Dyscarbia is common in out-of-hospital cardiac arrest (OHCA) patients and its association to neurological outcome is undetermined.2) and neurological outcome at 6 months, defined by the Cerebral Performance Category (CPC) scale, dichotomized to good outcome (CPC 1 and 2) and poor outcome (CPC 3–5). The effects of hypercapnia and hypocapnia, and the time-weighted mean PaCO2 and absolute PaCO2 difference were analyzed. Additionally, the association between mild hypercapnia (6.0–7.30 kPa) and neurological outcome, its interaction with target temperature (33 °C and 36 °C), and the association between PaCO2 and peak serum-Tau were evaluated.This is an exploratory post-hoc substudy of the Target Temperature Management (TTM) trial, including resuscitated OHCA patients, investigating the association between serial measurements of arterial partial carbon dioxide pressure . Mild hypercapnia was not associated with neurological outcome (P = 0.78) and there was no statistically significant interaction with target temperature (Pinteraction = 0.95). There was no association between PaCO2 and peak serum-Tau levels 48 or 72 h after return of spontaneous circulation (ROSC).Of the 939 patients in the TTM trial, 869 were eligible for analysis. Ninety-six percent of patients were exposed to hypocapnia or hypercapnia. None of the analyses indicated a statistical significant association between PaCO2 in the post-cardiac arrest phase and neurological outcome at 6 months after cardiac arrest was detected. There was no significant interaction between mild hypercapnia and temperature in relation to neurological outcome.Dyscarbia is common after ROSC. No statistically significant association between PaCOThe online version of this article (10.1186/s13054-018-2119-5) contains supplementary material, which is available to authorized users. Despie effect . PaCO2 ilkalosis . In rece outcome –17, whil outcome –21.As hypercapnia has been associated with good neurological outcome , the nov2 in serial measurements at predefined time points during the first hours after ROSC, to explore the role that PaCO2 may have in the neurological outcome of OHCA patients, and to specifically analyze the interaction between mild hypercapnia and targeted temperature management in relation to neurological outcome. To further strengthen the analyses, we investigated the association of PaCO2 to a surrogate marker of neurological outcome: peak levels of serum-protein Tau, a marker of neuronal damage that has shown to be more accurate than neuron specific enolase (NSE) in predicting outcome [We conducted this exploratory substudy of the TTM trial to describe the evolution of PaCO outcome .This study is based on the TTM trial conducted between 2010 and 2013 and was approved by the TTM trial steering group before completion of the trial . Ethical2 value to 1 h before T0 (T – 1). PaCO2 data were surveyed for physiological plausibility and in four of the measurements we corrected a misplaced decimal point. Corrections were conducted by FE and NN in accordance with other data registered on the same patient.Prehospital data were reported according to the Utstein criteria . BaselinThe primary outcome was overall neurological function at 6-month follow-up, using the Cerebral Performance Category (CPC) scale  –28. The 2 value during the observation period. The groups were defined as hypocapnia (< 4.5 kPa), normocapnia (4.5–6.0 kPa), and hypercapnia (> 6.0 kPa) in keeping with a previous investigation [As our main analysis, we studied the association of dyscarbia with neurological outcome by dividing the cohort into three groups according to the single highest or lowest PaCOtigation . The out2 (∆PaCO2) was calculated as a continuous variable, investigating an association of maximum amplitude in PaCO2 during the observation period and neurological outcome.The amplitude in PaCO2 over time. The AUC including eight PaCO2 values was analyzed, investigating an association with neurological outcome over the whole observation period, as well as the AUC of the first four PaCO2 values, in order to study the influence of early dyscarbia. To investigate nonlinear patterns, we divided the PaCO2 AUC for the whole exposure time into quintiles.We obtained an approximation of the time-weighted mean carbon dioxide exposure as an area under the curve (AUC) by integrating PaCO2 and lowest pH as continuous variables and neurological outcome was evaluated in univariable analyses. Thereafter, both variables were introduced into a combined logistic regression model.The association between maximum PaCO2 groups approximating the PaCO2 ranges employed by Eastwood et al. [2 and temperature in relation to outcome.From the time-weighted mean carbon dioxide exposure analysis, we extracted two PaCOd et al.   cohort including 100% of the patients, even those not surviving the full exposure period.For sensitivity analysis we used the complete case cohort consisting of 485 patients (56%) with blood gas samples registered from all measuring points and the total case , sex , chronic heart failure (yes/no), asthma/chronic obstructive pulmonary disease (yes/no), cardiac arrest witnessed (yes/no), bystander CPR (yes/no), first rhythm shockable (yes/no), time to ROSC (minutes), GCS-Motor score (1 versus 2–5), shock on admission (yes/no), and pH at admission (units). Whether pooling of the two temperature groups was feasible was established by a term of interaction model between the PaCO2 analysis groups and the two temperature groups. A significant term of interaction entailed subgroup analyses for the 33 °C and the 36 °C groups. A nonsignificant term of interaction entailed a combined group analysis.Proportions are expressed as percentages and continuous data as means with standard deviations (SDs). The association between PaCO2 and 1 unit for pH).Logistic regressions are presented as odds ratios (ORs) with 95% confidence intervals (CIs), with OR below 1 indicating better and OR above 1 indicating worse neurological outcome. ORs relating to continuous data describe the variation per one unit  where computed for each imputed dataset and evaluated using regression models. The estimates from the logistic and linear regression for each imputed sample were combined into one estimate with 95% CI including the uncertainty from the multiple imputations based on Rubin’s rule [P < 0.05 as significant. Analyses were conducted using IBM SPSS statistics for Windows version 22.0  and R: A language and environment for statistical Computing version 3.3.3 , with the package mice used for multiple imputations [Multiple imputation was used to compensate for missing values. We employed predictive mean matching, where information from nonmissing values of the same individual, all available TTM trial study variables, and values of matching patients were used to impute the missing values. In total, 20 imputed datasets were generated by chained equations and evaluated by graphical methods. The summary measures of PaCOn’s rule . The priutations .n = 62), patients with no PaCO2 data (n = 2), and patients with no data on neurological outcome (n = 6)  Fig. , leaving2 was 6.40 (SD 1.99) kPa and decreased over time in both the temperature and outcome groups  kPa. The PaCO2 AUC for the first four measuring points was mean 5.51 (SD 0.92) kPa while the PaCO2 AUC for all measurements was mean 5.37 (SD 0.62) kPa.On arrival, the mean PaCO 6.40 SD .99 kPa a2 groups showed no significant difference for the main outcome (Pmain outcome = 0.072–0.98) in all comparisons. The terms of interaction, except for ∆PaCO2 (P = 0.046), were also nonsignificant (Pinteraction = 0.255–0.947). For the ∆PaCO2 group, the 33 °C and the 36 °C subgroups were analyzed separately, showing no significant association with outcome in any of the groups which made pooling of temperature groups feasible.The analysis of interaction between temperature groups (33 °C or 36 °C) and PaCOMultivariable models with all covariables included in the analyses for hypercapnia and hypocapnia versus normocapnia are presented in Tables P = 0.13) or the hypercapnia and nonhypercapnia groups  in relation to poor neurological outcome. In a similar analysis, the hypocapnia group was compared to the normocapnia group and subsequently to the nonhypocapnia group with no significant differences . The ∆PaCO2 analysis did not reveal a statistically significant association with poor neurological outcome, either for the 33 °C or the 36 °C subgroup , or for the combined group . The PaCO2 AUC from admission to the end of intervention time and for the first four measured PaCO2 values (early dyscarbia) was not associated with poor neurological outcome . Furthermore, we were not able to detect an association pattern with neurological outcome after we divided the total exposure time PaCO2 AUC into quintiles (P = 0.73–0.96). Details of this investigation are presented in Additional file Our main analysis revealed no statistically significant difference between the hypercapnia and normocapnia groups . When analyzed in a combined logistic regression model, only the significant association between lowest pH and poor neurological outcome prevailed  per unit decrease in pH, while PaCO2 lost its significant association with neurological outcome . PaCO2 and pH were not highly correlated with a collinearity between the regression coefficients of − 0.64.When analyzed separately in univariable logistic regression models, maximum PaCOP = 0.79) between temperature and PaCO2 in relation to outcome; thus, we continued with a multivariable model without interaction. This analysis did not show a significant difference either between the mildly elevated (n = 121) and normocapnic (n = 675) PaCO2 groups  in relation to neurological outcome or between the temperature groups .The TTMH analysis, comparing a normocapnia group to a mild hypercapnia group, showed a nonsignificant term of interaction (n = 64) or missing peak s-Tau values (n = 36). The multivariable analysis of the remaining 589 patients showed no association between PaCO2 and s-Tau in our models (P = 0.12–1.00). The terms of interaction analysis were nonsignificant (Pinteraction = 0.11–0.83). Complete results are presented in Table Of the 689 patients in the s-Tau nested cohort analysis, 100 were excluded either due to our exclusion criteria (P = 0.32–0.96) and for the all-patient cohort with 939 patients (P = 0.15–0.98). For details concerning the sensitivity analyses, see Additional file Both sensitivity analyses revealed similar results as the analyses on the imputed dataset, for the complete sample cohort with 485 patients  were not available and, apart from a nested cohort analysis, only single PaCO2 values were analyzed. With the exception of the CCC trial [2, we are aware of only one study analyzing multiple PaCO2 values over time during the post-cardiac arrest phase [2 level was an independent predictor for good outcome at 12 months. We chose a comparable approach in our analysis of the PaCO2 AUC, but could not confirm this finding. In their study, blood gases were analyzed by either alpha-stat or pH-stat [2, arterial pH, and CBF when alpha-stat was compared to pH-stat. These findings might identify a source of error in studies using mixed blood gas management [2 was not. This confirms previous findings that pH is an independent outcome predictor after ROSC [Our results differ from a prospective single-center study by Roberts et al.  includinCC trial  randomizst phase . This pr pH-stat , while t pH-stat . Voicu e pH-stat  showed anagement  and whenter ROSC , 35.2 values , while the CCC trial randomized patients to specific PaCO2 ranges . Additionally, we widened the mild hypercapnia group for our analysis to 6.0–7.30 kPa for increased robustness of our results. Whether TTMH is indeed beneficial remains to be proven in a definitive clinical trial [2 in terms of outcome, supporting the possibility to coenroll in trials investigating carbon dioxide and temperature targets [Our study does not indicate benefit of TTMH as investigated by Eastwood et al. , but alsal trial . Importa targets .2 on biomarkers have to date only been evaluated in the aforementioned CCC trial where mild hypercapnia reduced NSE and S100 calcium-binding protein B (S100B) levels [2 showed no association with peak s-Tau levels, which is in line with the lack of association between PaCO2 and neurological outcome in our study. We have previously reported that s-Tau is superior to NSE in predicting outcome and that S100 does not add to a prediction model including NSE and clinical information [The effects of PaCO) levels . In our ormation , 36.2 at a certain point in time and we assumed that the PaCO2 between blood samples was linear. It is also important to point out that patients not surviving the analysis period were excluded from the analysis to allow a defined exposure period of carbon dioxide. Our data were corrected for strong covariables available at admission to hospital and no covariables that may have been available later during hospital stay; therefore we did not include, for example, PaO2 as a covariable in our analyses. There are, however, considerable strengths in our analysis as data were derived from a large, well-controlled cohort of OHCA patients with availability of important confounders. Physiological and biochemical data were collected prospectively at specified time points according to a predefined protocol and blood gases were analyzed in a uniform way. Measurements were serial and therefore likely to demonstrate the association of PaCO2 with outcome in the post-cardiac arrest phase more accurately than single measurements. Follow-up data were acquired with face-to-face interviews using a structured protocol and the loss of patients in the follow-up period was minimal [There is no consensus on how to report carbon dioxide levels in relation to outcome in cardiac arrest patients and previous studies have employed methods suited to the nature of their data . In this study we have employed many different analytic approaches and used different outcomes  in order to provide an investigation as robust as possible. It is important to emphasize that the study was conceived post hoc and with a definite exploratory approach. All results must be regarded as hypothesis generating, and due to the observational design we cannot make causality statements. Blood gases represent the PaCO minimal . We perf2 measured as extreme values and over time was not associated with neurological outcome at 6-month follow-up. Mild hypercapnia was not associated with adverse outcome and there was no interaction with temperature group affiliation.Dyscarbia after ROSC was common in OHCA patients, but PaCOAdditional file 1:Tables presenting detailed information on the excluded cohort, exposure group 6-month neurological outcome, and sensitivity analyses. (DOCX 23 kb)


In order to achieve high field sensitivities a magnetic bias field is necessary to operate at the maximum piezomagnetic coefficient of the MS phase, harnessing mechanical resonances further enhances this direct ME effect size. Despite being able to detect very small AC field amplitudes, exploiting mechanical resonances directly, implies a limitation to available signal bandwidth along with the inherent inability to detect DC or very low frequency magnetic fields. The presented work demonstrates converse ME modulation of thin film Si cantilever composites of mesoscopic dimensions (25 mm × 2.45 mm × 0.35 mm), employing piezoelectric AlN and magnetostrictive FeCoSiB films of 2 µm thickness each. A high frequency mechanical resonance at about 515 kHz leads to strong induced voltages in a surrounding pickup coil with matched self-resonance, leading to field sensitivities up to 64 kV/T. A DC limit of detection of 210 pT/Hz An emerging field, requiring much higher spatial resolution and compact sensor dimensions, is the contactless imaging or monitoring of biological entities using the magnetic field component of bioelectric currents5. This biomagnetic field was unveiled by early studies, proving the concept using giant pickup-coils6 later by sensitive yet very complex to operate SQUID magnetometers7. Finally, Wikswo8 coined the term “Nondestructive testing of humans” more than two decades ago.Magnetic field sensors are employed in a variety of industrial and electronic device applications, apart from widespread applications like linear position determination or shaft rotation speed sensing  there are applications where the magnetic field itself needs to be determined precisely. This holds true for geomagnetic sensing8. The permanent field of the earth is about six orders of magnitude higher, thus imposing a requirement towards dynamic range. The frequencies of interest range from DC to below 1 kHz8, which is typically the ELF (extremely low frequency) to VLF (very low frequency) frequency regime.The signals emitted from humans in form of magnetic stray fields are of very low amplitude, cardiac signals show amplitudes on the order of 10…100 pT, whereas brain signals are typically one to two orders of magnitude lower9, respectively. In magnetoencephalography (MEG) applications the use of multichannel arrays is anticipated in order to extract useful information10, thus imposing stringent spatial constraints on any proposed sensor system. xMR11 are very promising as they can readily be produced in volume, but yet suffer from excessive 1/f-noise levels12. Using flux concentration measures in order to enhance magnetometer sensitivity has proven quite effective14 but inherently brings a delicate trade-off between sensitivity and spatial resolution.Many available low-cost, high volume sensor technologies (e.g. Magnetoresistive (xMR) or Hall effect sensors) incrementally improve in performance yet to reach the threshold of being a viable candidate for widespread convenient biomedical sensing operation. Optically pumped magnetometers (OPM) as well as fluxgate magnetometers are promising candidates for the detection of biomagnetic signals. However, despite their room temperature operation and DC field capability they unfortunately exhibit bandwidth and scalability limitations16, especially as sensing elements for most demanding weak fields17 in biomagnetic signals are being made19. By exploiting mechanical resonance enhancement of the direct magnetoelectric effect, the sensitivity can be vastly enhanced for magnetic fields coinciding to the mechanical resonance21, at the expense of the sensor’s bandwidth. This straightforward approach is completely passive, thus scoring by simplicity, however, low frequency fields are intrinsically tedious to detect. By actively modulating the composite magnetically23, electrically24 or utilizing the delta-E effect25 one can up-convert off-resonance signals and thus benefit from resonances and be sensitive in the low frequency regime of interest. Using high frequency surface acoustic wave (SAW) sensors incorporating magnetostrictive material can similarly up convert the magnetic signal by phase modulation26.Continuing efforts of bringing magnetoelectric (ME) devices towards applicationsIn this study the converse ME effect in a thin film composite is exploited, exciting a high mechanical resonance mode showing a large vibration amplitude. The signals are detected by a pickup coil which is tuned in order to match its electromagnetic resonance with the excited mechanical resonance of the composite cantilever. Vibrometry measurements give insight to the nature of the mechanical oscillation, leading to periodic magnetisation modulation. The system performance towards small amplitude, low frequency magnetic fields as well as noise behaviour is analysed.90Co10)78Si12B10 at an argon pressure of 6*10−3 mbar, using a vonArdenne CS730s cluster sputtering tool. After subsequent depositions of 200 nm material, a pause of 10 minutes allows for cooling and prevents crystallization of the deposited material caused by plasma heating. To promote adhesion to the silicon and prevent ambient oxidation of the alloy, it is sandwiched between thin <10 nm sputtered tantalum layers. The highly textured PE AlN is deposited by reactive magnetron sputtering with nitrogen using a pulse DC source, details are extensively given in27. A platinum layer of 80 nm below the AlN serves a dual purpose of seeding the crystal growth as well as to enable electrical contact to the bottom electrode. A top electrode consisting of sputtered chromium (10 nm) and gold (80 nm) functions as a top contact in the PE plate capacitor arrangement. Electrical access to the buried bottom electrode is ensured by partial wet chemical etching of the AlN, for this standard photolithography is used in conjunction with phosphoric acid (H3PO4) at 80 °C for 20 minutes. The wafers are diced into 25 mm × 2.45 mm dies, which are then heat treated at 270 °C on a hot plate in ambient atmosphere in a magnetic field of 800 Oe directed along the short axis, provided by large permanent magnets. Finally, the silicon dies are bonded to a FR4 PCB board using cyanoacrylate glue thus creating a cantilever structure. The contacts are wire bonded to the carrier PCB. The formed AlN plate capacitor holds a capacity of 1.7 nF off-resonance. Magneto-optical Kerr effect (MOKE) microscopy is performed with a large view polarization sensitive microscope and high power LED illumination, allowing magnetic domain visualization of the magnetic layer. The exact configuration and working principle is described by McCord in28. Mechanical characterisation was performed in unshielded environment using a vibrometry system  and a 5x objective lens, the multicarrier signal in vibration spectroscopy was provided by a vector signal generator . The electrical characterization is carried out in a magnetically and electrically shielded environment comprising a multilayer mu-metal cylinder , further details are given in29. The magnetic test field is delivered using a calibrated cylinder coil in conjunction with a low noise AC and DC current source , saturation fields are provided by a bi-polar power supply . Analysis of the sensor system, with respect to excitation and readout is performed using a high frequency lock-in amplifier .Thin film ME composites based on silicon are fabricated at Kiel Nanolaboratory using standard microelectromechanical systems (MEMS) cleanroom processes including magnetron sputtering and photolithography. Both active layers, aluminium nitride (AlN) and amorphous iron–cobalt–silicon–boron (FeCoSiB) with a thickness of 2 µm are deposited on adjacent sides of a 350 µm thick double side polished silicon wafer. The FeCoSiB layer is RF magnetron sputtered at 200 W from a 200 mm target  with a nominal composition of  in parallel to the coil, its resonance frequency can be tuned downwards, in order to match the excited mechanical resonance. A similar approach has been implemented in fluxgate magnetometers32, essentially acting as a measure of low noise signal amplification. Figure tune, buffered by a low noise operational amplifier in unity gain configuration  in order to decouple the resonant circuit from subsequent readout electronics, i.e. the lock-in amplifier. The voltage Vcoil is fed to the digital lock-in amplifier for synchronous demodulation. Figure coil = 31 Ohm, Ccoil = 47 pF and Lcoil = 1.16 mH, which were determined using an Agilent 4294 A assuming a series RLC circuit, using Qcoil = 1/Rcoil * (Lcoil/Ccoil)1/2 resulting in a coil resonance quality factor of Qcoil ~ 160. Depending on the state of the magnetic material which is introduced into the coil, the Qcoil will decrease. The name U mode stems from the fact that a strong U-shaped curvature along the short cantilever axis is formed, as the reader will shortly find out. This U mode mechanical resonance is much sharper, holding a Q factor nearly an order of magnitude higher of QUM ~ 1000, tuning does not have to be overly accurate in order to benefit from this resonance convolution. Figure 33.The fabricated composite is immersed into a coil, which is wound on a polymeric bobbin using 750 windings of 110 µm thick enamelled copper wire. This assembly is in close proximity to a battery powered amplifier board, Fig. Ku. The observed in-plane stripe-like domains show a rather uniform width of about 60 µm throughout the length of the cantilever. Such a domain structure is typical for thin amorphous films exhibiting low total anisotropy28 after magnetic annealing. Externally applied magnetic test fields are directed orthogonally to the sample’s magnetic easy axis, denoted by H. In this axis a maximum of magnetoelastic coupling is achieved , this gives rise to the very high resonance frequency of about 515 kHz compared to widely studied flexural modes34, typically present in the audio frequency regime for mesoscopic silicon structures of millimetre dimensions. The dominant oscillation loss mechanism of flexural modes in long cantilevers lies in air damping36, thus seldom exceeding a Q of few 100 in ambient atmosphere. Due to much less displaced air molecules, this U mode shows inherently less damping, thus leading to a higher value of Q than that of low order flexural modes. The maximum displacement along the long y-axis amounts to about 25 nm, when excited by 100 mV, thus the resulting curvature contributes marginally to magnetoelastic effects. However, the deflection amplitude towards the free end reaches about 30 nm peak-to-peak displacement along the front most x-line which is about one tenth the cantilevers length, thus giving rise to a vastly increased curvature and accompanying strong magnetoelastic coupling. Note the ripple pattern along this axis, indicating a possibly simultaneously excited high order flexural mode33. The frequency of the U mode oscillation deviates by less than 0.7% through a set of five samples, irrespective of the rather large variance introduced by manual die mounting using adhesive glue. The high curvature along the x-axis leads to a very pronounced stress induced anisotropy (Kσ), uniaxially acting on the magnetostrictive material. This anisotropy is periodically changing its sign, leading to a directionality change of Kσ. For tensile stress of the film along the short axis, this results in Kσ being parallel to the thermally induced anisotropy Ku, leading to the addition of these two uniaxial anisotropies. While compressive stress leads to a configuration where Kσ lies along the y-axis of the cantilever, thus being orthogonal to Ku, which is quantified using Eq.  electromechanical spectrum, high speed vibrometry measurements were performed. Figure Ku acts along the x-axis of the cantilever, for values of Hk = 880 A/m and Ms= 1.12 MA/m a value of 620 J/m3 is obtained.x-axis line scan along the tip of the cantilever, for both extreme cases of oscillation as well as its resting position, along with a parabolic fit matching the shape. Using simple bending analysis after Ohring37 Eq. (σpiezo) can be made. For the data of an excitation level of 100 mV, a radius of curvature (R) along the x-axis at the free end of the cantilever of about 8.1 m is obtained, while the substrate to film thickness ratio of ts = 350 µm to 2 µm, respectively, makes the film negligibly thin. Therefore followingE is the Young’s modulus of 169 GPa, of the Silicon substrate38 a peak piezoelectrically induced stress of 3.7 MPa at 100 mV of excitation is estimated. From here, Kσ can be derived, depending on the sign of the piezoelectrically induced stress σpiezo and assuming an isotropic magnetostriction (λiso) of 30 ppm.Figure ng37 Eq.  and the Kσ of 165 J/m3 is generated at the tip. This is one quarter of the value of Ku meaning the ferromagnetic energy landscape is modulated, yet magnetisation reorientation is not expected. The addition of the two energy densities in the case of tensile stress may lead to domain wall motion already below the energy equilibrium of Kσ and Ku. Exciting the resonance at very high amplitudes in one case even lead to fracture through the entire composite along the centre of the y-axis, without facing dielectric breakdown  of 169 GPa and a Poisson’s ratio (ν) of 0.27 was chosen for silicon38. Figure y-axis towards the free end .A simple mechanical mode analysis was performed using Abaqus CAE 2018 using a uniform mesh size of 50 µm. As both active layers are expected to contribute only negligibly to the overall oscillation behaviour, making up only about 1% of the composite thickness, with no vastly differing Young’s moduli, a simple slab of silicon material having the same geometric dimensions is modelled. In order to best mimic the experimental study, a density of 2.330 g/cmH. When coming from negative saturation the induced voltage reaches above 800 mV at −30 µT, to vastly decrease until it reaches coercivity at 5 µT, where a phase reversal of the induced voltage takes place. This is attributed to the effective magnetic anisotropy switching direction along the y-axis of the cantilever, leading to a change of magnetisation and therefore flux direction change within the coil. The obtained loop is highly symmetric with respect to the saturation direction, owing to precise magnetic annealing. At very high magnetic fields, where a saturation of the magnetic material is inevidable, the voltage output again drops to near zero.The magnetoelectric sensor provides high dependency of the induced voltage towards low amplitude DC signals, which is presented in Fig. th order filter, of about 9 µV/Hz1/2, leading to an LOD of about 210 pT/Hz1/2 at DC, for a signal-to noise ratio of 1.Figure Q) and the frequency of vibration (f) a bandwidth (BW) can be estimated, within which the sensor will be able to detect signals above DC. For a Q = 1000 and f = 515 kHz, by using BW = f/(2·Q) a low pass characteristic with a −3 dB point of 260 Hz can be estimated40. This corner frequency is well within the requirements for most biomagnetic applications.From the mechanical  and 23 Hz (dashed). A spurious 50 Hz power line signal is also present symmetric to the carrier. The up-converted spectrum left and right of the carrier, contains the same informational content, hence why amplitude modulation has an inherent efficiency of 50%. The inset shows the linearity towards different AC magnetic field amplitudes. The slope of the fit equals a linear sensitivity of 30.4 kV/T, half of that given in Fig. 1/2 to 52 pT/Hz1/2 at a frequency of 2 Hz to 53 Hz, respectively. The low frequency performance is limited by the up-conversion of 1/f noise.Magnetic fields applied to the excited composite lead to an amplitude modulation at the excitation frequency, Fig. A cantilevered mesoscopic ME structure was electrically excited in a mechanical resonance lying in the medium wave regime, at 514.8 kHz exhibiting a mechanical quality factor of about 1000. High speed mechanical vibration spectroscopy reveals that this oscillation leads to large out-of-plane displacements of the structure along its short axis and to high stresses coupled into the magnetostrictive phase. These prove sufficiently strong to alter the magnetic energy landscape by inverse magnetostriction. The observed mechanical U mode is verified by a FEM mode analysis, quantitatively finding a matching resonance frequency at 517.3 kHz, which is within 0.5% of the experimentally determined value. The simulation furthermore suites the experimentally determined deformation pattern.Kσ is necessary in order to make converse ME interaction effective. Most determined modes lead to weak Kσ and are therefore unable to energetically balance or overcome the statically present induced anisotropy. Furthermore, symmetry of mechanical oscillation modes may lead to spatially localised voltages generated along the structure, which may lie out of phase, effectively cancelling the inductive signal.If placed within a pickup coil the ME oscillator responds strongly to only very few of the determined mechanical vibration modes by sharp induced voltage peaks. The reason for this discrepancy between purely mechanical and converse ME measurements may lie in the fact that a large 46. A magnetic LOD of 210 pT/Hz1/2 at DC is determined for a staircase test signal. For a 10 Hz signal an LOD of about 70 pT/Hz1/2 was achievable, decreasing noise with distance to the carrier, leads to an LOD of about 50 pT/Hz1/2 at 53 Hz. The presented setup enables array integration, as the pickup coil is operated entirely passive and no permanent magnetic bias field is required. Furthermore, a signal bandwidth of DC to 260 Hz meets the criteria of low frequency biomagnetic signals, additional improvement of the LOD is necessary in order to meet demands of biomagnetic signal amplitudes.If the PE excitation (carrier) signal is set to match the mechanical resonance at 514.8 kHz, its induced voltage amplitude at the excitation frequency is modulated by external magnetic fields. The magnetic field sensitivity increases nearly linearly with carrier signal amplitude, reaching 64 kV/T at 160 mV. Conversely, the noise strongly increases once above an excitation voltage of about 200 mV, leading to excessive stress induced remagnetisation, accompanied domain wall propagation creating a dominant source of noise. Introduction of more sophisticated magnetic layers may lead to restrain of random domain wall motion, i.e. introducing an exchange biased interlayer has proven helpfulSupplemental MaterialSupplemental 2, Video


HAT1, KAT5, HDAC6, MBD1 and DNMT3A affected by oxidative stress and hypoxia in an in vitro qPCR screening step performed on an osteoblast cell line was analysed in trabecular bone tissue samples from 96 patients. Their expression was significantly reduced in patients with postmenopausal osteoporosis and osteoarthritis as compared to autopsy controls and significantly correlated with bone mineral density and several bone histomorphometry-derived parameters of bone quality and quantity as well as indicators of oxidative stress, RANK/RANKL/OPG system and angiogenesis. Furthermore, oxidative stress increased DNA methylation levels at the RANKL and OPG promoters while decreasing histone acetylation levels at these two genes. Our study is the first to show that higher expression of HAT1, HDAC6 and MBD1 is associated with superior quantity as well as quality of the bone tissue having a more favourable trabecular structure.Epigenetic mechanisms including posttranslational histone modifications and DNA methylation are emerging as important determinants of bone homeostasis. With our case-control study we aimed to identify which chromatin-modifying enzymes could be involved in the pathology of postmenopausal osteoporosis and osteoarthritis while co-regulated by estrogens, oxidative stress and hypoxia. Gene expression of  Posttranslational histone modifications and DNA methylation are two of the most studied epigenetic mechanisms. Acetylation of histones is associated with transcriptional activation of target genes and is mediated by histone acetylases (HATs) and histone deacetylases (HDACs). DNA methylation is on the other hand usually associated with target gene repression. It is mediated by DNA methyltransferases (DNMTs) and facilitates binding of various DNA methyl-binding proteins to the DNA molecule2. However, any of these epigenetic mechanisms could be modified by different environmental factors, like oxidative stress, hypoxia and estrogens which are some of the known factors influencing bone remodelling in complex in vivo conditions.Epigenetic mechanisms represent an important ubiquitous group of gene expression regulators associated with normal and aberrant bone remodelling and homeostasis3. In addition, oxidative stress has been associated with decreased osteoblast and osteoclast numbers, decreased bone formation rate and increased osteoblast and osteocyte apoptosis. Reactive oxygen species (ROS) were shown to inhibit WNT/β-catenin signalling pathway in osteoblasts and increase production of adipocytes. In addition, age-related decline in estrogen levels substantially decreases defence against oxidative stress in bone thus further increasing bone resorption4. Another important aspect of bone health and homeostasis is angiogenic-osteogenic coupling. Hypoxia inducible factor 1α (HIF1α) signalling pathway and its target gene vascular endothelial growth factor A (VEGFA) are two of the most critical regulators of this coupling and are both substantially affected by hypoxia. Hypoxia has been associated with the inhibition of growth and differentiation of osteoblasts while strongly inducing osteoclast formation5. Conversely, selective activation of the HIF1α signalling pathway in osteoblasts showed protection from ovariectomy induced bone loss together with improved bone vascularity and angiogenesis. Furthermore, 17β-estradiol has been associated with HIF1α signalling pathway and modulation of angiogenesis7. This further underlines the importance of oxygen tension and HIF1α signalling pathway in osteogenesis.17β-estradiol reduces bone remodelling and reciprocally affects differentiation and apoptosis of osteoblasts and osteoclasts, thereby reducing bone resorption and increasing bone formation. These effects are at least in part mediated through WNT/β-catenin signalling pathway and RANK/RANKL/OPG system. Estrogen deficiency is thus considered to be one of the major causes of postmenopausal bone loss in women and age-related bone loss in both sexesin vitro study were measured in human bone tissue samples of PMO and OA patients and controls. Their expression was correlated with clinically relevant phenotype data like bone mineral density (BMD), micro computed tomography (µCT) and bone histomorphometric (BHM) measurements as well as the indicators of oxidative stress, RANK/RANKL/OPG system and angiogenesis. Finally, the impact of H2O2 on the DNA methylation and histone acetylation state of HOS cells was evaluated for the highly relevant receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) genes.Since the influence of estrogens, oxidative stress and hypoxia on the epigenetic mechanisms in bone is not elucidated yet the aim of our study was to identify which chromatin-modifying enzymes are substantially influenced by these factors and could be involved in the pathology of postmenopausal osteoporosis (PMO) and osteoarthritis (OA). We first determined the gene expression profile of a panel of 48 selected genes in a human osteoblast cell line HOS treated with 17β-estradiol, hydrogen peroxide and hypoxia mimetic deferoxamine (DFO). Next, the most exciting genes from the 2O2 and DFO on chromatin-modifying enzymes’ gene expression in osteoblasts we first prepared a suitable experimental in vitro model using HOS cells. Since estrogen receptor α (ERα) was surprisingly completely absent HOS cells were transfected with plasmid pCMV-ESR1. The efficiency of transfection was controlled with western blot and qPCR  gene expression 8. As expected, DFO also established hypoxic response in HOS cells  indicated transcriptional activity of HIF1α protein which is associated with its cellular accumulation and is a characteristic of hypoxia exposure. VEGFA (p = 3.56 × 10−2) expression was also transiently induced by H2O2  acetyltransferase 5 (KAT5) and lysine acetyltransferase 8 (MYST1) from the HAT genes, sirtuin 1 (SIRT1), SIRT6, HDAC6, HDAC7 and HDAC9 from the HDAC genes, and methyl-CpG binding domain protein 1 (MBD1) and DNMT3A from DNA-methylation associated genes exhibited the most pronounced changes in expression and were therefore selected for further analysis.Results from the qPCR screening array are presented in Supplementary Table HAT1 expression before inducing it at the later time point   and HDAC7 (p = 3.21 × 10−2) while inducing the expression of HDAC9 (p = 1.79 × 10−3) and SIRT1 (p = 1.99 × 10−2) genes at different time points  expression was also significantly induced by both H2O2 and DFO, only DFO showed negative effect on DNMT3A (p = 1.13 × 10−3)  , HDAC6  and MBD1  differed significantly between all three tested groups, wherein control samples again exhibited the highest levels of expression followed by OA patients and then PMO patients. DNMT3A expression was significantly lower in PMO patients than in either control samples or OA patients . Similarly, HDAC9 expression was also significantly lower in PMO patients as compared to OA patients (p = 1.16 × 10−5).The selection of genes was further refined to ly) Fig. . ExpressHIF1α, VEGFA and AOX1 in the same bone tissue samples. Expression of VEGFA was significantly higher in controls as compared to both patient groups  . Expression of HIF1α did not differ between the tested groups.We additionally looked at the expression of ly) Fig. . ConversKAT5 showed a substantial positive correlation with BMD at least at one of the measured sites, with MBD1 correlating substantially at all three  and concurrently a substantial negative correlation with trabecular separation (Tb.Sp) indicating a strong association of these three genes with bone microarchitecture   expression after 72 hours of treatment which resulted in significantly increased RANKL/OPG ratio (p = 1.96 × 10−2). DFO, on the other hand, significantly suppressed RANKL (p = 7.78 × 10−3) and OPG expression (p = 3.84 × 10−3 and 2.49 × 10−4), however no significant changes in the RANKL/OPG ratio occurred  Fig. . Converso p = 1.9 × 10−2. o p = 1.9 × 10−2. RANKL and OPG promoters, previously shown to influence their transcription10. In both cases a clear positive trend was evident after 24-hour exposure to H2O2  as well as bone quality . The latter are rarely available thus importantly contributing to the relevance of our study. Furthermore, HAT1, HDAC6 and MBD1 positive correlation with OPG and VEGFA expression indicate that their positive effect on bone could be mediated through their impact on the RANK/RANKL/OPG system and angiogenesis either by directly regulating the expression of OPG and VEGF or indirectly by regulating the expression of other important factors upstream from these two genes. In addition, their positive correlation with AOX1 expression clearly shows that oxidative stress present in bone tissue has a significant impact on the expression of chromatin-modifying enzymes which thus represent an additional mechanism by which oxidative stress affects bone metabolism. Limited information is available so far regarding HAT1, HDAC6 and MBD1 role in bone tissue. HAT1 has been reported to have a role in embryonic bone development, while HDAC6 could affect bone tissue through its involvement in angiogenesis and RUNX2 interaction14.We used three stimuli with an important role in bone biology, 17β-estrogen, oxidative stress and hypoxia to evaluate their influence on epigenetic regulation of bone remodelling in HOS cells before validating the significance of the affected chromatin-modifying enzymes on bone tissue samples from PMO and OA patients and healthy controls. Three genes in particular, i.e. HDAC9 and DNMT3A gene expressions were significantly lower in PMO than in OA patients, while their expressions in OA patients didn’t differ from that in controls. This indicates that their expressions are aberrant only in pathological changes associated with PMO and not OA thus linking their suppression to bone loss. In addition, their expression also significantly correlated with parameters of bone quantity as well as OPG and AOX1 linking them to the RANK/RANKL/OPG system and oxidative stress response in a similar way to the already described HAT1, HDAC6 and MBD1. Based on published data HDAC9 could also potentially impact the skeleton by directing the differentiation of mesenchymal stem cells towards osteoblasts instead of adipocytes while our study is one of the first pointing towards a significant role of DNMT3A in bone tissue16.Interestingly, KAT5 expression did not differ between the two patient groups however it was significantly lower than in controls. This indicates that there occur certain changes in epigenetic regulation in bone tissue that could be involved in the pathogenesis of both diseases. Interestingly, our results revealed a negative correlation between KAT5 and RANKL expression also linking it to the RANK/RANKL/OPG system and confirming its regulatory potential of bone remodelling similar to that of the already described five genes.Contrary to the described five genes VEGFA in normal bone tissue which would indicate good vascularization; however, there was no correlation between VEGFA expression and parameters of bone quality and quantity. Moreover, expression of VEGFA was similar in OA and PMO patients even though they have significantly different BMD and bone quality17. On the other hand, there was significant correlation between VEGFA expression and expression of HAT1 and MBD1, suggesting there is a certain relationship between angiogenesis and epigenetic mechanisms in bone tissue.We also observed a strong expression of AOX1 in OA bone tissue18. Oxidative stress is in fact a relevant part of pathogenesis of OA and is likely to be involved in cartilage destruction and bone cyst formation19. In line with that, HDAC9, shown to be induced by oxidative stress in our in vitro model, also exhibited the highest expression in OA bone.Even though oxidative stress has been primarily associated with bone loss in PMO patients, our results revealed the highest expression of RANKL and OPG as an example indicated, that oxidative stress increased DNA methylation and decreased histone acetylation at these two genes. While this could cause downregulation of target genes, additional regulatory mechanisms seem to be induced by oxidative stress representing a higher level of regulation. This however does not diminish the relevance of our results since regulation of bone remodelling is highly complex and involves numerous other factors that could be epigenetically regulated beside RANKL an OPG. In addition, involvement of oxidative stress-induced epigenetic changes has been reported for diseases such as chronic obstructive pulmonary disease20, ageing21 and cancer23.Finally, we have shown that changes in gene expression of chromatin-modifying enzymes are accompanied by alterations of DNA methylation and histone acetylation levels in HOS cells when exposed to oxidative stress. Using 26. In order to overcome this problem, we finally decided to transfect them with a ERα-carrying plasmid.There are some limitations to our study. Due to a very restricted availability and challenging collection of bone tissue samples, we were not able to completely match the number and gender structure of our autopsy control group of patients to the PMO and OA groups. However, because we didn’t observe any systemic bias in gene expression results for this patient group we think that having three groups of patients spanning a wide range of bone quality and quantity phenotypes adds an important validity to our study that is often missing in other studies using human bone tissue samples. The other limitation of our study is the use of HOS cells to study the effect of estrogen signalling. There are several reports in the literature describing ERα mRNA and protein expression in this cell line; however, we were unable to detect neither of them despite testing two different batches of HOS cells obtained from the ATCCin vitro observations of hypoxia and oxidative stress induced expression changes in chromatin-modifying enzymes to clinical samples demonstrating that HAT1, KAT5, HDAC6, MBD1 and DNMT3A expression was both significantly downregulated in PMO and OA. Moreover, higher expression of HAT1, HDAC6 and MBD1 was associated with superior quantity and quality of the bone tissue having a more favourable trabecular structure. This is the first report showing the importance of these enzymes in the maintenance of bone homeostasis and their possible involvement in the pathology of PMO and OA and certain other bone disorders.In conclusion we have translated our et al.27. Briefly, our study included 96 patients, 43 with a fragility fracture of the hip due to PMO, 41 with primary end-stage OA of the hip and 14 autopsy controls without any signs or history of disease or medication use known to affect bone or mineral metabolism  as described previously17. Briefly, bone samples were scanned using a Skyscan 1,172 mCT scanner  at two resolutions (10 and 20 mm voxel size). After scanning, undecalcified bone samples were embedded in methylmethacrylate, stained with von Kossa van Giesson. A semi-automated program, developed by dr. van’t Hof was used28. Total RNA was isolated from bone tissue samples of all patients and reverse transcribed as described previously27. Patients were grouped on the basis of their precisely determined bone phenotype. Due to low numbers of male patients and high phenotypic homogeneity within the groups we restrained from further gender-based subgrouping. The study was approved by Komisija republike Slovenije za medicinsko etiko and a signed informed consent form was obtained from each patient. All experiments were carried out in accordance with the approved guidelines and related regulations.The patient cohort was described and examined by Dragojevič 29. Cells were seeded in T25 cell culture flasks at a density of approximately 12000 and 7000 cells/cm2 depending on the duration of treatment. Transient transfection was performed with plasmid pCMV-ESR1  and X-tremeGENE HP DNA transfection reagent  according to manufacturer’s instructions; however the amount of plasmid DNA was reduced because of high ERα protein levels post transfection. Concomitantly cells were treated with 10 nM 17β-estradiol  or vehicle control for 24 hours. After transfection and pre-treatment cells were exposed to 500 μM H2O2 , 20 µM DFO  or vehicle control. Following 24 and 72 hours of treatment cells were lysed directly in the cell culture flask and total RNA was isolated using miRNeasy Mini Kit  according to manufacturer’s instructions. RNA integrity was checked using 2100 Bioanalyzer . 2 µg of RNA were reverse transcribed with Transcriptor First Strand cDNA Synthesis Kit  according to manufacturer’s instructions.Human osteosarcoma cell line HOS TE-85  was obtained from the American Type Culture Collection  and maintained as described previously29. A complete list and short description of the 48 selected genes is included in Supplementary Table −ΔΔCt method was used for analysis of relative gene expression data30. Ribosomal protein, large, P0 (RPLP0) and lysine acetyltransferase 6B (MYST4) were selected as reference genes based on their stability of expression determined with NormFinder software31.A RealTime ready custom panel 384 – 48  was designed and used for gene expression profiling as described previouslyKAT5, HAT1, MYST1, HDAC6, HDAC7, HDAC9, SIRT1, SIRT6, DNMT3A and MBD1), AOX1, HIF1α, VEGFA, AXIN2, RANKL, OPG, ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER1) were included in the analysis. Primer pairs .In addition to the genes selected in the screening step  was also performed on RNA isolated from bone tissue samples of PMO and OA patients and controls. qPCR analysis was performed as for cell culture experiments with certain exceptions; i.e. only 15 ng of cDNA was used per reaction and RPLP0 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as reference genes32. Gene expression data on RANKL and OPG used in the correlation analysis was already published by Dragojevič et al.27.A gene expression analysis of a subset of genes (post hoc test (age and BMI) or Student’s t test for independent samples . Gender differences between groups were assessed using Fisher’s test. Differences in gene expression and viability of HOS cells between treated groups and controls were assessed using one-way ANOVA with Dunnett’s post hoc test. Gene expression data obtained on bone tissue samples that exhibited normal distribution was analysed using analysis of covariance (ANCOVA) with age, gender and BMI as covariates and Bonferroni post hoc test. Data that didn’t meet the assumptions for parametric tests was first analysed using the Kruskal-Wallis and then a series of Mann-Whitney U tests with a p value corrected according to the Bonferroni method. Spearman’s rho correlation analysis and false discovery rate method to control for multiple testing were employed to examine relationships between gene expression data and bone quality parameters. All tests were performed two-tailed with accepted alpha level of 0.05. Results with a p value of 0.05 or less were considered statistically significant apart from Spearman’s rho correlation analysis where p value of 0.009 was used. All statistical analyses were performed using SPSS Statistics software 22 .Differences in anthropometric parameters between patients were analysed using one-way analysis of variance (ANOVA) with Tukey’s Detailed descriptions of quantitative methylation-specific PCR, chromatin immunoprecipitation, metabolic activity assay and western blot analysis are provided in the Supplementary Information.Supplementary information


The European Water Framework Directive has been adopted by Member States to assess and manage the ecological integrity of surface waters. Specific challenges include harmonizing diverse assessment systems across Europe, linking ecological assessment to restoration measures and reaching a common view on ‘good’ ecological status.2 = 0.32), total nitrogen (r2 = 0.22) as well as chlorophyll-a (r2 = 0.35–0.38) gradients, providing a link between ecological data, stressors and management decisions. Despite differing assessment approaches and initial differences in classification, a consensus was reached on how type-specific macrophyte assemblages change across the ecological status gradient and where ecological status boundaries should lie.In this study, nine national macrophyte-based approaches for assessing ecological status were compared and harmonized, using a large dataset of 539 European lakes. A macrophyte common metric, representing the average standardized view of each lake by all countries, was used to compare national methods. This was also shown to reflect the total phosphorus . Our findings establish a ‘guiding image’ of the macrophyte community at ‘good’ ecological status in hard-water lakes of the Central-Baltic region of Europe.A marked decline in submerged vegetation, especially Charophyta (characterizing ‘good’ status), and an increase in abundance of free-floating plants (characterizing ‘less than good’ status) were the most significant changes along the ecological status gradient. Macrophyte communities of ‘good’ status lakes were diverse with many charophytes and several  In shalIn most European lakes the composition and abundance of macrophytes has changed because of various human pressures . MacrophMany European countries have therefore included macrophytes in their ecological assessment tool-kit, for example, Austria , DenmarkTo ensure comparability of ecological assessment and promote shared levels of ambition among EU member states, the Water Framework Directive  stipulatHowever, ecological assessment is not a panacea that will single-handedly ensure ‘good’ ecological status of European waters. The next steps toward this challenging goal include diagnosing causes , defining management targets  and suggesting restoration measures to remedy the situation. Pressure-response relationships between stressors and biota are one means towards these ends . HoweverLast but not least, it is necessary to communicate about the health of lake ecosystems to the public and decision makers . Great eIn this study, we seek to provide a simple overview of the process of intercalibration performed on assessments of lake ecological status based on macrophytes and then demonstrate that the result has ecological relevance for lake management in Europe.First, we briefly describe macrophyte assessment methods including the process of intercalibration (i.e. comparison of assessment methods and harmonisation of class boundaries). Next, we establish relationships between the common macrophyte assessment and indicators of the focal pressure, eutrophication. Further, we explore the change of macrophyte communities along the ecological status gradient. Finally, we define those taxa characteristic of ‘high’ and ‘good’ status and contrast them with indicators of ‘less than good’ status. Our findings establish a ‘guiding image’ of the macrophyte community at ‘good’ ecological status in hard-water lakes of the Central-Baltic region of Europe.22.1Lake macrophyte assessment methods from nine countries were included in this analysis . The macAll assessment systems follow the WFD approach and are thus based on a change from reference condition with status expressed as an Ecological Quality Ratio (EQR), an approach where natural variability is taken into account by assigning lakes to “types”. Therefore, all Member States have developed type-specific reference values that describe the expected value of their index under near-natural conditions for each lake type in their territory. The most common approaches, mostly used in combination, include the use of data from near-natural sites  or historical data ; only a few countries used modelling  or palaeolimnological data (DE). All indices are expressed as an EQR ranging from 1  to 0 (heavily impacted) which are divided into five classes of biological quality . Various approaches were adopted to define these ecological boundaries, ranging from uniform division of the EQR scale  to more ecological approaches based on shifts in macrophyte communities, for example, changes in dominance from sensitive to tolerant species .2.2a, Secchi depth) as well as macrophyte data. Total phosphorus and chlorophyll-a values were averaged over the growing season, which was defined separately by each country to reflect local climate. Macrophyte data were collected by each country using methods shown in Biological and environmental data from 539 lakes from nine countries were analyzed. The dataset contained descriptive data , physical–chemical data  and depth (0.3–15 m), but all were lowland , hard-water  water bodies. Lakes were allocated to two common intercalibration lake types: LCB1, defined as shallow  and LCB2, defined as very shallow   .−1, total nitrogen (TN) from 0.03 to 11.9 mg L−1, chlorophyll a (chl-a) from 0.5 to 361.6 µg L−1.Lakes represented the entire spectrum of ecological status (macrophyte EQR 0.14–0.90) and trophic conditions, with mean growth season total phosphorus (TP) in the global dataset ranging from 0.006 to 1.46 mg L2.3Step 1: A common dataset was established and all national methods were applied to the datasets of all other countries ;Step 2: Assessments of lakes by all member states methods were corrected for country effects by continuous benchmark standardization ;Step 3: An ‘Intercalibration Common Metric’ (ICM) was calculated. This is the average EQR of all Member State assessment methods except the native one. Thus, in an exercise involving five countries, for country A the ICM is calculated as the average EQR of countries B, C, D and E; for country B – as the average of countries A, C, D and E, etc.;Step 4: In order to be retained in the exercise, regression between the ICM and the national EQR should be significant (P < 0.05) with r values > 0.5 and slopes between 0.5 and 1.5 . If thesStep 5: The boundary values of the different assessment methods were transformed to the common scale and compared with the global mean view of all countries. National boundaries were adjusted so as not to exceed a deviation of ±0.25 class widths from the global mean of all countries . This meThe intercalibration process followed a well-established methodology outlined in the WFD Guidance document  and descWhere low correlations with the common metric were observed and/or where boundaries deviated strongly from the global view of all countries an iterative series of steps were performed to (i) ensure adequate relationship between national method and intercalibration common metric; (ii) to ensure that the boundaries of assessment systems complied with comparability criteria. For several countries  class boundaries were adjusted where necessary to ensure compliance with comparability criteria (+/− 0.25 class widths) for HG and GM boundary bias.2.4Statistical analyses were performed using the R software package .a) as well as against average EQR (EQRavg). The EQRavg is the average EQR of all intercalibrated assessment methods after benchmark standardization and assumes a consistent view of ecological status between member states.The response of national macrophyte indices to eutrophication was tested by calculating linear regressions between national indices and water quality indicators  and EQRavg at ‘moderate’, ‘poor’ and ‘bad’ status (further referred to as ‘less than good status’).To explore the shift in macrophyte composition with lake degradation, the frequency distribution of taxa over the gradient of the global mean EQRavg gradient are considered to be insensitive.Taxa with more than 75% of their occurrences in lake-years above the ‘good’-‘moderate’ status intercalibration boundary are considered indicative for ‘good’ status. Taxa occurring over the whole EQR33.1All methods include abundance and compositional metrics; however the designs differ among countries . Macroph3.2p < 0.001; r2 ranging from 0.4 to 0.7).After harmonization, Member States assessment indices (expressed as EQR) were strongly and significantly related to the Intercalibration Common Metric (ICM), (a for (r = −0.59 LCB1 and r = −0.62 LCB2), all significant with P < 0.001  was also significantly correlated to TP (r = −0.57 both types), TN (r = −0.47 both types) and chl- < 0.001 .Fig. 2Re3.3Species richness of aquatic vegetation  showed aSubmerged macrophytes declined markedly along the ecological status gradient (significant difference among most abundance classes). Lakes without submerged vegetation were associated only with moderate and worse status, whilst lakes with abundant submerged vegetation were associated with ‘good’ and ‘high’ status .Fig. 4BoCharophytes declined along the ecological gradient a; there For rooted floating-leaved vegetation b, there For very shallow lakes (LCB2), the decrease in ecological status was also manifested by increases in non–rooted floating-leaved plants; a high abundance of this group was characteristic for ‘less than good’ status lakes c. A simi3.4The list of potential indicator taxa consists of 53 aquatic macrophyte taxa for LCB1 lakes and 50 for LCB2 lakes . The taxa list was dominated by elodeids (51%) and charids (23%), followed by nymphaeids (12%), lemnids (9%), filamentous algae and bryophytes.Chara contraria, C. hispida, C. rudis, C. tomentosa, C. vulgaris, Nitella flexilis, Nitellopsis obtusa, Potamogeton filiformis and P. friesii) and six taxa for LCB2  can be considered strong indicators, with >90% occurrence in ‘high’ and ‘good’ status (Twenty-six taxa (across both lake types) were considered to be characteristic of good status, as these taxa occurred mainly (>75% of observations) in high and good status lakes. Nine taxa for LCB1 type .Boxplots show 25 and 75% percentiles, medians and ranges of taxa distribution. Sensitive species: 25th percentile > EQRPotamogeton species, Myriophyllum verticillatum, Najas marina (only for LCB1 type), Stratiotes aloides, Utricularia sp. and U. vulgaris (for both types).Good status indicators are taxa that disappear from communities moving down the ecological status gradient. The ‘good’ status indicator list is dominated by charophytes and elodeids , severalP. praelongus, P. friesii and P. gramineus) but some taxa (e.g. Najas marina) were characteristic of good status in only one lake type.The majority of the taxa characteristic of good status were the same for LCB1 and LCB2  can thus be considered as indifferent to the EQR gradient and hence for the pressures to which the assessment methods are designed to be responsive. In terms of numbers of indicator species typically present LCB1 lakes supported 4.5 ± 0.2 sensitive species at good or better status compared with 0.9 ± 0.1 in less than good status lakes, while LCB2 lakes supported 4 ± 0.2 sensitive species at good or better status compared with 0.5 ± 0.1 at less than good status.44.1Macrophyte-based assessment methods have proved indispensable in lake management, especially in lake restoration projects directed at recovery of macrophytes . HoweverSolutions to these problems can be fostered by compiling and analyzing large transnational databases , detectiIn our study, we applied these approaches to a large database of 539 lake-years from nine countries to harmonize their macrophyte assessment systems for two common lake types of Central Europe. This was a complex task, in part because of the surprising variety of ways macrophyte communities are measured and assessed among Member states. Considerable differences exist in survey methods, recording of species abundance, inclusion of certain groups , identification level  and metrics  reflecting different national traditions . Do we nNevertheless, a clear common view emerges for LCB1 and LCB2 lakes from the different national assessment methods. Initially, some relatively large disagreements were experienced by the member states but these were resolved through the harmonization exercise. The remaining disagreement largely results from different views on metrics, focus on different pressures and differences in regional species pools and is unlikely to be solved in the near future.4.2Chara phytocenoses  are significant and similar to those recorded in other studies  or <0.03 mg L−1 . In our study, we used a similar principle, with an important difference in that species are linked not to nutrient concentrations, but to an ecological status assessment harmonised among member states. Therefore, the selected taxa reflect the common view of member states on what macrophyte communities in ‘good’ status lakes should include.The indicator species concept  is widela . For insChara, Nitella, Nitellopsis), several Potamogeton and elodeid species. The dominance of charophytes, their role in maintaining a clear-water state, and their decline due to lake degradation is well described and understood  and Utricularia vulgaris (both types), as well as Stratiotes aloides (both types). Elodeids are typically regarded as eutrophication indicators  and nymphaeids (Persicaria amphibia) which tolerate high turbidity by concentrating their biomass at the water surface during summer. Filamentous algae were also indicators of ‘less than good’ status – several studies have shown positive associations between their development and nutrient enrichment  for defining ‘less than good’ status, in part because poorer status was associated with a reduction in macrophyte abundance and richness. The few taxa characteristic of ‘less than good’ status  react porichment , in spitrichment . Severalnditions , and thecapacity , typicalcapacity .4.5There is an extensive body of work on how macrophyte communities respond to anthropogenic pressures that underpins macrophyte-based assessment methods . HoweverIt is widely accepted that target setting should be based on pressure-response relationships  and thatPotamogeton spp. and several other taxa   . In the ria sp.) .Fig. 7ShWhat are the benefits of setting a ‘guiding image’ for ‘good’ ecological status? Firstly, it helps to convey ecological data to the public and non-technical stakeholders in a more visual way . Secondl


After 2005, consumption of forage fish increased for all predator species, suggesting diet flexibility with changing abiotic and biotic conditions. An associated temporal shift from a trophically diverse to a trophically redundant predator assemblage occurred where predators now play similar trophic roles by consuming prey primarily from the pelagic energy pathway. Overall, these long-term ecological changes signify that trophic shifts of a high trophic-level predator assemblage associated with climate change have occurred in the Arctic food web.Climate change is leading to northward shifts in species distributions that is altering interspecific interactions at low- and mid-trophic levels. However, little attention has been focused on the effects of redistributions of species on the trophic ecology of a high trophic-level predator assemblage. Here, during a 22-year period (1990–2012) of increasing sea temperature (1.0°C) and decreasing sea ice extent (12%) in Cumberland Sound, Nunavut, Canada, we examined the trophic structure of a near-apex predator assemblage before (1990–2002) and after (2005–2012) an increase in the availability of capelin—generally an indicator species in colder marine environments for a warming climate. Stable isotopes (δ The standard reference material was Pee Dee Belemnite carbonate for CO2 and atmospheric nitrogen N2. A triplicate was run for every 10th sample, and a measurement precision for δ13C and δ15N was 0.1‰ and 0.1‰, respectively. The instrumentation accuracy was determined based on NIST standards 8573, 8547 and 8548 for δ15N values and 8542, 8573, 8574 for δ13C values . The mean differences from the certified values were ≤0.1‰ for δ15N values and ≤0.1‰ for δ13C values.Owing to the presence of lipids affecting fish and mammal tissue δC values ,52, frozC values . Subsequ2.4.13C and δ15N values of each prey and electronic supplementary material, figures S1 and S2 for stable isotope bi-plots). Prior to analysis, we assessed normality of δ13C and δ15N using a χ2 quantile–quantile plot for each predator species. As capelin and Arctic cod are a part of the same functional group (i.e. forage fish) and δ13C and δ15N values for the two species were similar , these prey items were combined to reduce the total number of prey sources to four and allow a more constrained, diffuse solution [13C: 1.3‰, Δ15N: 2.4‰ [13C: 1.3‰, Δ15N: 1.2 ‰ [13C and δ15N values relative to diet isotope values; 1.2‰ for Δ13C and 2.1‰ for Δ15N [13C and δ15N) for each species . As well, few individuals who did not fall within simulated mixing polygons  were removed prior to mixing model analysis  were performed. Alpha was set to 0.05. To quantify prey contributions to focal predators  over the two defined study periods, we used Bayesian mixing model analysis in SIAR v. 4.2.2  in R v. solution . For rin5N: 2.4‰ ) and cetN: 1.2 ‰ ), respecfor Δ15N ,60. Follfor Δ15N , we incosis  for statistical comparisons among predators. To examine differences in SEAB between both time periods, we calculated percentage of estimates from the posterior probability distribution for SEAB that were lower in 2005–2012 versus 1990–2002 relative to the total number of estimates from the posterior probability distribution (10 000).To examine variation in isotopic niche sizes and relative niche position for each predator species over the two study time periods, standard ellipses were estimated using the SIBER package v. 2.0.3  in R whital data . The nic13C–δ15N bi-plot space and the relative trophic position of the predator assemblage to provide a measure of trophic diversity and redundancy. The δ13C and δ15N ranges measure the distance between the two individuals with highest and lowest values, and thus represent the variability in basal carbon source and relative trophic position of the predator assemblage. Mean distance to centroid is the mean Euclidean distance of each species' niche to the δ13C–δ15N centroid of the predator assemblage and represents the overall degree of trophic diversity. Mean nearest neighbour distance is the mean Euclidean distance to each species’ nearest neighbour in isotopic space, thereby representing density of species packing where species with similar trophic ecologies (i.e. trophic redundancy) exhibit smaller mean nearest neighbour distances. Standard deviation of the nearest neighbour distance is the standard deviation of Euclidean distance of each species to its nearest neighbour and thus represents packing of species in isotopic space and trophic redundancy. Total community area was modified by calculating the total isotopic area among the means of each species' niche and thus is less biased to convex hull extremities [13C and δ15N of selected prey sources between both time periods were similar allowing a comparison of the community-wide metrics between both time periods  occurred and an increase in temperature of 1°C was found .Over the entire study period (1990–2012), there were marked shifts in sea ice concentrations in the Davis Strait and summer sea surface temperatures within Cumberland Sound; a 12% decline in sea ice extent  and δ15N (i.e. relative trophic position) ranges for the predator assemblage in 2005–2012 was greater than 98% (figures All six community-wide metrics of trophic structure were lower in the most recent sampling period 2005–2012) than the 1990–2002 time period . By anal005–2012 4.With a warming ocean and continuing reduction in sea ice extent, Arctic marine ecosystems continue to face multiple abiotic and biotic stressors that are impacting species interactions and overall ecosystem structure and function ,64. WhilBeluga whales, ringed seals, Greenland halibut and Arctic char exhibited flexible foraging behaviour over the study period with consumption of higher proportions of forage fish in the 2005–2012 time period. Flexible foraging behaviour is pervasive in nature and allows opportunistic predators to exploit shifting prey diversity and abundance in response to seasonal and inter-annual variations in environment . For exaThe most probable explanation for the increase of forage fish consumption among the four near-apex predator species is the increased availability of capelin since the mid-2000s. Cumberland Sound beluga whales have been previously documented to consume both Arctic cod and capelin, though with an increasing reliance on capelin over time ,30. In aGiven that capelin are highly adapted to survive across a broad range of temperatures (2°C to 12°C), and their northward expansion in distribution is linked with increasing sea temperatures , CumberlComparison of the metrics measuring the trophic structure of the predator assemblage between 1990–2002 and 2005–2012 in Cumberland Sound revealed the latter having less variability in basal carbon source use and trophic position, such that the community exhibited less trophic diversity and more trophic redundancy. Warming waters and phenological changes to sea ice over time strongly influence the seasonal pulse of algal productivity during the summer allowing more prolonged availability of this energy pathway to consumers . In turnClimate-driven community shifts in the Arctic will probably accelerate with warming temperatures and decreasing sea ice leading to more subarctic and temperate species, from phytoplankton to predators, invading Arctic waters . This no


Vernonia amygdalina and Chromolaena odorata and were applied as follows: 10 tonnes ha−1 vernonia + 0 tonnes ha−1 chromolaena (T1), 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2), 5.0 tonnes ha−1 vernonia + 5.0 tonnes ha−1 chromolaena (T3), 2.5 tonnes ha−1 Vernonia + 7.5 tonnes ha−1 chromolaena (T4), 0 tonnes ha−1 vernonia + 10 tonnes ha−1 chromolaena (T5) while in-organic fertilizer (NPK 20:10:10) was applied at 200 kg NPK ha−1 (T6) and there was a control plot (T7). The experiment was laid out in a Randomized Complete Block Design (RCBD) replicated four times. Vegetative, yield and quality parameters of radish were taken. Data collected were subjected to Analysis of Variance (ANOVA) using Statistical Analysis Software (S.A.S), 2000. Treatment means were compared using Duncan Multiple Range Test (DMRT) at 0.05 level of probability. The study showed that application of green manures increased vegetative, yield and yield parameters and were comparable with application of NPK fertilizer while there was a significant increase in the nutritional composition of radish with application of green manures when compared with NPK and control. It can therefore be concluded that application 10 tonnes ha−1 vernonia + 0 tonnes ha−1 chromolaena (T1) as green manure increased vegetative, yield and yield parameters while application 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2) improved radish quality.Field experiments were carried out during the 2016 and 2017 cropping seasons at the Teaching and Research Farm, Landmark University, Omu-Aran, Kwara State (latitude 8.9°N and longitude 50°61 E.), Nigeria, to study the effect of some green manures as an alternative to inorganic fertilizer on growth, yield, mineral and proximate composition of radish. Green manure composed of leaves of  Raphanus sativus L.), a cruciferous and nutritious root vegetable which is a good source minerals and vitamins belongs to the family Brassicaceae. It originated from Europe or Asia and is presently cultivated all over the world. Depletion in soil nutrients is considered as one of the most serious challenges facing agricultural sector in recent times resulting in significant reduction in crop growth and yield1. Continuous cropping on the same piece of land over time reduces the soil nutrients thereby causing a reduction in the yield of crop2.Radish  of the United StatesThe pre-planting soil analysis for the two years is as shown in Table The meteorological data for the periods of the experiment is as shown in Table Table 6) resulted in taller plants which was similar to the application 10 tonnes ha−1 vernonia + 0 tonnes ha−1 chromolaena (T1) in both years and 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2) in 2016. There was no significant difference in the application of other treatments except the control (T7) which gave the least value for plant height.The effect of vernonia and chromolaena leaves on plant height and number of leaves of radish is as shown in Table 7) which gave the least number of leaves in 2016 and 2017 cropping seasons, more number of leaves was observed with the application of NPK (T6). The values were statistically similar with other treatment values in 2017 and 10 tonnes ha−1 vernonia + 0 tonnes ha−1 chromolaena (T1) alone in 2016.Compared with the control (T6) and 10 tonnes ha−1 vernonia + 0 tonnes ha−1 chromolaena (T1) on yield and yield attributes of radish  which gave the least values for yield and all its attributes in both years, application of NPK fertilizer (T6) and 10 tonnes ha−1 vernonia + 0 tonnes ha−1 chromolaena (T1) significantly increased values for all the parameters. Application of 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2), 5.0 tonnes ha−1 vernonia + 5.0 tonnes ha−1 chromolaena (T3), 2.5 tonnes ha−1 vernonia + 7.5 tonnes ha−1 chromolaena (T4) and 0 tonnes ha−1 vernonia + 10 tonnes ha−1 chromolaena (T5) resulted in varying and non-significant values for yield and its attributes.The significant influence of NPK fertilizer , application of NPK (T6) significantly increased moisture content (M.C) while sole and combined application of green manures resulted in varying but statistically similar values. Percentage protein, fat and carbohydrate (CHO) increased with application of 10 tonnes ha−1 vernonia + 0 tonnes ha−1 chromolaena (T1) and 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2) though the values were not significant with percentage fat when other green manures applied. Application of NPK (T6) and control significantly reduced values for protein, fat and CHO. Control produced higher values for fibre and ash while application of sole and combined green manures gave statistically similar values.Application of NPK fertilizer, sole and combined green manures significantly influenced proximate composition of radish Fig. . Compare−1 vernonia + tonnes ha−1 chromolaena (T1) and 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2) having higher and statistically similar values  fertilizer and control (T7) which gave statistically similar but lower values. Application of 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2) significantly increased values for P. K and Mg though statistically similar with the application of NPK (T6) only in P and K. There was no significant difference in P, K and Mg values when 5.0 tonnes ha−1 vernonia + 5.0 tonnes ha−1 chromolaena (T3), 2.5 tonnes ha−1 vernonia + 7.5 tonnes ha−1 chromolaena (T4) and 0 tonnes ha−1 vernonia + 10 tonnes ha−1 chromolaena (T5) were applied. Control plots showed least values for P, K and Mg.Mineral composition of radish root also varied significantly with the application of green manures and NPK fertilizer Table . CalciumLaboratory determination of the nutrient elements contained in the leaves of vernonia and chromolaena revealed that they both contained mineral elements in varying proportions suitable for the cultivation of radish. There was quick response of radish when both the green manures and inorganic fertilizer were applied in both years of the experiments. The response of radish to the amendments could be as a result of continuous cropping and the type of dominant weeds that were found on the experimental field which have depleted most of the essential nutrient elements required for the cultivation of radish.−1 vernonia + 0 tonnes ha−1 chromolaena (T1) alone and 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2). Increase in the rate of application of vernonia was also found to increase values for plant height and number of leaves. The increase in the vegetative parameters as a result of application of green manures could be attributed to the chemical composition of the green manures which increased soil availability of the nutrients most especially Mg which is part of chlorophyll molecule located in the thylakoid membrane of the leaves that are essential for increased photosynthesis16. Also found that incorporation of green manures increased soil availability of OM, N, P, K and Mg. Increase in plant height and number of leaves of radish could also be adduced to high moisture content and low C:N ratio contained in the leaves of vernonia and chromolaena leading to early mineralization and fast release of nutrients17. Similar result was obtained by18, who reported that the application of organic fertilizers to soil supply plant nutrients for increased plant height and more leaves of shallot.Vegetative parameters were significantly affected by the application of green manures of vernonia and chromolaena. Plant height and number of leaves were higher in plants treated with NPK fertilizer which was comparable with plots treated with 10 tonnes ha6) and 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2) increased vegetative and yield parameters of radish. This could be as a result of nitrogen that was present in both amendments which helped to increase plant growth and development. Nitrogen is frequently the most limiting essential nutrient in natural ecosystems19. The increase in these parameters as a result of application of NPK fertilizer could be due to early availability of the nutrients culminating into enhanced nutrient uptake and hence faster growth. This is in line with the work of some researchers20 who found that while nutrient supplied by in-organic fertilizer was readily available, the nutrient supplied by organic fertilizer was released slowly.Results of this study showed that application of NPK (T−1 vernonia + 0 tonnes ha−1 chromolaena (T1) increased yield and yield parameters  of radish and it was comparable with application of NPK fertilizer.Incorporation of 10 tonnes ha21 where they found that biomass yield, root weight, root length and root diameter of radish increased with application of tithonia up to 10 tonnes ha−1. Increase plant vigour and yield as a result of application of green manures could be adduced to its ability to improve soil physical and chemical properties22.Taller plants and more number of leaves may have contributed to increased yield and yield parameters. This is because plant height and increased number of leaves could accumulate more environmental resources thereby transporting them to the sinks (roots) of the plant. Similar result was by23.Sole application of vernonia performed better than sole application of chromolaena in terms of vegetative growth, yield and yield parameters and in the same vein, results showed that, as the level of vernonia increased in the combination of the green manures, the better the performance of radish. This could be as a result of the inherent chemical composition of vernonia. It could also be as a result of its high organic carbon when compared with chromolaena. High organic matter in the soil as a result of application of green manure must have increased microbial activities in the soil thereby increasing soil physical characteristics and porosity. Presence of green manures increased activities of beneficial soil fauna and organic matter decomposition, which could lead to enhanced soil porosity and reduced soil bulk density24. In their experiments found that rate of litter decomposition depends on the C:N ratio and/or nitrogen content of the leaf litter. There was a consistent increase in growth, yield and yield parameters of radish in 2016. This could be as a result of higher rainfall experienced during the weeks of the experiment which facilitated decomposition of the green manures thereby releasing its nutrients for plant use.Result of the study also showed that the rate of mineralization of vernonia and chromolaena leaves varied significantly with vernonia having higher degree of mineralization. This could be attributed to higher N value and lower C:N ratio of vernonia−1 vernonia + 0 tonnes ha−1 chromolaena (T1) and NPK (T6). Increase in the values for these parameters could be as a result of one of the functions of phosphorus in facilitating the production of more roots or that the soil was low in the nutrient. Like nitrogen, phosphorous is an essential constituent of the genetic material and augments cell division22.Phosphorus fertilizer facilitates the production of more roots and increased number of branches. Both the green manure and NPK contained suitable quantity of phosphorus. Total biomass and root yield increased with application of 10 t ha−1 vernonia + 0 tonnes ha−1 chromolaena (T1) competes favorably with NPK (T6). This could be as a result of presence of calcium in vernonia which function in plant root and tip elongation25. Reported that soil applied with tithonia enhanced stem girth better than NPK applied soils.Application of 10 tonnes ha26. Concluded that compost application to nutrient limiting soil improved quality and quantity of both biomass and proximate constituents of maize.Application of vernonia and chromolaena leaves as green manures either as sole or in combination with each other increased the proximate composition of radish root. This increase could be as a result of availability of sufficient nutrient for plant uptake which was later translocated into the appropriate sources where they were partitioned into their respective sinks−1 vernonia + 0 tonnes ha−1 chromolaena (T1) and 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2) having higher values when compared with application of NPK (T6) and the control (T7). This could also be attributed to increased availability of nutrients in soil as a result of the mineralization of the manures leading to increased uptake by the plants. Similar result was by27 who found that conventional production system of lettuce resulted in lower vitamin C content28. Also reported that the vegetables produced under organic systems frequently had higher contents of vitamin C, when compared with those produced conventionally.Sole and combined application of green manures increased vitamin C content and Mg of radish roots with application of 10 tonnes haLaboratory analysis of vernonia and chromolaena showed that there was no significant difference in Ca values of both leaves. This could possibly explain why Ca values of radish root did not differ significantly among sole and combined application of green manures. Application of green manures and NPK fertilizer increased P and K contents of radish roots significantly compared with the control. This could be as a result of reasonable amount of the nutrients present in the leaves of the green manures and the in-organic fertilizer used for the study. The reason for low level of P, K and Mg in radish root in the control plots could be that the soil has been depleted of the nutrients as a result of continuous cropping and the type of dominant weeds found on the field before the start of the experiment.−1 vernonia + 0 tonnes ha−1 chromolaena (T1) as green manure is recommended for increased vegetative, yield and yield parameters, 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2) is recommended for improved vitamin C, protein, carbohydrate and mineral content of radish.The observed response of radish to sole and combined application of vernonia and chromolaena suggests that they can be used as an alternative to in-organic fertilizer because they contained mineral nutrients in varying proportions and are capable to improved soil physical and chemical properties. It can therefore be concluded that while 10 tonnes ha0 9′ N and long 50 61′ E.) located in the derived savanna ecological zone of Nigeria to study the effect of fresh leaves of vernonia and chromolaena as green manures on the growth, yield and nutritional composition of radish. The ecological zone has an annual rainfall pattern which extends between the months of April and October and it ranges between 600–1500 mm, with peak rain in May-June and September-October, while the dry season is between November and March. The experimental sites had been under continuous cropping for more than five years while the dominant weeds were itch grass (Rottboellia cochinchinensis (Lour) Claton), goose grass (Eleusine indica (L.) Gaertn), sour millet (Echinochloa colona (L.) Link), Milkweed (Euphorbia heterophylla L) and goat weed (Ageratum conyzoides L.).The experiment was conducted during 2016 and 2017 cropping seasons at the Teaching and Research Farm of Landmark University, Omu-Aran, Kwara State  = 122 m2.Pre-cropping soil samples were collected from each plot during the cropping seasons and bulk to determine the physicochemical properties of the soil. Mechanical land preparation was adopted using the tractor drawn disc plough and harrow. The land was ploughed once and harrowed twice to give a well pulverized soil. Thereafter the field layout was carried out to mark out the appropriate number of treatment plots. The size for each bed was (2 m × 2 m) = 4mGreen tender leaves and stem of vernonia were collected from the Research Farm of the Biofuels Alternatives and Renewable Energy Ltd, Edidi, Kwara State Nigeria while chromolaena was collected on a fallow field at Landmark University Teaching and Research Farm, Omu-Aran. The leaves were picked from the stem, chopped into pieces with a sharp knife for easier decomposition, weighed and applied to a depth of about 10 cm two weeks before seed sowing using hand held hoes according to the layout.−1 vernonia + 0 tonnes ha−1 chromolaena (T1), 7.5 tonnes ha−1 vernonia + 2.5 tonnes ha−1 chromolaena (T2), 5.0 tonnes ha−1 vernonia + 5.0 tonnes ha−1 chromolaena (T3), 2.5 tonnes ha−1 vernonia + 7.5 tonnes ha−1 chromolaena (T4), 0 tonnes ha−1 vernonia + 10 tonnes ha−1 chromolaena (T5), 200 kg NPK ha−1 (T6) and no fertilizer/green manure control (T7). There were seven (7) plots in each replicates, making a total of twenty eight (28) experimental units. Treatments were laid out in Randomised Complete Block Design and replicated four times.The followings are the treatment combinations which were applied as green biomass two weeks before seed sowing to give room for decomposition: − 10 tonnes ha−1. Inorganic fertilizer (NPK 20:10:10) was manually applied to the assigned plots at the rate of 200 kg NPK ha−1  two weeks after sowing by side placement 5–8 cm away from the base of the plant.The variety of radish used for the experiment was ‘French breakfast’. At two weeks after incorporation of green manures, seventy two seeds were sown second week of June during the experimental years at a depth of 5 cm on a prepared 2 m by 2 m bed at two seeds per hole with 30 cm by 30 cm inter and intra-row spacing. Plants were thinned to one plant per stand one week after sowing leaving a total number of 36 plants per bed i. e 11, 111 stands haManual weeding was done 2 weeks after sowing (WAS) and it was supplemented with hand-picking of weeds 4 WAS. Radish roots were harvested 6 WAS by hand pulling the roots from the ground. The following vegetative, yield and yield component parameters were taken in both years of the experiment, plant height, number of leaves, total biomass, root weight, root length, root diameter while proximate, minerals and vitamin C values were collected for the roots as post-harvest parameters.oC and grinded in a Willey mill. Leaf samples of vernonia and chromolaena were analysed for leaf N, P, K, Ca and Mg while radish root samples were analysed for P, K, Ca and Mg as described by29. Leaf N was determined by the micro-Kjeldahl digestion method. Ground samples were digested with nitric-perchloric-sulphuric acid mixture for the determination of P, K, Ca and Mg. Phosphorus was determined colorimetrically using the vanadomolybdate method, K was determined using a flame photometer and Ca and Mg were determined by the EDTA titration method30. The percentage of organic carbon in the sample was determined by the Walkley and Black procedure using the dichromate wet oxidation method31. Sample pH was determined by using a soil–water medium at a ratio of 1:2 using Jenway digital electronic pH meter model 352032. Vitamin C content of radish root was determined by using the indophenol dye method33.Samples of vernonia, chromolaena and radish root were collected fresh and oven-dried for 24 h at 80Proximate analysis of radish root was carried out at the central laboratory, Landmark University, Nigeria. Root samples were grinded and analyzed for their proximate composition under the Parten D analyzer. The root samples were analyzed to determine the Moisture content, crude fiber content, carbohydrate content, protein content, ash content and the Fat content.34. The significant treatment means were compared using Duncan Multiple Range Test (DMRT) at 0.05 level of probability.Data collected were subjected to statistical analyses of variance (ANOVA) using statistical Analysis Software


Scientific Reports 10.1038/s41598-018-33124-4, published online 12 November 2018Correction to: Part of the paper presents results  for temporal “clusters,” or time periods associated with anomalously high incidence rates of Kawasaki disease in the San Diego region. In the Article, these clusters were defined using a threshold density of at least 4 Kawasaki disease cases in a 7-day period. However, the analysis was conducted using a definition of 5 or more cases in a 7-day period. As a result, this Article contains errors.The legend of Figure 1 is incorrect,“Blue vertical bars show temporal clusters, defined as 7-day windows including onsets of 4 or more KD cases .”should read:“Blue vertical bars show temporal clusters, defined as 7-day windows including onsets of 5 or more KD cases .”In the Results section, under the subheading ‘Seasonality and temporal clustering’,“In addition to the seasonal pattern of KD in San Diego, there are also anomalous high-incidence periods of shorter duration . Temporal clusters were defined as seven consecutive days encompassing the onset of four or more KD cases .”should read:“In addition to the seasonal pattern of KD in San Diego, there are also anomalous high-incidence periods of shorter duration . Temporal clusters were defined as seven consecutive days encompassing the onset of five or more KD cases .”Figure In the Methods section, under the subheading ‘Temporal cluster construction’,“By using a 7-day moving window over the time period from January 2002 to April 2017, we first defined all of the days in the analysis period as “cluster” days if they fell within a 7-day window that contained 4 or more cases, or “non-cluster” days if they were not part of a 7-day window with at least 4 KD cases.”should read:“By using a 7-day moving window over the time period from January 2002 to April 2017, we first defined all of the days in the analysis period as “cluster” days if they fell within a 7-day window that contained 5 or more cases, or “non-cluster” days if they were not part of a 7-day window with at least 5 KD cases.”In the Methods section, under the subheading ‘Monte Carlo Methods’,“For the temporal clustering, we randomly distributed the same number of cases over the time period, with the observed seasonality, and counted the number of clusters using the method described above.”should read:“For the temporal clustering, we randomly distributed the same number of cases over the time period, with the observed seasonality and a linear trend, and counted the number of clusters using the method described above.”Finally, Table 1 should correspond to the temporal cluster construction (at least 5 cases in 7 days) that is used throughout the paper. As a result, the legend,“An illustrative example of temporal cluster construction, with a simulated set of 10 KD case onsets (second row) over a period of 22 consecutive days. By our definitions, this would be coded as 2 temporal clusters ; 9 of the 10 cases here would be Cluster KD cases, and 1 (occurring on Day 9) would be a Non-Cluster KD case. The “Cluster Day” row indicates whether a given day in the time series falls within any 7-day window that contains at least 4 KD onsets. “Cluster KD Day” indicates a KD onset day defined as within a temporal cluster. These are the designations used in the construction of climate anomalies, and in all analyses grouped by temporal cluster membership . Although the initial definition is at least 4 KD case onsets in a period of 7 days, the final cluster length can extend beyond 7 days and final cluster size can extend beyond 4 cases, since a cluster is extended until a “break” occurs and there is a non-cluster day . This is why the vertical axis of Fig. should read:“An illustrative example of temporal cluster construction, with a simulated set of 11 KD case onsets (second row) over a period of 25 consecutive days. By our definitions, this would be coded as 2 temporal clusters ; 10 of the 11 cases here would be Cluster KD cases, and 2 (occurring on Days 9 and 25) would be a Non-Cluster KD cases. The “Cluster Day” row indicates whether a given day in the time series falls within any 7-day window that contains at least 4 KD onsets. “Cluster KD Day” indicates a KD onset day defined as within a temporal cluster. These are the designations used in the construction of climate anomalies, and in all analyses grouped by temporal cluster membership . Although the initial definition is at least 5 KD case onsets in a period of 7 days, the final cluster length can extend beyond 7 days and final cluster size can extend beyond 5 cases, since a cluster is extended until a “break” occurs and there is a non-cluster day . This is why the vertical axis of Fig. The correct Table