated piglets with a tidal volume of 13 ml/kg body weight for 48 hours and did not observe histopathological changes compared to lungs of healthy piglets .An impact related to a reduced kidney perfusion was mainly excluded by using advanced hemodynamic monitoring. This was crucial since an impairment of systemic circulation can result in a reduction of renal blood flow (RBF), a decreased glomerular filtration rate (GFR) and a decreased renal clearance rates–regardless of the underlying pathomechanism. Direct measurements of RBF are hard to perform in critical ill patients and as a severe limitation we were not able to conduct direct measurements of RBF in this complex experimental setting. Nevertheless, it is known that even short warm ischemia due to cardiac arrest does not result in AKI  and expeAn impact related to local or systemic inflammation was not likely since we did not use an animal model based on inflammation like a sepsis model. Furthermore, we did not find a distinct pattern of proinflammatory mediators–neither local nor systemic–which would support the hypothesis of an inflammatory response of the kidneys to an insult.It is well known that the exposure of blood to an extracorporeal circuit triggers cascades of proinflammatory mediators . On the Nevertheless, it is difficult to interpret the relevance of proinflammatory mediators as long as organ function is not compromised. Novel biomarkers of AKI, such as IL-18 and NGAL, may detect AKI within a few hours of the incipient renal pathology, even before ‘traditionally’ used markers like serum creatinine or diuresis rate detect AKI , 32. Int+-ATPase) are the focus of recent research concerning acid-base regulation [+-pump rich intracellular vesicles fuse with the luminal plasma membrane when the renal tubules of rabbits were exposed to an increased partial pressure of CO2 for just 15 minutes. Their work elegantly demonstrated that the major effect happened within the first minute of exposure and was accompanied by an increase in H+ secretion [Regardless of these findings, significantly more kidney cell damage was observed in the acidemic groups compared to groups without acidemia. The additional hypoxemia worsened the histopathological scores even further. It is well known that kidneys increase the excretion of acid equivalents during respiratory as well as metabolic acidosis. The kidneys’ response is ‘slow’ and three to five days pass before a new steady state is reached , 35. Nevgulation , 37. Schecretion . These pThus, the data presented here support the following hypothesis:Kidneys are exposed to an energy consuming compensatory workload to regulate acid base-balance in case of acidemia, when regulation by the respiratory system is impairedCell damage will appear at an early stage–within hours–before clinically used markers detect AKIThis etiology may work as a ‘first hit’-insult potentiating further insults from ongoing biotrauma due to ventilation, impaired perfusion and inflammation in critically ill patients.aCO2 above 50 mmHg within the first 48 hours of mechanical ventilation, was associated with an increased ICU-mortality as well as a higher incidence of AKI in their work [This hypothesis is supported by clinical data from a retrospective pooled analysis of three ARDS trials by Nin et al. Hypercapnia, defined as a maximum Peir work .Furthermore, Jaber et al. demonstrated, that critically ill patients with a preexisting chronic kidney disease (Acute Kidney Injury Network score of 2 to 3 at study enrollment) profit from buffering a metabolic acidemia  with sodium bicarbonate with respect to days alive and days without RRT in a recent RCT .This study has several limitations. In order to assess the possible impact of acidemia/hypoxemia on kidney function, the experiments were performed in healthy pigs without preexisting diseases and the experimental time was limited to seven hours. Contrary to the clinical situation, the experimental setting supported the harvest of tissue samples. However, tissue samples just reveal morphological and immunohistochemical changes at the time point of sampling. Furthermore, in our experiments kidney slices were scored for reversible markers of cell damage like cell granulation as well as irreversible changes like the loss of cell barriers. It remains unclear whether areas of the kidneys with potentially reversible changes would have developed irreversible changes over time. Some of the potentially reversible changes may present a physiological response to the energy consuming insult rather than a pathophysiological finding. Nevertheless, some cell changes like the loss of cell barrier are a permanent destruction of the tubules’ anatomic units.At present, it is speculative to what extent the histological findings correlate with a clinically relevant compromised kidney function. Existing data suggests that healthy kidneys will sustain a considered amount of damage before kidney function is compromised at a clinical relevant level. Further research is needed to evaluate the role of acidemia as ‘first insult’ or contribution factor to AKI in a multifactorial setting.An exposure to a mixed acidemia of 5 hours resulted in histomorphological changes like a tubular loss of cell barriers in the kidneys of anesthetized healthy pigs. An exposure to an additional hypoxemia aggravated these histomorphological changes. The experimental model did not result in a systemic or local inflammation which could be correlated to histomorphology or kidney function. Kidney function as well as systemic perfusion were not compromised in this experimental setup. Instead, the additional energy-depending secretion of protons through the kidneys may have caused the histomorphological changes. Further investigations are needed about the exact pathomechanism and its possible relevance in critically ill patients.S1 Methods(DOCX)Click here for additional data file.S1 Fig1; results and pictures concerning transmission electron microscopy not published). For this, anesthized pigs were exposed to CVVH and infusion of either 4% gelatine 30 kDa (The kidney slices were obtained in experiments investigating possible biocompatibiltiy reactions between continuous veno-venous hemofiltration (CVVH) and infusion of different colloidse 30 kDa  or 10% he 30 kDa  and 3150(TIF)Click here for additional data file.S2 FigKidney slices were stained in hematoxylin eosin (HE) and photographed at 400:1 enlargement. A shows a slice with little cell damage, arrows indicate proximal tubular cell vacuolization. B and C are exemplary for high tubular cell damage with nucleus pycnosis (B) and cell barrier loss (C) highlighted with arrows. D shows normal morphology in comparison. * renal corpuscles, # proximal tubules, + distal tubules.(TIF)Click here for additional data file.S3 FigA) Intensity of the signal score 1.B) Intensity of the signal score 2.C) Intensity of the signal score 3.D) Intensity of the signal score 4.E) Intensity of the signal score 0.All stained biomarkers occur in a brown colour as indicated by an arrow. The intensity of IHC staining was compared using as semiquantative score ranging from no IHC staining visible (0) to staining visible in more than 50% of the visual field (4).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableScoring criteria for analyzing kidney cell damage in haematoxylin-eosin (HE) staining.(DOCX)Click here for additional data file.S3 TableThe table summarizes which change in percent of a visual field for a certain score criteria equals which score value.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableThe table summarizes the renal structures which were analyzed for immunohistochemical (IHC) staining.(DOCX)Click here for additional data file.S6 TableThe table provides details what qualitative IHC staining intensity equals which score value.(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.